CN114808276B

CN114808276B - Preparation method of nerve conduit with three-layer structure and nerve conduit

Info

Publication number: CN114808276B
Application number: CN202110086162.1A
Authority: CN
Inventors: 薛佳佳; 余逸玲; 张立群; 张馨丹; 龚博文
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2021-01-22
Filing date: 2021-01-22
Publication date: 2023-05-26
Anticipated expiration: 2041-01-22
Also published as: CN114808276A

Abstract

The invention discloses a preparation method of a nerve conduit with a three-layer structure and the nerve conduit. The method comprises the following steps: (1) Adding soluble aliphatic polyester or soluble aliphatic polyester and natural high molecular polymer into the solvent A, and fully dissolving to obtain a solution D; (2) Carrying out electrostatic spinning on the solution D by using a roller rotating at a high speed as a receiver to obtain an electrospun fiber membrane with an orientation structure; (3) Coaxially and electrostatically spraying the shell layer solution E and the core layer solution F onto the electrospun fiber membrane of the orientation structure; step (4), carrying out electrostatic spinning deposition on the solution D to obtain an electrospun fiber film loaded with coaxial electrospun particles, so as to form the electrospun fiber film with a three-layer structure; (5) And crimping the electrospun fiber membrane with the three-layer structure into a tube, and bonding the tube with the solution D to obtain the nerve conduit with the three-layer structure. The nerve conduit can effectively accelerate the repair of peripheral nerves and improve the repair effect.

Description

Preparation method of nerve conduit with three-layer structure and nerve conduit

Technical Field

The invention relates to the technical field of biological materials, in particular to a preparation method of a nerve conduit with a three-layer structure and the nerve conduit.

Background

Since the clinical peripheral nerve injury cases in China reach 2000 tens of thousands, and the peripheral nerve injury increases at a speed of about 200 tens of thousands of cases each year, the peripheral nerve injury has become a serious scientific problem for the study in the field of nerve repair. The clinical gold standard is autograft; however, autografts have limited sources, damage to donor areas, size mismatch, secondary surgery, and the like, limiting their use. Therefore, development of an artificial nerve conduit stent as a substitute is desired to solve the problem of the lack of autograft.

The artificial nerve conduit stent material which is clinically available at present mainly plays a role in guiding broken ends of injured peripheral nerves to bridge and degrade after repair is completed, but has low biological activity and little loading of growth factors or medicines, so that the repair speed of the peripheral nerve injury is slower. Therefore, development of a nerve conduit having a microenvironment necessary for repair of peripheral nerve injury is extremely important for repair of peripheral nerve injury.

In recent years, a great deal of innovative research is carried out on the development of nerve conduits by researchers, however, the preparation method of the bioactive nerve conduit which is loaded with growth factors or medicines and has a slow release function is less, the nerve conduit with a three-layer structure is constructed, microparticles with a core-shell structure containing the growth factors are loaded, the cell microenvironment is regulated and controlled while the peripheral nerve regeneration is guided, the schwann cells are recruited, the axon extension is guided, and the peripheral nerve repair process is accelerated, so that the peripheral nerve regeneration is promoted; the nerve conduit may be degraded and replaced by a new nerve or may be degraded and separated from the surface of the peripheral nerve after healing.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a preparation method of a nerve conduit with a three-layer structure and the nerve conduit. The effects of slow release of growth factors or medicines and the like are realized by regulating and controlling the spinning fiber topological structure and combining electrospray particles with a core-shell structure, and the difficulties existing in the current research can be solved, so that various induction signals for promoting the repair of the peripheral nerve are combined efficiently, the repair of the peripheral nerve is accelerated effectively, the repair effect is improved, and a foundation is laid for further developing nerve conduit materials which can be used in clinical transformation.

In the selection of the type of nerve conduit used to construct the electrospun particles, electrospun fibers are of great interest due to the nature of their extracellular matrix-like structure. Various materials can be prepared into nerve conduits for repairing peripheral nerve injury by using an electrostatic spinning technology, for example, natural materials such as collagen, gelatin, chitosan and the like, and synthetic materials such as polycaprolactone, polylactic acid, polyurethane and the like.

