A rigid-flexible composite nerve conduit with staged release and its preparation and application

Abstract

The application provides a rigid-flexible composite nerve conduit with a staged release type and a preparation method and application thereof. The rigid-flexive composite nerve conduit with the staged release type is prepared by using a double-layer nanofiber membrane structure polymer fiber membrane as a tube wall, using a multi-section hydrogel including a short-section hydrogel containing an anti-inflammatory substance and a long-section hydrogel containing a growth factor as an inner core, and depositing bioactive substance particles containing a growth factor on the inner wall of the polymer fiber membrane. The composite nerve conduit provided by the application can satisfy the requirements of each stage of injury by spatiotemporally controllable delivery of the rigid-flexible composite nerve conduit of the anti-inflammatory substance and the growth factor, and can jointly regulate the microenvironment at each stage after spinal cord injury and promote subsequent functional recovery by using multiple induction signals such as topological structure and chemotactic induction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological materials, and particularly relates to a rigid-flexible composite nerve conduit with staged release and a preparation method and application. BACKGROUND

[0002] Spinal cord injury caused by mechanical injury, disease, etc. is a serious trauma occurring in the central nervous system, and the clinical manifestations include loss of sensation below the damaged spinal cord segment, motor ability and sphincter dysfunction, etc., with a very high disability rate and mortality rate. As the most serious injury among spinal cord injuries, the repair of spinal cord transection has become a major medical problem in the field of trauma repair. At present, the main treatment methods in clinical practice include decompression surgery and drug therapy, but these treatment methods often have single effect and short-acting time, which cannot meet the needs of the microenvironment for regeneration at each stage after spinal cord injury, and sometimes are accompanied by serious secondary injury and sequelae. In order to solve the above problems, it is necessary to start from the mechanism of spinal cord injury and design appropriate repair materials according to the needs.

[0003] Spinal cord injury is divided into primary injury and secondary injury, and the early stage of secondary injury will form an inflammatory microenvironment in the injury area, causing a large number of cell deaths, and a large number of cell reductions of growth factors secreted by the injury area; in the middle and late stages of secondary injury, due to the lack of growth factors in the microenvironment, the differentiation of neural stem cells into neurons is inhibited, and the regenerating axons cannot pass through the injury area and establish nerve connections with the descending fiber bundle, so that functional recovery cannot be achieved. The current research on biomaterials applied to the spinal cord focuses on rebuilding a suitable microenvironment for repair after spinal cord injury, but the current material design can only regulate the microenvironment at a specific stage after spinal cord injury, and cannot achieve the regulation of the microenvironment at different stages to adapt to the needs of regeneration at each stage of injury, for example, the research of Shen H, Liao S, etc. (Shen H, Xu B, Yang C, et al. A DAMP-scavenging, IL-10-releasing hydrogel promotes neural regeneration and motor function recovery after spinal cord injury [J]. Biomaterials, 2022, 280: 121279; Liao S, Liu Y, Kong Y, et al. A bionic multichannel nanofiber conduit carrying Tubastatin A for repairing injured spinal cord [J]. Materials Today Bio, 2022, 17: 100454). SUMMARY

[0004] The present application provides a rigid-flexible composite nerve conduit with a staged release mode, comprising a polymer nanofiber nerve conduit and a multi-section hydrogel, wherein the polymer nanofiber nerve conduit comprises two layers of an inner layer and an outer layer, the inner layer is a unidirectional ordered fiber layer, and the surface is deposited with nanoparticles with a core-shell structure, the shell layer is a bioactive substance, and the core layer is a growth factor; the outer layer structure is a randomly arranged polymer fiber layer; the multi-section hydrogel is a modified biological macromolecule with different crosslinking degrees, comprising two parts of a short section and a long section, the short section hydrogel has a lower crosslinking degree, and is internally dispersed with an anti-inflammatory drug, can degrade rapidly in the body first, and is used for regulating early inflammatory response of spinal cord injury; the long section hydrogel has a higher crosslinking degree, and is internally dispersed with a growth factor, and degrades slowly in the body later, and is used for tissue regeneration after spinal cord injury.

[0005] The present application realizes the spatiotemporal controllable delivery of various anti-inflammatory substances and growth factors by regulating the topological structure and gradient structure of the spun polymer fiber, and combining the multi-section hydrogel delivery system, regulates the early spinal cord injury microenvironment, mediates the cell migration and axon extension in the middle stage of spinal cord repair, promotes the neural connection reconstruction and functional recovery in the later stage of spinal cord injury, and provides a new way and method for the design of spinal cord injury repair materials.

[0006] One of the purposes of the present application is to provide a rigid-flexible composite nerve conduit with a staged release mode, which takes a polymer fiber membrane as a tube wall and a multi-section hydrogel as an inner core, the inner wall of the polymer fiber membrane is deposited with bioactive substance particles containing growth factors, and the multi-section hydrogel comprises a short section hydrogel containing anti-inflammatory substances and a long section hydrogel containing growth factors.

[0007] The rigid-flexible composite nerve conduit with a staged release mode provided by the present application has the following advantages:

[0008] The polymer fiber membrane is a double-layer nanofiber membrane structure, wherein the inner layer is a unidirectional ordered nanofiber membrane, and the outer layer is a randomly arranged nanofiber membrane;

[0009] The polymer in the polymer fiber is selected from at least one of degradable aliphatic polyesters, preferably at least one of polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid), and poly(lactic-co-glycolic acid-co-caprolactone);

[0010] The anti-inflammatory substance is selected from at least one of catalase, superoxide dismutase, interleukin, and a reducing molecule;

[0011] The growth factor has a repairing effect, and preferably, the growth factor is at least one selected from the group consisting of vascular endothelial growth factor, fibroblast growth factor, epidermal cell growth factor, brain-derived neurotrophic factor, neurotrophic factor-3, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, and nerve growth factor; the same or different growth factors can be used in the biological active material particles and the long hydrogel, and one or more growth factors can be used;

[0012] The hydrogel precursor compound used in the hydrogel is at least one selected from the group consisting of acrylated biological macromolecules, and preferably at least one selected from the group consisting of methacrylated gelatin, methacrylated hyaluronic acid, methacrylated silk fibroin, methacrylated chitosan, and methacrylated sodium alginate; the same hydrogel precursor compound is used in the short hydrogel and the long hydrogel;

[0013] The growth factor-containing biological active material particle has a core-shell structure, wherein the shell layer is the biological active material, and the core layer is the growth factor;

[0014] The biological active material simulates the composition of the extracellular matrix, and specifically, the biological active material is at least one selected from the group consisting of protein compounds, and preferably at least one selected from the group consisting of collagen, laminin, fibronectin, and collagen protein;

[0015] The thickness of the polymeric fiber membrane tube wall is 50-800 μm, and preferably 100-500 μm;

[0016] The length ratio of the short hydrogel to the long hydrogel is 1:(1.5-10), and preferably 1:(1.5-5); the length of the hydrogel can be adjusted by the volume of the injected hydrogel precursor solution;

[0017] The cross-linking degree of the short hydrogel is lower than that of the long hydrogel, and the cross-linking degree of the hydrogel can be obtained by adjusting different cross-linking times.