The spinning fiber scaffold with a single structure often cannot repair damage of peripheral nerves at a longer distance, so that the surface topology structure and the functionality of the spinning fiber are regulated, the growth factors for promoting repair are added, the bioactivity of the scaffold material is endowed, and slow release is realized, so that the scaffold is a key and current research hot spot for improving the repair effect of the peripheral nerve damage. The surface morphology of the nanofiber scaffold prepared by electrostatic spinning has a regulating and controlling effect on cell behaviors including adhesion, migration, proliferation and differentiation. The oriented fiber structure can induce the migration of nerve cells along the orientation direction of fibers, and is beneficial to the adhesion and proliferation of cells. The nanofibers of the oriented structure are more advantageous in promoting the extension of axons and the ordered release of active particles than the nanofibers of the random structure. Therefore, the nanofiber using the electrostatic spinning can be used as a nerve tissue engineering scaffold, and provides a proper microenvironment for the regeneration of peripheral nerves.

The invention aims at providing a preparation method of a nerve conduit with a three-layer structure.

The method comprises the following steps:

step (1), adding soluble aliphatic polyester or soluble aliphatic polyester and natural high molecular polymer into a solvent A, and fully dissolving to obtain a solution D;

step (2), carrying out electrostatic spinning on the solution D by adopting a roller rotating at a high speed as a receiver to obtain an electrospun fiber membrane with an orientation structure;

step (3), adding a solvent B into the natural high molecular polymer to obtain a shell layer solution E; adding a growth factor into the solvent C to obtain a nuclear layer solution F; coaxially and electrostatically spraying the shell layer solution E and the core layer solution F onto the oriented electrospun fiber membrane obtained in the step (2) to obtain an electrospun fiber membrane loaded with coaxial electrospun particles;

step (4), carrying out electrostatic spinning deposition on the solution D on the electrospun fiber membrane loaded with the coaxial electrospun particles and obtained in the step (3), so as to form a electrospun fiber membrane with a three-layer structure;

and (5) crimping the electrospun fiber membrane with the three-layer structure, and bonding the electrospun fiber membrane by using the solution D to prepare the nerve conduit with the three-layer structure.

In a preferred embodiment of the present invention,

the mass concentration of the solution D is 3-20%; preferably 5 to 15%;

the use amount of the natural high molecular polymer is 0 to 100 weight parts based on 1 weight part of the aliphatic polyester;

the solvent A is at least one selected from hexafluoroisopropanol, trifluoroethanol, chloroform, methanol, dichloromethane and N, N' -dimethylformamide; and/or the number of the groups of groups,

the degradable aliphatic polyesters are: one or a combination of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid-glycolic acid-caprolactone copolymer; and/or the number of the groups of groups,

the natural high molecular polymer is: collagen, gelatin, chitosan, starch, cellulose, elastin, or a combination thereof.

In a preferred embodiment of the present invention,

the growth factor is one or the combination of vascular endothelial growth factor, nerve growth factor, platelet derived growth factor, epidermal growth factor, fibroblast growth factor and hepatocyte growth factor;

the natural high molecular polymer is one or a combination of collagen, gelatin, chitosan, starch, cellulose and elastin;

in a preferred embodiment of the present invention,

step (3),

the solvent B is at least one of acetic acid, hexafluoroisopropanol, trifluoroethanol, chloroform, methanol and dichloromethane;

the concentration of the shell layer solution E is 5-100 ug/mL; preferably 10-50mg/mL

The solvent C is at least one selected from acetic acid, water, ethanol, methanol and physiological saline;

the concentration of the nuclear layer solution F is 1-100 ug/mL; preferably 20-50ug/mL.

In a preferred embodiment of the present invention:

the dosage ratio of the shell layer solution to the core layer solution is (1-10): 1, a step of; preferably (2-5): 1, a step of;

the dosage ratio of the electrospun fiber membrane to the sum of the shell solution and the core-shell solution is as follows: 1g (5-100 mL); preferably 1g (50-100 mL).

In a preferred embodiment of the present invention,

step (4),

the dosage ratio of the electrospun fiber film loaded with the coaxial electrospun particles to the solution D is as follows: 1g: (1-50 mL); preferably 1g: (20-50 mL).

In a preferred embodiment of the present invention,

spinning process conditions of the step (2): the advancing speed of the solution D is 0.1-5 mL/h, the voltage is 8-25 kV, the rotating speed of the roller is 100-3000rpm, the receiving distance is 10-30 cm, and the spinning is 10-720 min.