[0018] The second object of the present application is to provide a preparation method of the above-mentioned rigid-flexible composite nerve conduit with a staged release, comprising: obtaining a plurality of hydrogels by cross-linking a hydrogel precursor solution containing an anti-inflammatory substance and a hydrogel precursor solution containing a growth factor through ultraviolet light, and then wrapping the plurality of hydrogels with a polymeric fiber membrane in which biological active material particles containing a growth factor are deposited in the inner layer, to obtain the rigid-flexible composite nerve conduit with a staged release.

[0019] The present application provides a preparation method of a rigid-flexible composite nerve conduit with a staged release, specifically comprising the following steps:

[0020] Step (1) electrospinning a polymer solution A to obtain a double-layer polymeric fiber membrane;

[0021] Step (2) coaxially electrostatically spraying the growth factor solution B and the bioactive substance solution C to the inner layer of the polymer fiber membrane obtained in step (1) to prepare a polymer fiber membrane with gradient density deposited bioactive substance particles;

[0022] Step (3) mixing the growth factor solution D and the anti-inflammatory substance solution E with initiators respectively to obtain initiator solution F and initiator solution G, and then adding the hydrogel precursor compound respectively to obtain hydrogel precursor solution H and hydrogel precursor solution I;

[0023] Step (4) injecting the hydrogel precursor solution H into a mold, crosslinking under ultraviolet light, and then continuously injecting the hydrogel precursor solution I, crosslinking under ultraviolet light to obtain a multi-section hydrogel;

[0024] Step (5) contacting the inner layer of the polymer fiber membrane with gradient density deposited bioactive substance particles obtained in step (2) with the multi-section hydrogel obtained in step (4), and wrapping into a tube, and finally using the polymer solution A to adhere, thereby obtaining the staged release type rigid-flexible composite nerve conduit.

[0025] In step (1) of the above preparation method:

[0026] The solvent in the polymer solution A is selected from at least one of hexafluoroisopropanol, trifluoroethanol, trichloromethane, methanol, dichloromethane, and N,N'-dimethylformamide;

[0027] The mass percentage concentration of the polymer solution A is 5-25%, preferably 8-15%;

[0028] The electrospinning includes: uniaxial electrospinning of the polymer solution A, with a roller or a U-shaped groove as a receiver, a spinning solution flow rate of 0.1-3 mL / h, a voltage of 10-20 kV, and a receiving distance of 10-20 cm; preferably, when the electrospinning uses a roller as a receiver, first spinning with a high-speed rotating roller as a receiver for 2-10 h to obtain a unidirectional ordered fiber membrane, and then spinning at a reduced roller speed for 2-10 h; or, when the electrospinning uses a U-shaped groove as a receiver, first spinning with a U-shaped groove as a receiver for 2-10 h to obtain a unidirectional ordered fiber membrane, and then removing the U-shaped groove to obtain a unidirectional ordered fiber membrane as a collector for spinning for 2-10 h.

[0029] In step (2) of the above preparation method:

[0030] The solvent in the growth factor solution B is selected from at least one of phosphate buffered saline (PBS), water, ethanol, polyvinyl alcohol, polyacrylamide, and polyacrylic acid;

[0031] The concentration of the growth factor solution B is 0.1 μg / mL-5 mg / mL, preferably 1 μg / mL-1 mg / mL;

[0032] The solvent in the bioactive substance solution C is selected from at least one of hydrochloric acid, aqueous acetic acid, ethyl acetate, glycerol, trifluoroethanol, and hexafluoroisopropanol;

[0033] The concentration of the bioactive substance solution C is 1-50 mg / mL, preferably 10-30 mg / mL;

[0034] The coaxial electrostatic spraying includes: gradient concentration electrostatic spraying in a mask mode, regulating the electrostatic spraying time of different positions on the fiber surface by moving the mask, and depositing bioactive substance particles in a gradient increasing manner along the ordered arrangement direction of the fiber, specifically, depositing bioactive substance particles in a gradient increasing manner along the ordered arrangement direction of the fiber, injecting the bioactive substance solution C into the shell layer injector, injecting the growth factor solution B into the core layer injector, using the inner layer of the polymer fiber membrane obtained in step (1) as a receiver, the shell layer electrospinning solution flow rate is 0.5-3 mL / h, the core layer electrospinning solution flow rate is 0.2-1 mL / h, the voltage is 10-20 kV, the receiving distance is 10-20 cm, and the electrostatic spraying time is 1-60 min; preferably, the shell layer electrospinning solution flow rate is higher than the core layer electrospinning solution flow rate; the density of the growth factor nanoparticles in the obtained polymer fiber membrane increases in a gradient along the arrangement direction of the fiber;

[0035] The polymer fiber membrane obtained after electrostatic spraying still needs to be removed from the solvent, which can be removed by a common solvent removal method, or can be volatilized by standing, such as: placing in a fume hood at room temperature for 2-7 days, and packaging and sterilizing after removing the solvent.

[0036] In step (3) of the above preparation method:

[0037] The solvent in the growth factor solution D is selected from at least one of phosphate buffered saline (PBS), water, ethanol, polyvinyl alcohol, polyacrylamide, and polyacrylic acid; the concentration of the growth factor solution D is 0.1 μg / mL-5 mg / mL, preferably 1 μg / mL-1 mg / mL;

[0038] The solvent in the anti-inflammatory substance solution E is selected from at least one of phosphate buffered saline (PBS), water, chloroform, glycerol, trifluoroethanol, and hexafluoroisopropanol; the concentration of the anti-inflammatory substance solution E is 1 μg / mL-5 mg / mL, preferably 0.1-2 mg / mL;

[0039] The initiator in the initiator solution F and the initiator in the initiator solution G are independently selected from at least one of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP), 2,4,6-trimethylbenzoylphenyl phosphinic acid ethyl ester (TPO-L);

[0040] The mass percentage of the initiator in the initiator solution F is 0.1-0.5%, preferably 0.15-0.3%; the mass percentage of the initiator in the initiator solution G is 0.1-0.5%, preferably 0.15-0.3%;

[0041] The mass percentage of the hydrogel precursor compound in the hydrogel precursor solution H is 5-15%, preferably 8-12%; the mass percentage of the hydrogel precursor compound in the hydrogel precursor solution I is 5-15%, preferably 8-12%;

[0042] In step (3), when the growth factor solution D and the anti-inflammatory substance solution E are mixed with the initiator, the mixing is carried out at 37℃ in the dark, and after sufficient shaking, the initiator solution F and the initiator solution G are obtained, respectively, and the hydrogel precursor compound is added to the initiator solution, and the hydrogel precursor solution is obtained by fully shaking at 37℃.