In a preferred embodiment of the present invention,

in the coaxial electrostatic spraying, the propelling speed of the shell layer electrospray solution is 0.3-6 mL/h, the propelling speed of the core layer electrospray solution is 0.1-3 mL/h, the voltage is 10-30 kV, the receiving distance is 10-30 cm, and the spraying time is 1-360 min.

In a preferred embodiment of the present invention,

and (4) electrostatic spinning is deposited on the electrospun fiber film loaded with the coaxial electrospun particles, the advancing rate of the solution D is 0.1-6 mL/h, the voltage is 8-25 kV, the receiving distance is 10-30 cm, and the spinning is carried out for 10-720 min.

Another object of the present invention is to provide a nerve conduit with a three-layer structure.

The nerve conduit is of a three-layer structure, the inner layer is an electrospun fiber layer of an orientation structure, the middle layer is an electrospun particle loading layer, and the outer layer is a random electrospun fiber layer.

The inner diameter of the nerve conduit is 0.5-2.0mm.

The invention adopts the following technical scheme:

the nerve conduit with the three-layer structure comprises an inner layer, an intermediate layer and an outer layer structure, wherein the inner layer structure is an oriented electrospun fiber layer, the intermediate layer structure is an electrospun particle loading layer, the electrospun particle loading layer of the core-shell structure comprises electrospun particles, the electrospun particles are of a core-shell structure, a shell layer of the electrospun particles is a solution prepared by a natural high polymer, and the core layer is a solution prepared by a growth factor.

The deposition concentration of the electrospray particles containing the growth factors on the surface of the intermediate layer structure is uniformly distributed.

The polymer is degradable aliphatic polyester or a blend of the degradable aliphatic polyester and natural high molecular materials.

The polymer fiber is degradable aliphatic polyester with uniform thickness or blend fiber of the degradable aliphatic polyester and natural high polymer material.

The inner layer structure is an oriented electrospun fiber layer;

the outer layer structure is a random electrospun fiber layer;

the invention relates to a preparation method of a nerve conduit with a three-layer structure for repairing peripheral nerve injury, which comprises the steps of forming a fiber layer with an inner layer orientation structure by electrostatic spinning of a solution containing degradable aliphatic polyester or a blend fiber of the degradable aliphatic polyester and a natural high polymer, forming a particle load layer with a core-shell structure by preparing a solution with a core layer as a growth factor by using a solution prepared by natural high polymer material or the degradable aliphatic polyester, and forming a random orientation fiber layer with an outer layer structure on the load layer by electrostatic spinning of the solution containing the degradable aliphatic polyester, thereby obtaining the nerve conduit with the three-layer structure for repairing peripheral nerve injury.

The preparation method specifically comprises the following steps:

step (2), carrying out electrostatic spinning on the solution D obtained in the step (1) by using a rotating speed of a roller rotating at a high speed of 100-3000rpm to obtain an electrospun fiber film with an orientation structure;

step (4), using the electrospun fiber membrane loaded with the coaxial electrospun particles as a receiver to deposit the solution D obtained in the step (1) onto the electrospun fiber membrane loaded with the coaxial electrospun particles obtained in the step (3) in an electrostatic spinning manner, so as to form the electrospun fiber membrane with a three-layer structure;

the electrospun fiber membrane with the three-layer structure also needs to stand the sample for 2-3 days to remove the solvent;

and (5) crimping the electrospun fiber membrane with the three-layer structure, and bonding the solution D to obtain the nerve conduit with the three-layer structure, wherein the inner diameter of the nerve conduit is 0.5-2.0mm.

According to the preparation method of the nerve conduit with the three-layer structure and the nerve conduit, provided by the invention, peripheral nerve injury is repaired, and electrospray particles with core-shell structures and uniform deposition density are contained, so that cell recruitment and migration are promoted; microparticles containing a core-shell structure that promotes peripheral nerve repair growth factors to regulate the behavior of different cells through the release of growth factors. Has excellent biocompatibility and degradability, can mediate migration, proliferation and differentiation of cells and extension of axons, can accelerate the axons from the proximal end to the distal end, and can improve the repair effect of peripheral nerve injury.

Drawings

FIG. 1 is an SEM photograph of a polycaprolactone nanofiber membrane of the inner layer orientation structure obtained in the step (2) of example 1.

FIG. 2 is an SEM photograph of a polycaprolactone nanofiber membrane of an outer random structure of a nerve conduit prepared in the step (4) of example 1.