[0043] In step (4) of the above preparation method:

[0044] The mold is a tubular mold; the conditions for ultraviolet light crosslinking are that the crosslinking temperature is 18-37℃, preferably 20-30℃; and the crosslinking time is 4-60s, preferably 4-30s;

[0045] The ultraviolet light crosslinking includes: first, the hydrogel precursor solution H and the hydrogel precursor solution I are warmed to 37℃ in a constant-temperature water bath in the dark, then the hydrogel precursor solution H is added to a syringe and quickly injected into a tubular mold through a 0.22μm filter, the hydrogel precursor solution in the mold is crosslinked under light, the light crosslinking time is 4-60s, finally the hydrogel precursor solution I is added to a syringe and quickly injected into the same tubular mold through a 0.22μm filter, and the hydrogel and the hydrogel precursor solution in the mold are crosslinked again under light, the light crosslinking time is 4-60s, and different degrees of crosslinking of the hydrogel can be obtained by adjusting the crosslinking time.

[0046] The third object of the present application is to provide the use of the above-mentioned staged-release rigid-flexible composite nerve conduit or the staged-release rigid-flexible composite nerve conduit prepared by the above-mentioned preparation method in a biological-based repair material.

[0047] The application of the rigid-flexible composite nerve conduit capable of releasing anti-inflammatory substances and growth factors in stages as a spinal cord injury repair material provided by the application can realize the time and space controllable delivery of anti-inflammatory substances and growth factors in the hydrogel, and match the process of spinal cord injury repair. The continuous degradation and release of the loaded factors solve the problem of short-acting of drugs. The short hydrogel loaded with anti-inflammatory drugs will degrade and release anti-inflammatory drugs to regulate the inflammatory microenvironment in the early stage of spinal cord injury, so as to provide a suitable microenvironment for subsequent repair. Subsequently, the long hydrogel loaded with multiple growth factors degrades in the middle and late stages of spinal cord injury to supply multiple growth factors to the injury area, promote the extension of axons in the injury area, and establish nerve connection with the descending fiber bundle. In addition, while realizing the repair of each stage of spinal cord injury through the time and space controllable delivery of multiple factors, the nanofiber with a one-way ordered arrangement topology, the gradient density signal of the surface biological active substance particles and the introduction of the repair-promoting growth factor also provide physical guidance for the extension of axons. The rigid-flexible composite nerve conduit capable of releasing anti-inflammatory substances and growth factors in stages can better meet the needs of each stage after spinal cord injury, and the nerve conduit can be degraded and metabolized by the body after the completion of the spinal cord injury repair process.

[0048] The application can achieve the following beneficial effects:

[0049] 1. The nanofiber conduit inner surface adopted in the application realizes the efficient integration of multiple induction signals such as ordered topological structure and growth factor gradient density signal;

[0050] 2. The fiber conduit part in the rigid-flexible composite nerve conduit in the application not only plays a role in stabilizing the hydrogel form, but also can promote the extension of axons and cell migration for a long time after the degradation of the hydrogel due to the one-way ordered fiber arrangement structure and the gradient deposition of biological active substance particles on the inner layer conduit; the randomly arranged fibers on the outer layer provide sufficient elasticity and toughness to meet the needs of spinal column activity during the operation and after the operation;

[0051] 3. The rigid-flexible composite nerve conduit capable of releasing anti-inflammatory substances and growth factors in stages adopted in the application has excellent biocompatibility and degradation performance, and can provide the required microenvironment for each stage of injury through the time and space controllable delivery of different factors, promote the extension of axons and the reconstruction of nerve connection;

[0052] 4. The rigid-flexible composite nerve conduit capable of releasing anti-inflammatory substances and growth factors in stages has the ability to release multiple factors in time and space, can meet the needs of the microenvironment required by each stage of spinal cord injury, has the effects of promoting cell migration, axon extension and nerve connection reconstruction, and has a biomimetic structure similar to extracellular matrix (ECM) and good biocompatibility, and is expected to become an ideal spinal cord injury repair material;

[0053] 5. This invention addresses spinal cord transection injuries by designing a nerve conduit to bridge the two ends of the injury. The rigid-flexible composite design provides a favorable environment for nerve regeneration in terms of the elasticity and toughness of the matrix.

[0054] 6. This invention employs a multi-segment hydrogel spatiotemporally controllable delivery strategy. By degrading and releasing anti-inflammatory substances in the early stages of injury through short-segment hydrogels, the damaged microenvironment is regulated, providing a suitable microenvironment for subsequent repair. In the middle and late stages of injury, the degradation of long-segment hydrogels continuously replenishes growth factors to the damaged area, promoting axonal extension and establishing neural connections with descending fiber bundles, which can effectively promote the functional recovery of the damaged spinal cord. Attached Figure Description

[0055] Figure 1 This is a schematic diagram of a staged release rigid-flexible composite nerve conduit formed by wrapping a polymer fiber membrane containing inner layer deposited growth factor particles obtained in Examples 1-4 around a multi-segment hydrogel loaded with anti-inflammatory drugs and growth factors.

[0056] Figure 2 These are SEM images of the polymer fiber membrane prepared in Example 1 before and after gradient density deposition of growth factor particles. Figure 2 It can be seen that the polymer fiber membrane has a distinct bilayer structure, with the inner layer of fibers arranged in a unidirectional ordered manner and the outer layer arranged randomly. Furthermore, the bioactive material particles are densely deposited on the surface of the ordered fibers in the inner layer.

[0057] Figure 3 The image shows a SEM image of a polymer fiber membrane containing gradient density deposited growth factor particles obtained in Example 1, wound into a fiber conduit without a mold.

[0058] Figure 4 These are SEM images of the short-segment and long-segment hydrogels prepared in Example 1. Figure 4 It can be seen that the hydrogel loaded with anti-inflammatory drugs has a larger pore size and a faster degradation rate, while the hydrogel loaded with growth factors has a smaller pore size and a slower degradation rate. This difference in degradation rate between different segments of the hydrogel allows for spatiotemporally controlled delivery of the loaded drugs.