Detailed Description

The present invention is described in detail below with reference to the specific drawings and examples, and it is necessary to point out that the following examples are given for further illustration of the present invention only and are not to be construed as limiting the scope of the present invention, since numerous insubstantial modifications and adaptations of the invention to those skilled in the art will still fall within the scope of the present invention.

The raw materials used in the examples are all commercially available.

Example 1

Step (1), dissolving polycaprolactone in dichloromethane, and magnetically stirring at room temperature for 12 hours to obtain a solution D with the mass concentration of 11%;

step (2), carrying out electrostatic spinning by using the solution D, using a roller with the rotating speed of 1500rpm as a receiver, wherein the advancing speed of the spinning solution is 2.0mL/h, the voltage is 18kV, the receiving distance is 20cm, and spinning is carried out for 720min to obtain the orderly oriented polycaprolactone nanofiber membrane;

step (3), dissolving gelatin in acetic acid water solution, magnetically stirring at room temperature for 8h, and fully mixing to obtain shell solution E with the concentration of 15mg/mL; adding vascular endothelial growth factor into deionized water, and stirring thoroughly to obtain nuclear layer solution F with a concentration of 40ug/mL;

replacing spinning solution into a shell layer solution E and a core layer solution F, replacing a uniaxial spinning needle with a coaxial needle, injecting the shell layer solution E into a shell layer injector, and injecting the core layer solution F into the core layer injector; taking the orderly oriented polycaprolactone nanofiber membrane obtained in the step (2) as a receiver, carrying out coaxial electrostatic spraying, wherein the shell electrospraying solution advancing rate is 3.0mL/h, the core electrospraying solution advancing rate is 1.0mL/h, the voltage is 16.0kV, the receiving distance is 15.0cm, and the spraying is carried out for 60min, so as to obtain the polycaprolactone nanofiber membrane loaded with coaxial electrospraying particles; 1.0g of the fiber membrane requires 20mL of core layer solution F and 50mL of shell layer solution E.

Step (4), replacing spinning solution with solution D, replacing a coaxial needle with a single-shaft spinning needle, and carrying out electrostatic spinning deposition on the particles with the core-shell structure obtained in the step (3), wherein the advancing rate of the spinning solution is 2.0mL/h, the voltage is 18kV, the receiving distance is 20cm, and spinning is carried out for 360min to obtain a polycaprolactone fiber membrane with a three-layer structure;

the dosage ratio of the electrospun fiber film loaded with the coaxial electrospun particles to the solution D is 1g:30mL.

And (5) placing the polycaprolactone fiber membrane with the three-layer structure in a fume hood for 3 days at room temperature to fully volatilize the residual solvent. And curling the polycaprolactone fiber membrane with the three-layer structure, and bonding the polycaprolactone fiber membrane with the three-layer structure by using the solution D to prepare the nerve conduit with the three-layer structure, wherein the inner layer is a polycaprolactone fiber layer with an orientation structure, the middle layer is an electrospraying particle loading layer, and the outer layer is a random polycaprolactone fiber layer. The diameter within the nerve conduit was 1.3mm.

Example 2

Step (1), dissolving polylactic acid in trifluoroethanol, and magnetically stirring at room temperature for 8 hours to obtain a solution D with the mass concentration of 9%;

step (2), carrying out electrostatic spinning by using the solution D, using a roller with the rotating speed of 1500rpm as a receiver, wherein the advancing speed of the spinning solution is 2.0mL/h, the voltage is 18kV, the receiving distance is 20cm, and spinning is carried out for 720min to obtain the orderly oriented polylactic acid nanofiber membrane;

step (3), dissolving collagen in an acetic acid aqueous solution, magnetically stirring at room temperature for 8 hours, fully mixing to obtain a 35mg/mL solution E, adding nerve growth factor into sterile water, fully stirring and dissolving to obtain a 45ug/mL solution F, wherein 1g of polylactic acid fiber membrane needs 50mL of a shell layer solution E, and the dosage of the core layer solution F is 25mL;

replacing spinning solution into a shell layer solution E and a core layer solution F, replacing a uniaxial spinning needle with a coaxial needle, injecting the shell layer solution E into a shell layer injector, and injecting the core layer solution F into the core layer injector; taking the ordered oriented fiber obtained in the step (2) as a receiver, carrying out coaxial electrostatic spraying, wherein the advancing rate of the shell electrospray solution is 5.0mL/h, the advancing rate of the core electrospray solution is 2.0mL/h, the voltage is 17.0kV, the receiving distance is 15.0cm, and the spraying is carried out for 60min, so as to obtain the polylactic acid nanofiber membrane loaded with coaxial electrospray particles;