[0059] Figure 5 The figures represent the cell proliferation test results for the blank group, the polymer fiber membrane with gradient density deposition of pure collagen particles in Comparative Example 1, the polymer fiber membrane with uniform density deposition of pure collagen particles in Comparative Example 2, the polymer fiber membrane with no deposited bioactive substance particles in Comparative Example 3, and the polymer fiber membrane with gradient density deposition of growth factor particles in Example 1. The horizontal axis represents the number of days of cell culture, and the vertical axis represents the absorbance value. Figure 5 It can be seen that the density gradient signal and growth factor deposition on the fiber membrane in Example 1 have a promoting effect on cell proliferation. Detailed Implementation

[0060] The present invention will now be described in detail with reference to specific embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are still within the scope of protection of the present invention.

[0061] The testing instruments and conditions used in this embodiment are as follows:

[0062] Electrospinning equipment: Model: TEADFS-103, Beijing Xinrui Baina Technology Co., Ltd.; (Roller is the receiver)

[0063] Electrospinning equipment: Model: NANO 01, MECC Co., Ltd., Japan; (U-shaped groove is the receiver)

[0064] Electrostatic spraying equipment: Model: NANO 01, MECC Co., Ltd., Japan.

[0065] Unless otherwise specified, the raw materials used in the examples and comparative examples are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0066] The biodegradable aliphatic polyester raw materials used in the examples were purchased from Sigma Reagents, organic solvents from Aladdin, and growth factors from Thermo Fisher Scientific. Modified biomolecules and initiators were purchased from EFL, and anti-inflammatory drugs were purchased from Yuanye Company.

[0067] Example 1

[0068] Step (1) Polycaprolactone was added to a mixed solution of dichloromethane and N,N-dimethylformamide (volume ratio of dichloromethane to N,N-dimethylformamide was 4:1), and magnetically stirred at room temperature for 24 h to obtain a polymer solution A with a mass concentration of 10%. Uniaxial electrospinning was performed using solution A at a flow rate of 1 mL / h, a voltage of 16 kV, and a receiving distance of 15 cm. A high-speed rotating roller was used as the receiver. Spinning was performed for 5 h to obtain unidirectional ordered fibers. Then, the roller speed was gradually reduced, and random fibers were collected for 5 h to obtain a double-layer polymer fiber membrane. The polymer fiber membrane was placed in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate.

[0069] Step (2) neurotrophin-3 is dissolved in PBS, mixed well to obtain a growth factor solution B with a concentration of 100 μg / mL; collagen is dissolved in 70% acetic acid aqueous solution, mixed well to obtain a bioactive substance solution C with a concentration of 20 mg / mL; the electrospinning liquid is replaced by the growth factor solution B and the bioactive substance solution C, the single-axis spinning needle is replaced by a coaxial needle, the growth factor solution B is injected into the core layer syringe, and the bioactive substance solution C is injected into the shell layer syringe; the inner layer of the polymer fiber membrane obtained in step (1) is used as a receiver to perform coaxial electrostatic spraying, a mask plate strategy is adopted to deposit bioactive substance particles with increasing particle density along the unidirectional ordered arrangement direction, the flow rate of the growth factor solution B is 0.5 mL / h, the flow rate of the bioactive substance solution C is 1.5 mL / h, the voltage is 20 kV, and the electrostatic spraying is performed for 30 min to deposit growth factor particles on the inner surface of the polymer fiber membrane in a gradient density;

[0070] Step (3) epidermal growth factor, glial cell-derived neurotrophic factor, and neurotrophin-3 are dissolved in PBS, mixed well to obtain a growth factor solution D with a concentration of 100 μg / mL; catalase and superoxide dismutase are added to PBS, mixed well to obtain an anti-inflammatory substance solution E with a concentration of 100 μg / mL; LAP is added to the growth factor solution D and the anti-inflammatory substance solution E respectively, and the mixture is mixed well under oscillation at 37°C in the dark to obtain an initiator solution F and an initiator solution G with a mass fraction of 0.25%; methacrylated gelatin is added to the initiator solution F and the initiator solution G respectively, and the mixture is mixed well under oscillation at 37°C in the dark to obtain a hydrogel precursor solution H and a hydrogel precursor solution I with a mass fraction of 10%;

[0071] Step (4) the hydrogel precursor solution H and the hydrogel precursor solution I obtained in step (3) are warmed to 37°C, the hydrogel precursor solution H is then added to a syringe, rapidly injected into a 15-μL tubular mold through a 0.22-μm filter, and the hydrogel precursor solution in the mold is crosslinked under irradiation of 405-nm blue light, with a light crosslinking time of 8 s; finally, the hydrogel precursor solution I is added to a syringe, rapidly injected into a 5-μL tubular mold through a 0.22-μm filter, and the hydrogel and the hydrogel precursor solution in the mold are crosslinked again under irradiation of 405-nm blue light, with a light crosslinking time of 8 s, to obtain a multi-segment filling hydrogel, wherein the length ratio of the short segment to the long segment is 1:3;

[0072] Step (5) using the multi-sectioned hydrogel obtained in step (4) as a mold, the inner layer of the gradient density growth factor particle-containing polymer fiber membrane obtained in step (1) is contacted with the hydrogel and wrapped into a tube, a polymer solution A is applied to the adhesion site at room temperature, and after adhesion, the adhesion site is dried in an ultra-clean bench, and finally the mold is removed to obtain a rigid-flexible composite nerve conduit capable of releasing anti-inflammatory substances and growth factors in stages.

[0073] Example 2

[0074] Step (1) polylactic acid is added to dichloromethane, and magnetic stirring is performed at room temperature for 24 h to obtain a polymer solution A with a mass concentration of 10%; uniaxial electrospinning is performed using the solution A, the flow rate of the solution A is 1 mL / h, the voltage is 16 kV, the receiving distance is 15 cm, a high-speed rotating roller is used as a receiver, and spinning is performed for 5 h to obtain unidirectionally ordered fibers, and then the rotation speed of the roller is gradually reduced, and random arrangement fibers are continuously collected for 5 h to obtain a polymer fiber membrane with a double-layer structure; the polymer fiber membrane is placed in a fume hood at room temperature for 3 days to fully volatilize the residual solvent;

[0075] Step (2) nerve growth factor is dissolved in PBS, and is fully mixed and uniform to obtain a growth factor solution B with a concentration of 100 μg / mL; collagen is dissolved in a 70% volume fraction acetic acid aqueous solution, and is fully mixed and uniform to obtain a bioactive substance solution C with a concentration of 20 mg / mL; the electrospinning liquid is replaced with the growth factor solution B and the bioactive substance solution C, the uniaxial spinning needle is replaced with a coaxial needle, the growth factor solution B is injected into the core layer syringe, and the bioactive substance solution C is injected into the shell layer syringe; the inner layer of the polymer fiber membrane obtained in step (1) is used as a receiver, and coaxial electrospinning is performed, a mask plate strategy is adopted, and bioactive substance particles with a gradient density increasing along the unidirectionally ordered arrangement direction are deposited, the flow rate of the growth factor solution B is 0.5 mL / h, the flow rate of the bioactive substance solution C is 1.5 mL / h, the voltage is 20 kV, and electrospinning is performed for 10 min to obtain a polymer fiber membrane with growth factor particles deposited in the inner surface with a gradient density;