step (4), replacing spinning solution with solution D, replacing a coaxial needle with a single-shaft spinning needle, and carrying out electrostatic spinning deposition on the electrospun particles with the core-shell structure obtained in the step (5), wherein the advancing rate of the spinning solution is 1.0mL/h, the voltage is 18.0kV, the receiving distance is 20.0cm, and spinning is carried out for 300min to obtain a polylactic acid fiber membrane with a three-layer structure;

the ratio of electrospun fiber film loaded with coaxial electrospun particles to solution D was 1.0g:25mL;

and (5) placing the polylactic acid fiber membrane with the three-layer structure in a fume hood for 3 days at room temperature to fully volatilize residual solvents, curling the polylactic acid fiber membrane with the three-layer structure, and bonding the polylactic acid fiber membrane with the three-layer structure by using the solution D to prepare the nerve conduit with the three-layer structure, wherein the inner layer is a polylactic acid fiber layer with an orientation structure, the middle layer is an electrospraying particle loading layer, the outer layer is a random polylactic acid fiber layer, and the diameter in the nerve conduit is 1.0mm.

Example 3

Step (1), dissolving polylactic acid-glycolic acid copolymer in chloroform, and magnetically stirring at room temperature for 12 hours to obtain solution D with the mass concentration of 10%;

step (2), carrying out electrostatic spinning by using the solution D, using a roller with the rotating speed of 2000rpm as a receiver, wherein the advancing speed of the spinning solution is 1.5mL/h, the voltage is 16.0kV, the receiving distance is 16.0cm, and spinning for 720min to obtain the orderly oriented polylactic acid-glycolic acid copolymer fiber membrane;

step (3), dissolving gelatin in chloroform, magnetically stirring at room temperature for 8 hours, and fully mixing to obtain a shell solution E with the concentration of 35.0 mg/mL; adding vascular endothelial growth factor and nerve growth factor into ethanol, stirring thoroughly to dissolve for 12h, and mixing thoroughly to obtain nuclear layer solution F of 35.0 ug/mL;

replacing spinning solution into a shell layer solution E and a core layer solution F, replacing a uniaxial spinning needle with a coaxial needle, injecting the shell layer solution E into a shell layer injector, and injecting the core layer solution F into the core layer injector; taking the ordered oriented fiber obtained in the step (3) as a receiver, carrying out coaxial electrostatic spraying, wherein the advancing rate of a shell electrospray solution is 3.0mL/h, the advancing rate of a core electrospray solution is 1.0mL/h, the voltage is 19.0kV, the receiving distance is 13.0cm, and spraying is carried out for 30min, so as to obtain an electrospray particle-loaded fiber membrane with a core-shell structure; 1.0g of the oriented fiber film requires 25mL of core layer solution F and 40mL of shell layer solution E.

Step (4), replacing spinning solution with solution D, replacing a coaxial needle with a single-shaft spinning needle, and carrying out electrostatic spinning deposition on the electrospun particles with the core-shell structure obtained in the step (3), wherein the advancing speed of the spinning solution is 2.5mL/h, the voltage is 18.0kV, the receiving distance is 20.0cm, and spinning is carried out for 360min, so as to obtain an electrospun fiber membrane with a three-layer structure;

the ratio of electrospun fiber film loaded with coaxial electrospun particles to solution D was 1.0g:35mL;

and (5) placing the electrospun fiber membrane with the three-layer structure in a fume hood for 3 days at room temperature to fully volatilize residual solvents, curling the electrospun fiber membrane with the three-layer structure, and bonding the electrospun fiber membrane with the three-layer structure by using the solution D to prepare the nerve conduit with the three-layer structure, wherein the inner layer is a polylactic acid-glycolic acid copolymer fiber layer with an orientation structure, the middle layer is an electrospun particle loading layer, and the outer layer is a random polylactic acid-glycolic acid copolymer fiber layer. The diameter within the nerve conduit was 1.4mm.