[0076] Step (3) reconstitute human basic fibroblast growth factor, epidermal cell growth factor, brain-derived neurotrophic factor in PBS, mix well to get growth factor solution D with concentration of 100 μg / mL; add IL-10 and IL-4 into PBS, mix well to get anti-inflammatory substance solution E with concentration of 100 μg / mL; add LAP into growth factor solution D and anti-inflammatory substance solution E respectively, mix well at 37℃ in dark to get initiator solution F and initiator solution G with mass fraction of 0.25%, add methacrylated gelatin into initiator solution F and initiator solution G respectively, mix well at 37℃ in dark to get hydrogel precursor solution H and hydrogel precursor solution I with mass fraction of 10%;

[0077] Step (4) warm hydrogel precursor solution H and hydrogel precursor solution I obtained in step (3) to 37℃, inject 16 μL of hydrogel precursor solution H into the tubular mold through 0.22 μm filter in a syringe, crosslink the hydrogel precursor solution in the mold under 405 nm blue light, the light crosslinking time is 8 s, finally inject 4 μL of hydrogel precursor solution I into the same tubular mold through 0.22 μm filter in a syringe, crosslink the hydrogel and hydrogel precursor solution in the mold again under 405 nm blue light, the light crosslinking time is 8 s, to get multi-segment filling hydrogel, the length ratio of the short segment and the long segment is 1:4;

[0078] Step (5) use the multi-segment filling hydrogel obtained in step (4) as mold, contact the inner layer of the polymer fiber membrane of gradient density deposited growth factor particles obtained in step (1) with the hydrogel and wrap into a tube, smear polymer solution A at the adhesion site at room temperature, dry in the super-clean bench after adhesion, finally demold to get rigid-flexible composite nerve conduit which can release anti-inflammatory substances and growth factors in stages.

[0079] Example 3

[0080] Step (1) prepare polylactic acid-glycolic acid copolymer (PLGA) solution with volume ratio of dichloromethane:N,N-dimethylformamide = 4:1, magnetically stir at room temperature for 24 h to get polymer solution A with mass concentration of 10%; perform uniaxial electrospinning with solution A, the flow rate of solution A is 1 mL / h, the voltage is 16 kV, the receiving distance is 15 cm, use high-speed rotating roller as receiver, spin for 5 h to get unidirectional ordered arranged fibers, then gradually reduce the roller speed, continue to collect randomly arranged fibers for 5 h to get polymer fiber membrane with double-layer structure; place the polymer fiber membrane in the fume hood at room temperature for 3 days to fully volatilize the residual solvent;

[0081] Step (2) dissolve brain-derived neurotrophic factor in PBS, mix well to get growth factor solution B with a concentration of 100 μg / mL; dissolve collagen in 70% acetic acid aqueous solution, mix well to get bioactive substance solution C with a concentration of 20 mg / mL; replace the electrostatic liquid with growth factor solution B and bioactive substance solution C, replace the single-axis spinning needle with a coaxial needle, inject growth factor solution B into the core layer syringe and bioactive substance solution C into the shell layer syringe; use the inner layer of the polymer fiber membrane obtained in step (1) as a receiver to perform coaxial electrostatic spraying, adopt a mask strategy to deposit bioactive substance particles with increasing particle density along the unidirectional ordered arrangement direction, the flow rate of growth factor solution B is 0.5 mL / h, the flow rate of bioactive substance solution C is 1.5 mL / h, the voltage is 20 kV, and the electrostatic spraying is performed for 30 min to deposit growth factor particles on the inner surface of the polymer fiber membrane in a gradient density;

[0082] Step (3) dissolve recombinant human basic fibroblast growth factor, nerve growth factor, and glial cell-derived neurotrophic factor in PBS, mix well to get growth factor solution D with a concentration of 100 μg / mL; add curcumin and resveratrol to PBS, mix well to get anti-inflammatory substance solution E with a concentration of 100 μg / mL; add LAP to growth factor solution D and anti-inflammatory substance solution E respectively, mix well at 37°C in the dark, get initiator solution F and initiator solution G with a mass fraction of 0.25%, and add methacrylated gelatin to initiator solution F and initiator solution G respectively, mix well at 37°C in the dark, get hydrogel precursor solution H and hydrogel precursor solution I with a mass fraction of 10%;

[0083] Step (4) warm hydrogel precursor solution H and hydrogel precursor solution I obtained in step (3) to 37°C, then inject 12 μL of hydrogel precursor solution H into the tubular mold through a 0.22 μm filter in a syringe, crosslink the hydrogel precursor solution in the mold under 405 nm blue light irradiation, the light crosslinking time is 8 s, finally inject 8 μL of hydrogel precursor solution I into the same tubular mold through a 0.22 μm filter in a syringe, crosslink the hydrogel and hydrogel precursor solution in the mold again under 405 nm blue light irradiation, the light crosslinking time is 8 s, get a multi-segment filling hydrogel, the length ratio of the short segment and the long segment in it is 1:1.5;

[0084] Step (5) The inner layer of the gradient density deposited growth factor particle polymer fiber membrane obtained in step (1) is contacted with the multi-section filled hydrogel obtained in step (4) and wrapped into a tube, polymer solution A is applied at the adhesion site at room temperature, and after adhesion, the adhesion site is dried in an ultra-clean bench, and finally the hydrogel is removed to obtain a rigid-flexible composite nerve conduit capable of releasing anti-inflammatory substances and growth factors in stages.

[0085] Example 4

[0086] Step (1) A polylactic acid-glycolic acid copolymer (PLGA) solution is prepared with dichloromethane: N,N-dimethylformamide = 4:1 by volume ratio, magnetically stirred at room temperature for 24 h to obtain a polymer solution A with a mass concentration of 12%; uniaxial electrospinning is performed with solution A, the flow rate of solution A is 0.8 mL / h, the voltage is 15 kV, the receiving distance is 15 cm, a U-shaped groove is used as the receiver, and the spinning is performed for 6 h to obtain a unidirectionally ordered fiber membrane, then the U-shaped groove is removed, and the obtained unidirectionally ordered fiber membrane is used as a collector to collect randomly arranged fibers for 6 h to obtain a double-layer structured polymer fiber membrane; the polymer fiber membrane is placed in a fume hood at room temperature for 3 days to fully volatilize the residual solvent;

[0087] Step (2) The brain-derived neurotrophic factor is dissolved in PBS and mixed uniformly to obtain a growth factor solution B with a concentration of 150 μg / mL; collagen is dissolved in a 70% by volume aqueous acetic acid solution and mixed uniformly to obtain a bioactive substance solution C with a concentration of 20 mg / mL; the electrospinning liquid is replaced with the growth factor solution B and the bioactive substance solution C, the uniaxial spinning needle is replaced with a coaxial needle, the growth factor solution B is injected into the core layer syringe, and the bioactive substance solution C is injected into the shell layer syringe; the inner layer of the polymer fiber membrane obtained in step (1) is used as the receiver to perform coaxial electrospinning, a mask plate strategy is adopted to deposit bioactive substance particles with increasing particle density along the unidirectionally ordered arrangement direction, the flow rate of the growth factor solution B is 0.8 mL / h, the flow rate of the bioactive substance solution C is 1.6 mL / h, the voltage is 18 kV, and the electrospinning is performed for 10 min to obtain a polymer fiber membrane with gradient density deposited growth factor particles on the inner surface;

[0088] Step (3) VEGF, NGF-3, GDNF were dissolved in PBS, mixed well to obtain growth factor solution D with a concentration of 150 μg / mL; curcumin and resveratrol were added to PBS, mixed well to obtain anti-inflammatory substance solution E with a concentration of 150 μg / mL; LAP was added to growth factor solution D and anti-inflammatory substance solution E respectively, mixed well at 37℃ in dark, to obtain initiator solution F and initiator solution G with a mass fraction of 0.25%; methacrylated gelatin was added to initiator solution F and initiator solution G respectively, mixed well at 37℃ in dark, to obtain hydrogel precursor solution H and hydrogel precursor solution I with a mass fraction of 12%;

[0089] Step (4) hydrogel precursor solution H and hydrogel precursor solution I obtained in step (3) were warmed to 37℃, hydrogel precursor solution H was added to a syringe and injected into the tubular mold at a rate of 18 μL through a 0.22 μm filter, the hydrogel precursor solution in the mold was crosslinked under 405 nm blue light irradiation, the light crosslinking time was 10 s, finally hydrogel precursor solution I was added to a syringe and injected into the same tubular mold at a rate of 2 μL through a 0.22 μm filter, the hydrogel and hydrogel precursor solution in the mold were crosslinked again under 405 nm blue light irradiation, the light crosslinking time was 10 s, to obtain a multi-segment filling hydrogel, the length ratio of the short segment and the long segment in the hydrogel was 1:9;

[0090] Step (5) the multi-segment filling hydrogel obtained in step (4) was used as a mold, the inner layer of the polymer fiber membrane of the gradient density deposited growth factor particles obtained in step (1) was contacted with the hydrogel and wrapped into a tube, polymer solution A was applied at the adhesion site at room temperature, and the adhesion site was dried in a super-clean bench, and finally the mold was removed to obtain a rigid-flexible composite nerve conduit capable of releasing anti-inflammatory substances and growth factors in stages.

[0091] Comparative Example 1

[0092] Step (1) polycaprolactone was added to a mixed solution of dichloromethane and N,N-dimethylformamide (volume ratio of dichloromethane to N,N-dimethylformamide was 4:1), and magnetic stirring was carried out at room temperature for 24 h to obtain polymer solution A with a mass concentration of 10%; uniaxial electrospinning was carried out using solution A, the flow rate of solution A was 1 mL / h, the voltage was 16 kV, the receiving distance was 15 cm, a high-speed rotating roller was used as the receiver, and spinning was carried out for 5 h to obtain unidirectionally ordered fibers, then the roller speed was gradually reduced, and random arrangement fibers were continuously collected for 5 h to obtain a polymer fiber membrane with a double-layer structure; the polymer fiber membrane was placed in a fume hood at room temperature for 3 days to allow the residual solvent to volatilize sufficiently;

[0093] Step (2) collagen was dissolved in 70% acetic acid aqueous solution, mixed well to obtain a biological active substance solution C with a concentration of 20 mg / mL; the electrospinning liquid was replaced by the biological active substance solution C, a uniaxial spinning needle was used, and the biological active substance solution was injected into a syringe; the inner layer of the polymer fiber membrane obtained in step (1) was used as a receiver to perform uniaxial electrostatic spraying, a mask plate strategy was adopted to deposit biological active substance particles with an increasing particle density gradient along the unidirectional ordered arrangement direction, the flow rate of the biological active substance solution C was 1.5 mL / h, the voltage was 20 kV, the electrostatic spraying was performed for 30 min, and the inner surface of the polymer fiber membrane was gradient density deposited with pure collagen particles.

[0094] This comparative example prepared a polymer fiber membrane with an inner surface gradient density deposited with pure collagen particles corresponding to Example 1.

[0095] Comparative Example 2

[0096] Step (1) polycaprolactone was taken into a mixed solution of dichloromethane and N,N-dimethylformamide (the volume ratio of dichloromethane and N,N-dimethylformamide was 4:1), and was magnetically stirred at room temperature for 24 h to obtain a polymer solution A with a mass concentration of 10%; uniaxial electrospinning was performed using the solution A, the flow rate of the solution A was 1 mL / h, the voltage was 16 kV, the receiving distance was 15 cm, a high-speed rotating roller was used as a receiver, and the spinning was performed for 5 h to obtain unidirectionally ordered arranged fibers, and then the roller speed was gradually reduced, and randomly arranged fibers were continuously collected for 5 h to obtain a polymer fiber membrane with a double-layer structure; the polymer fiber membrane was placed in a fume hood at room temperature for 3 days to allow the residual solvent to be fully volatilized;

[0097] Step (2) collagen was dissolved in 70% acetic acid aqueous solution, mixed well to obtain a biological active substance solution C with a concentration of 20 mg / mL; the electrospinning liquid was replaced by the biological active substance solution C, a uniaxial spinning needle was used, and the biological active substance solution was injected into a syringe; the inner layer of the polymer fiber membrane obtained in step (1) was used as a receiver to perform uniaxial electrostatic spraying, a mask plate strategy was adopted to deposit biological active substance particles with an increasing particle density gradient along the unidirectional ordered arrangement direction, the flow rate of the biological active substance solution C was 1.5 mL / h, the voltage was 20 kV, the electrostatic spraying was performed for 30 min, and the inner surface of the polymer fiber membrane was gradient density deposited with pure collagen particles.

[0098] This comparative example prepared a polymer fiber membrane with an inner surface gradient density deposited with pure collagen particles corresponding to Example 1.