Example 4

Step (1), dissolving polylactic acid-glycolic acid copolymer in chloroform, and magnetically stirring at room temperature for 12 hours to obtain a solution D with the mass concentration of 7%;

step (2), carrying out electrostatic spinning by using the solution D, using a roller with the rotating speed of 1000rpm as a receiver, wherein the advancing speed of the spinning solution is 3.0mL/h, the voltage is 18.0kV, the receiving distance is 15.0cm, and spinning for 600min to obtain the orderly oriented polylactic acid-glycolic acid copolymer fiber membrane;

step (3), dissolving gelatin and collagen in chloroform, magnetically stirring at room temperature for 8 hours, and fully mixing to obtain a shell solution E with the concentration of 25.0 mg/mL; adding vascular endothelial growth factor and nerve growth factor into ethanol, stirring thoroughly to dissolve for 12h, and mixing thoroughly to obtain a nuclear layer solution F of 40.0 ug/mL;

replacing spinning solution into a shell layer solution E and a core layer solution F, replacing a uniaxial spinning needle with a coaxial needle, injecting the shell layer solution E into a shell layer injector, and injecting the core layer solution F into the core layer injector; taking the ordered oriented fiber obtained in the step (3) as a receiver, carrying out coaxial electrostatic spraying, wherein the advancing rate of a shell electrospray solution is 4.0mL/h, the advancing rate of a core electrospray solution is 2.0mL/h, the voltage is 17.0kV, the receiving distance is 16.0cm, and spraying is carried out for 30min, so as to obtain an electrospray particle-loaded fiber membrane with a core-shell structure; 1.0g of the oriented fiber film requires 20mL of core layer solution F and 30mL of shell layer solution E.

Step (4), replacing spinning solution with solution D, replacing a coaxial needle with a single-shaft spinning needle, and carrying out electrostatic spinning deposition on the electrospun particles with the core-shell structure obtained in the step (3), wherein the advancing speed of the spinning solution is 3.0mL/h, the voltage is 16.0kV, the receiving distance is 20.0cm, and spinning is carried out for 300min, so as to obtain an electrospun fiber membrane with a three-layer structure;

the ratio of electrospun fiber film loaded with coaxial electrospun particles to solution D was 1.0g:40mL;

and (5) placing the electrospun fiber membrane with the three-layer structure in a fume hood for 3 days at room temperature to fully volatilize residual solvents, curling the electrospun fiber membrane with the three-layer structure, and bonding the electrospun fiber membrane with the three-layer structure by using the solution D to prepare the nerve conduit with the three-layer structure, wherein the inner layer is a polylactic acid-glycolic acid copolymer fiber layer with an orientation structure, the middle layer is an electrospun particle loading layer, and the outer layer is a random polylactic acid-glycolic acid copolymer fiber layer. The diameter within the nerve conduit was 1.2mm.

Example 5

Step (1), dissolving polylactic acid-glycolic acid-caprolactone copolymer in dichloromethane and N, N' -dimethylformamide, and magnetically stirring at room temperature for 24 hours to obtain a solution D with the mass concentration of 7%;

step (2), carrying out electrostatic spinning by using the solution D, using a roller with the rotating speed of 2500rpm as a receiver, wherein the advancing speed of the spinning solution is 2.0mL/h, the voltage is 20kV, the receiving distance is 20cm, and spinning is carried out for 720min to obtain the orderly oriented polylactic acid-glycolic acid-caprolactone copolymer nanofiber membrane;

step (3), dissolving chitosan in acetic acid water solution, magnetically stirring at room temperature for 24 hours, and fully mixing to obtain shell solution E with the concentration of 25mg/mL; adding platelet-derived growth factor into deionized water, and stirring thoroughly to obtain nuclear layer solution F with concentration of 30ug/mL;

replacing spinning solution into a shell layer solution E and a core layer solution F, replacing a uniaxial spinning needle with a coaxial needle, injecting the shell layer solution E into a shell layer injector, and injecting the core layer solution F into the core layer injector; taking the orderly oriented polylactic acid-glycolic acid-caprolactone copolymer nanofiber membrane obtained in the step (2) as a receiver, carrying out coaxial electrostatic spraying, wherein the shell electrospray solution advancing rate is 5.5mL/h, the core electrospray solution advancing rate is 2.0mL/h, the voltage is 16.0kV, the receiving distance is 15.0cm, and spraying is carried out for 80min, so as to obtain the polylactic acid-glycolic acid-caprolactone copolymer fiber membrane loaded with coaxial electrospray particles; 1.0g of the fiber membrane requires 30mL of core layer solution F and 60mL of shell layer solution E.