[0099] Comparative Example 3

[0100] Step (1) : Polycaprolactone was added into a mixed solution of dichloromethane and N,N-dimethylformamide (volume ratio of dichloromethane to N,N-dimethylformamide was 4:1), and stirred magnetically at room temperature for 24 h to obtain a polymer solution A with a mass concentration of 10% ; uniaxial electrospinning was performed using the solution A, the flow rate of the solution A was 1 mL / h, the voltage was 16 kV, the receiving distance was 15 cm, a high-speed rotating roller was used as the receiver, and the spinning was performed for 5 h to obtain unidirectionally ordered arranged fibers, then the rotation speed of the roller was gradually reduced, and randomly arranged fibers were continuously collected for 5 h to obtain a polymer fiber membrane with a double-layer structure; the polymer fiber membrane was placed in a fume hood at room temperature for 3 days to fully volatilize the residual solvent;

[0101] This comparative example prepared a polymer fiber membrane corresponding to Example 1, but without depositing particles of bioactive substances on the inner surface.

[0102] Test Example:

[0103] CCK-8 method was used to perform cell proliferation test to verify the proliferation activity of cells on the polymer fiber membrane materials prepared in the examples and comparative examples.

[0104] The material treated by ultraviolet disinfection sterilization and other treatments was cut into a material with a diameter of 1.5 cm and laid on the bottom of a 24-well plate. A 1.5 cm diameter ring treated by high pressure sterilization and ultraviolet disinfection was used to fix the material so that the inner layer was upwardly embedded in the bottom. PBS buffer was added to the well plate and soaked for 20 minutes. A shaking table was used for shaking, and the surface residues of the material were washed away. After the shaking was completed, the PBS was poured out, and fresh PBS buffer was added for repeated washing twice, and then Schwann cell culture solution was used for washing once. The Schwann cells grown to cover the bottom of the T25 culture bottle were used for plate seeding. 1 mL of trypsin-EDTA solution with a mass ratio of 0.25% was added to the T25 culture bottle full of Schwann cells, and incubated in a 37°C cell incubator for 3 minutes. After being taken out, the side wall of the culture bottle was gently tapped, 3 mL of Schwann cell culture solution was added, and after being mixed uniformly, the liquid in the bottle was sucked out, and the cells were centrifuged at a speed of 1000 rpm for 2 minutes. After the centrifugation was completed, the supernatant was poured out, and the cells after centrifugation were blown and centrifuged with 10 mL of cell culture solution. The cell suspension was transferred to the well plate at 100 μL / well, seeded on the surface of the polymer fiber membrane material, and then 900 μL of cell culture solution was added to each well. The 24-well plate after seeding was placed in the cell culture box. The first day after seeding was recorded as 0d, and the time was counted from 0d. In the cell proliferation experiment of the fiber membrane, the cells proliferated on the blank group (not seeded on the polymer fiber membrane), the polymer fiber membrane without deposition of bioactive substance particles in Comparative Example 3, the polymer fiber membrane with uniform density deposition of pure collagen particles in Comparative Example 2, the polymer fiber membrane with gradient density deposition of pure collagen particles in Comparative Example 1, and the polymer fiber membrane with gradient density deposition of growth factor particles in Example 1 were tested for absorbance (OD) value at 1d, 3d, 5d, and 7d. All materials were changed at 1d, 3d, 5d, and 7d. After the original culture solution in each well was sucked out, 1 mL of cell culture solution was added. The CCK-8 test was performed on the wells participating in the test. After the original culture solution in each well was sucked out, CCK-8 solution was prepared by mixing cell culture solution and 10% CCK-8 reagent by volume, and 500 μL / well was added. The well plate was placed in the cell culture box for 4 hours, and then the OD value at 450 nm wavelength was tested. The OD value reflects the cell proliferation and is proportional to the cell number. The test results are shown in Table 1. Figure 5 As shown in Table 1, the OD value increased with the increase of days, indicating that the cells proliferated on the material, the cells could adhere and grow on the fiber, the cell growth state was good, the cell morphology was clear, and the excellent biocompatibility of the material was proved. Figure 5 As can be seen from Table 1, the gradient density signal and the deposition of growth factors on the fiber membrane in Example 1 have a promoting effect on cell proliferation.

Claims

1. A staged release rigid-flexible composite nerve conduit, comprising a polymer fiber membrane as the wall and a multi-segment hydrogel as the inner core, wherein the inner wall of the polymer fiber membrane is deposited with bioactive particles containing growth factors, and the polymer fiber membrane is a bilayer nanofiber membrane structure, wherein... The inner layer is a unidirectionally ordered nanofiber membrane, and the outer layer is a randomly arranged nanofiber membrane. The multi-segment hydrogel includes short-segment hydrogels containing anti-inflammatory substances and long-segment hydrogels containing growth factors. The short-segment hydrogels containing anti-inflammatory substances degrade and release anti-inflammatory drugs in the early stage of spinal cord injury to regulate the inflammatory microenvironment. The long-segment hydrogels containing growth factors degrade and replenish various growth factors in the damaged area in the middle and late stages of spinal cord injury.

2. The rigid-flexible composite nerve conduit according to claim 1, characterized in that, The polymer in the polymer fiber is selected from at least one of biodegradable aliphatic polyesters; and / or, The anti-inflammatory substance is selected from at least one of catalase, superoxide dismutase, interleukin, and reducing molecules; and / or, The growth factor is selected from at least one of vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, brain-derived neurotrophic factor, neurotrophic factor-3, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, and nerve growth factor; and / or, The hydrogel precursor compound used in the hydrogel is selected from at least one of acrylamide biomacromolecules; and / or The bioactive substance particles containing growth factors have a core-shell structure, wherein the shell layer is the bioactive substance and the core layer is the growth factor; and / or, The bioactive substance is selected from at least one of protein compounds.

3. The rigid-flexible composite nerve conduit according to claim 2, characterized in that, The polymer in the polymer fiber is selected from at least one of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, and polylactic acid-glycolic acid-caprolactone copolymer; and / or, The hydrogel precursor compound used in the hydrogel is selected from at least one of methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide silk fibroin, methacrylamide chitosan, and methacrylamide sodium alginate; and / or, The bioactive substance is selected from at least one of collagen, laminin, fibronectin, and collagen protein.

4. The rigid-flexible composite nerve conduit according to claim 1, characterized in that, The thickness of the polymer fiber membrane tube wall is 50~800 µm; and / or, The length ratio of the short hydrogel segment to the long hydrogel segment is 1:(1.5~10); and / or, The cross-linking degree of the short-segment hydrogel is lower than that of the long-segment hydrogel.

5. The rigid-flexible composite nerve conduit according to claim 4, characterized in that, The thickness of the polymer fiber membrane tube wall is 100~500 µm; and / or, The length ratio of the short hydrogel to the long hydrogel is 1:(1.5~5).