Step (4), replacing spinning solution with solution D, replacing a coaxial needle with a single-shaft spinning needle, and carrying out electrostatic spinning deposition on the particles with the core-shell structure obtained in the step (3), wherein the advancing speed of the spinning solution is 2.5mL/h, the voltage is 20kV, the receiving distance is 20cm, and spinning is carried out for 360min to obtain the polylactic acid-glycolic acid-caprolactone copolymer fiber membrane with a three-layer structure;

the ratio of electrospun fiber film loaded with coaxial electrospun particles to solution D was 1.0g:35mL.

And (5) placing the polylactic acid-glycolic acid-caprolactone copolymer fiber membrane with the three-layer structure in a fume hood for 3 days at room temperature to fully volatilize the residual solvent. And curling the polylactic acid-glycolic acid-caprolactone copolymer fiber membrane with the three-layer structure, and bonding the polylactic acid-glycolic acid-caprolactone copolymer fiber membrane with the three-layer structure by using the solution D to prepare the nerve conduit with the three-layer structure, wherein the inner layer is a polylactic acid-glycolic acid-caprolactone copolymer fiber layer with an orientation structure, the middle layer is an electrosprayed particle load layer, and the outer layer is a random polylactic acid-glycolic acid-caprolactone copolymer fiber layer. The diameter within the nerve conduit was 1.2mm.

Example 6

step (2), carrying out electrostatic spinning by using the solution D, using a roller with the rotating speed of 1800rpm as a receiver, wherein the advancing speed of the spinning solution is 2.0mL/h, the voltage is 20kV, the receiving distance is 20cm, and spinning is carried out for 720min to obtain the orderly oriented polycaprolactone nanofiber membrane;

dissolving cellulose in an acetic acid aqueous solution, magnetically stirring at room temperature for 24 hours, and fully mixing to obtain a shell layer solution E with the concentration of 30mg/mL; adding an epidermal growth factor and a platelet-derived growth factor into deionized water, and fully stirring and dissolving to obtain a nuclear layer solution F with the concentration of 25ug/mL;

replacing spinning solution into a shell layer solution E and a core layer solution F, replacing a uniaxial spinning needle with a coaxial needle, injecting the shell layer solution E into a shell layer injector, and injecting the core layer solution F into the core layer injector; taking the orderly oriented polylactic acid-glycolic acid-caprolactone copolymer nanofiber membrane obtained in the step (2) as a receiver, carrying out coaxial electrostatic spraying, wherein the advancing rate of a shell electrospray solution is 6.0mL/h, the advancing rate of a core electrospray solution is 3.0mL/h, the voltage is 16.0kV, the receiving distance is 15.0cm, and spraying is carried out for 60min, so as to obtain the polylactic acid-glycolic acid-caprolactone copolymer nanofiber membrane loaded with coaxial electrospray particles; 1.0g of the fiber membrane requires 40mL of core layer solution F and 70mL of shell layer solution E.

Step (4), replacing spinning solution with solution D, replacing a coaxial needle with a single-shaft spinning needle, and carrying out electrostatic spinning deposition on the particles with the core-shell structure obtained in the step (3), wherein the advancing rate of the spinning solution is 3.0mL/h, the voltage is 20kV, the receiving distance is 20cm, and spinning is carried out for 360min to obtain the polylactic acid-glycolic acid-caprolactone copolymer fiber membrane with a three-layer structure;

the ratio of electrospun fiber film loaded with coaxial electrospun particles to solution D was 1.0g:50mL.

And (5) placing the polylactic acid-glycolic acid-caprolactone copolymer fiber membrane with the three-layer structure in a fume hood for 3 days at room temperature to fully volatilize the residual solvent. And curling the polylactic acid-glycolic acid-caprolactone copolymer fiber membrane with the three-layer structure, and bonding the polylactic acid-glycolic acid-caprolactone copolymer fiber membrane with the three-layer structure by using the solution D to prepare the nerve conduit with the three-layer structure, wherein the inner layer is a polylactic acid-glycolic acid-caprolactone copolymer fiber layer with an orientation structure, the middle layer is an electrosprayed particle load layer, and the outer layer is a random polylactic acid-glycolic acid-caprolactone copolymer fiber layer. The diameter within the nerve conduit was 1.35mm.