6. A method for preparing a staged release rigid-flexible composite nerve conduit according to any one of claims 1 to 5, comprising: A multi-segment hydrogel was obtained by cross-linking a hydrogel precursor solution containing anti-inflammatory substances and a hydrogel precursor solution containing growth factors with ultraviolet light. Then, the multi-segment hydrogel was wrapped with a polymer fiber membrane with bioactive material particles containing growth factors deposited in the inner layer, thus obtaining the staged release rigid-flexible composite nerve conduit.

7. The preparation method according to claim 6, characterized in that, The preparation method includes the following steps: Step (1) Electrospinning polymer solution A to obtain a bilayer polymer fiber membrane; Step (2) The growth factor solution B and the bioactive substance solution C are coaxially electrostatically sprayed onto the inner layer of the polymer fiber membrane obtained in step (1) to prepare a polymer fiber membrane with gradient density deposition of bioactive substance particles. Step (3) Mix growth factor solution D and anti-inflammatory substance solution E with initiator respectively to obtain initiator solution F and initiator solution G, and then add hydrogel precursor compound respectively to obtain hydrogel precursor solution H and hydrogel precursor solution I; Step (4) Inject the hydrogel precursor solution H into the mold, crosslink it with ultraviolet light, and then continue to inject the hydrogel precursor solution I. After ultraviolet light crosslinking, multi-segment hydrogel is obtained. Step (5) The inner layer of the polymer fiber membrane containing the gradient density deposited bioactive material particles obtained in step (2) is contacted with the multi-segment hydrogel obtained in step (4) and wrapped into a tube. Finally, polymer solution A is used for adhesion to obtain the staged release rigid-flexible composite nerve conduit.

8. The preparation method according to claim 7, characterized in that, In step (1): The solvent in the polymer solution A is selected from at least one of hexafluoroisopropanol, trifluoroethanol, chloroform, methanol, dichloromethane, and N,N'-dimethylformamide; and / or, The polymer solution A has a mass percentage concentration of 5-25%; and / or, The electrospinning process includes: uniaxial electrospinning of polymer solution A, using a roller or U-shaped groove as a receiver, with a spinning solution flow rate of 0.1~3 mL / h, a voltage of 10~20 kV, and a receiving distance of 10~20 cm.

9. The preparation method according to claim 8, characterized in that, In step (1): The polymer solution A has a mass percentage concentration of 8-15%; and / or, When the electrospinning uses a roller as the receiver, the spinning is first performed for 2-10 hours using a high-speed rotating roller as the receiver to obtain a unidirectionally ordered fiber membrane, and then the roller speed is reduced for 2-10 hours of spinning; or, when the electrospinning uses a U-shaped groove as the receiver, the spinning is first performed for 2-10 hours using the U-shaped groove as the receiver to obtain a unidirectionally ordered fiber membrane, and then the U-shaped groove is removed, and the obtained unidirectionally ordered fiber membrane is used as the collector for 2-10 hours of spinning.

10. The preparation method according to claim 7, characterized in that, In step (2): The solvent in the growth factor solution B is selected from at least one of phosphate buffer solution, water, ethanol, polyvinyl alcohol, polyacrylamide, and polyacrylic acid; and / or, The concentration of the growth factor solution B is 0.1 μg / mL to 5 mg / mL; and / or, The solvent in the bioactive substance solution C is selected from at least one of hydrochloric acid, aqueous acetic acid solution, ethyl acetate, glycerol, trifluoroethanol, and hexafluoroisopropanol; and / or, The concentration of the bioactive substance solution C is 1~50 mg / mL; and / or, The coaxial electrostatic spraying includes: depositing bioactive material particles in a gradient manner along the orderly arrangement direction of the fibers using a mask pattern; injecting bioactive material solution C into the shell injector; injecting growth factor solution B into the core injector; using the inner layer of the polymer fiber membrane obtained in step (1) as the receiver; the flow rate of the shell electrospray solution is 0.5~3 mL / h; the flow rate of the core electrospray solution is 0.2~1 mL / h; the voltage is 10~20 kV; the receiving distance is 10~20 cm; and the electrostatic spraying time is 1~60 min.

11. The preparation method according to claim 10, characterized in that, In step (2): The concentration of the growth factor solution B is 1 μg / mL to 1 mg / mL; and / or, The concentration of the bioactive substance solution C is 10~30 mg / mL.

12. The preparation method according to claim 7, characterized in that, In step (3): The solvent in the growth factor solution D is selected from at least one of phosphate buffer solution, water, ethanol, polyvinyl alcohol, polyacrylamide, and polyacrylic acid; and / or, The concentration of the growth factor solution D is 0.1 μg / mL to 5 mg / mL; and / or, The solvent in the anti-inflammatory solution E is selected from at least one of phosphate buffer solution, water, chloroform, glycerol, trifluoroethanol, and hexafluoroisopropanol; and / or, The concentration of the anti-inflammatory substance solution E is 1 μg / mL to 5 mg / mL; and / or, The initiator in initiator solution F and the initiator in initiator solution G are independently selected from at least one of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and ethyl 2,4,6-trimethylbenzoylphenylphosphonate; and / or, The initiator solution F contains 0.1% to 0.5% by mass of the initiator; and / or, The initiator solution G contains 0.1% to 0.5% by mass of the initiator; and / or, The hydrogel precursor solution H contains 5-15% by mass of the hydrogel precursor compound; and / or, The mass percentage of the hydrogel precursor compound in the hydrogel precursor solution I is 5-15%.

13. The preparation method according to claim 12, characterized in that, In step (3): The concentration of the growth factor solution D is 1 μg / mL to 1 mg / mL; and / or, The concentration of the anti-inflammatory substance solution E is 0.1~2 mg / mL; and / or, The initiator solution F contains 0.15% to 0.3% by mass of initiator; and / or, The initiator solution G contains 0.15% to 0.3% by mass of initiator; and / or, The hydrogel precursor solution H contains 8-12% by mass of the hydrogel precursor compound; and / or, The mass percentage of the hydrogel precursor compound in the hydrogel precursor solution I is 8-12%.

14. The preparation method according to claim 7, characterized in that, In step (4): The mold is a tubular mold; and / or, The conditions for ultraviolet crosslinking are: crosslinking temperature of 18~37 ℃ and crosslinking time of 4~60 s.

15. The preparation method according to claim 14, characterized in that, In step (4): The conditions for ultraviolet crosslinking are: crosslinking temperature of 20~30 ℃ and crosslinking time of 4~30 s.

16. The application of a staged release rigid-flexible composite nerve conduit according to any one of claims 1 to 5, or a staged release rigid-flexible composite nerve conduit obtained by the preparation method according to any one of claims 6 to 15, in the preparation of bio-based repair materials.

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