Claims

1. A method for preparing a nerve conduit of three-layer structure, the method comprising:

step (1), adding soluble aliphatic polyester or soluble aliphatic polyester and natural high molecular polymer into a solvent A, and fully dissolving to obtain a solution D; the mass concentration of the solution D is 3-20%;

step (2), taking the solution D as spinning solution, and carrying out electrostatic spinning by taking a roller rotating at a high speed as a receiver to obtain a fiber membrane with an orientation structure;

step (3), adding natural high molecular polymer into the solvent B to obtain a shell layer solution E; adding the growth factors into the solvent C to obtain a nuclear layer solution F; taking the shell layer solution E and the core layer solution F as electro-spray liquid, taking the fiber membrane with the orientation structure obtained in the step (2) as a receiver, and obtaining a loaded coaxial electro-spray particle fiber membrane through electrostatic spraying;

the concentration of the shell layer solution E is 5-100 mg/mL; and/or the number of the groups of groups,

the concentration of the nuclear layer solution F is 1-100 ug/mL;

the dosage ratio of the shell layer solution to the core layer solution is (1-10): 1, a step of;

the dosage ratio of the electrospun fiber membrane to the sum of the shell layer solution and the core layer solution is as follows: 1g (5-100 mL);

step (4), taking the fiber film loaded with the coaxial electrospun particles obtained in the step (3) as a receiver, taking the solution D as a spinning solution, and depositing random fibers through electrostatic spinning to form a fiber film with a three-layer structure;

and (5) crimping the fiber membrane with the three-layer structure into a tube, and bonding the tube with the solution D to obtain the nerve conduit with the three-layer structure.

2. The method of preparing as claimed in claim 1, wherein:

the method comprises the following steps of (1), wherein the mass concentration of the solution D is 5-15%;

the natural high molecular polymer is used in an amount of 0-100 parts by weight based on 1 part by weight of the aliphatic polyester;

3. The method of preparing as claimed in claim 1, wherein:

step (3),

the growth factor is one or the combination of vascular endothelial growth factor, nerve growth factor, platelet derived growth factor, epidermal growth factor, fibroblast growth factor and hepatocyte growth factor; and/or the number of the groups of groups,

the natural high molecular polymer is one or the combination of collagen, gelatin, chitosan, starch, cellulose and elastin.

4. The method of preparing as claimed in claim 1, wherein:

step (3),

the concentration of the shell layer solution E is 10-50 mg/mL; and/or the number of the groups of groups,

the concentration of the core layer solution F is 20-50ug/mL.

5. The method of manufacturing according to claim 4, wherein:

the dosage ratio of the shell layer solution to the core layer solution is (2-5): 1, a step of;

the dosage ratio of the electrospun fiber membrane to the sum of the shell layer solution and the core layer solution is as follows: 1g (50-100 mL).

6. The method of manufacturing according to claim 1, wherein:

the dosage ratio of the electrospun fiber membrane loaded with the coaxial electrospun particles to the solution D in the step (4) is as follows: 1g: (1-50 mL).

7. The method of manufacturing according to claim 6, wherein:

the dosage ratio of the electrospun fiber membrane loaded with the coaxial electrospun particles to the solution D in the step (4) is as follows: 1g: (20-50 mL).

8. The method of manufacturing according to claim 1, wherein:

spinning process conditions of the step (2): the advancing speed of the solution D is 0.1-10 mL/h, the rotating speed of the roller is 100-3000rpm, the voltage is 10-30 kV, the receiving distance is 10-30 cm, and the spinning is performed for 30-720 min.

9. The method of manufacturing according to claim 1, wherein:

and (3) in the electrostatic spraying, the propelling speed of the shell layer electrospray solution is 0.1-10 mL/h, the propelling speed of the core layer electrospray solution is 0.1-5 mL/h, the voltage is 10-25 kV, the receiving distance is 10-25 cm, and the spraying time is 1-360 min.

10. The method of manufacturing according to claim 1, wherein:

and (4) depositing the electrostatic spinning on an electrospun fiber membrane loaded with coaxial electrospun particles, wherein the advancing rate of the solution D is 0.1-10 mL/h, the voltage is 10-20 kV, the receiving distance is 10-25 cm, and the spinning is performed for 10-360 min.

11. A three-layer nerve conduit obtainable by the method of any one of claims 1 to 10, wherein:

the nerve conduit is of a three-layer structure, the inner layer is an electrospun fiber layer of an orientation structure, the middle layer is an electrospun particle load layer, and the outer layer is a random electrospun fiber layer;

the inner diameter of the nerve conduit ranges from 0.5 to 2.0. 2.0mm.