A nerve regeneration guide conduit for promoting neural cell differentiation and a preparation method and application thereof

Abstract

The application discloses a nerve regeneration guide conduit for promoting nerve cell differentiation and a preparation method and application thereof. The nerve regeneration guide conduit can realize stable and long-acting release of bioactive factors, effectively reduce the risk of tissue adhesion, efficiently induce nerve cell differentiation, promote axon growth and function recovery, and provides a novel and effective solution for nerve regeneration and repair. In addition, the nerve regeneration guide conduit has excellent conductivity, biocompatibility, mechanical adaptability and the like, and therefore has a wide application prospect in the field of nerve regeneration and repair.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and in particular relates to a nerve regeneration guiding conduit for promoting nerve cell differentiation, its preparation method and application. Background Technology

[0002] Peripheral nerve injury is one of the most common traumatic diseases in clinical practice, severely affecting the recovery of patients' sensory and motor functions. Statistics show that millions of cases of traumatic peripheral nerve injury occur globally each year, commonly due to traffic accidents, industrial accidents, intraoperative injuries, or chronic compressive diseases. Traditional treatments mainly include autologous nerve transplantation, but limitations such as limited donor sources, secondary trauma to the surgical site, and graft rejection make it difficult to meet the needs of widespread clinical application. Therefore, the development of high-performance nerve guidance conduits (NGCs) to replace autologous nerve transplantation has become an important direction in current tissue engineering and neuroregenerative medicine research.

[0003] Current research on nerve conduits largely focuses on the synergistic delivery of material conductivity and bioactive factors. For example, biodegradable conductive composite materials are used to construct the conduit wall and load it with bioactive factors to promote nerve repair. However, such conduit structures are relatively dense, easily triggering adhesion to surrounding tissues and inflammatory responses. Furthermore, the lack of design for regulating hydration at the conduit interface can easily lead to postoperative nerve adhesion. Nerve adhesion not only physically compresses and restricts axonal extension but also induces chronic inflammation and nerve ischemia, and may even cause secondary loss of nerve conduction function due to scarring.

[0004] Therefore, existing neural conduit materials still have significant shortcomings in terms of conductivity, biocompatibility, anti-adhesion, and controlled release of biological factors. There is an urgent need to develop a novel neural regeneration guiding conduit with a reasonable structure and synergistic function to better adapt to the complex neural regeneration microenvironment. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, the present invention provides a neural regeneration guiding conduit for promoting neural cell differentiation, a method for its preparation, and its applications. Specifically, the present invention includes the following:

[0006] A first aspect of the present invention provides a neural regeneration guiding conduit for promoting neural cell differentiation, comprising a conduit body and a conductive gel located inside the conduit, wherein: The catheter body is made from polylactic acid-containing raw materials, and the wall of the catheter body is modified to include an anti-adhesion layer; The conductivity of the conductive gel is 10. -2 -10 2 S / m, and contains nerve growth factor or its precursor.

[0007] In some embodiments, the nerve regeneration guiding conduit for promoting nerve cell differentiation according to the present invention has a length of 10-15 mm, an inner diameter of 1.5-2.5 mm, and a wall thickness of 0.4-0.5 mm.

[0008] In some embodiments, the neural regeneration guiding conduit for promoting nerve cell differentiation according to the present invention, wherein the anti-adhesion layer comprises a hydrophilic polymer and has a contact angle of 0-10°.

[0009] In some embodiments, the neural regeneration guiding conduit for promoting nerve cell differentiation according to the present invention, wherein the anti-adhesion layer is connected to the conduit wall via polydopamine.

[0010] In some embodiments, the neural regeneration guiding conduit for promoting nerve cell differentiation according to the present invention, wherein the conductive gel is a methacryloyl hyaluronic acid hydrogel comprising poly(3,4-ethylenedioxythiophene):dextran sulfate.

[0011] A second aspect of the present invention provides a method for preparing a neural regeneration guiding conduit for promoting neural cell differentiation, comprising the following steps: (1) Electrospinning was performed using polylactic acid in an organic solvent to obtain a polylactic acid film with oriented arrangement. The obtained polylactic acid film was then annealed. (2) The annealed polylactic acid film is treated with dopamine, and then polyethylene glycol is grafted onto the treated polylactic acid film to obtain a hydration layer. Next, the obtained polylactic acid film is rolled up to form a nerve conduit. (3) The conductive gel precursor is infused into the interior of the nerve conduit to obtain the nerve regeneration guiding conduit for promoting nerve cell differentiation.

[0012] In some embodiments, according to the preparation method of the present invention, the organic solvent includes at least one selected from trifluoroethanol, dichloromethane, N,N-dimethylformamide, chloroform, ethanol, isopropanol, 2-pyrrolidone, glycerol, propylene glycol, polyethylene glycol, tetraethylene glycol, and acetone.

[0013] In some embodiments, according to the preparation method of the present invention, the conductive gel precursor comprises poly(3,4-ethylenedioxythiophene):dextran sulfate, nerve growth factor or a precursor thereof, a curing agent, and methacrylamide hyaluronic acid.

[0014] In some embodiments, according to the preparation method of the present invention, the step of promoting the solidification of the conductive gel precursor is further included after the conductive gel precursor is infused into the interior of the nerve conduit.

[0015] A third aspect of the present invention provides an in vitro nerve regeneration method for promoting nerve cell differentiation, comprising the step of contacting the nerve regeneration guiding conduit described in the first aspect of the present invention with nerve cells in vitro.

[0016] In some embodiments, the in vitro neural regeneration method for promoting neural cell differentiation according to the present invention further includes the step of applying ultrasound treatment.

[0017] A fourth aspect of the invention provides the use of the nerve regeneration guiding conduit according to the first aspect of the invention in the preparation of medical products that promote nerve cell differentiation and / or nerve regeneration.

[0018] The beneficial effects of this invention include: (1) Bionic guiding structure design: Through longitudinal electrospun nanofiber network, the orderly growth of nerve axons is effectively induced, thereby improving the quality of nerve regeneration.

[0019] (2) Significant anti-adhesion function: By modifying the anti-adhesion layer, a stable hydration layer is formed on the surface of the nanofiber, which inhibits non-specific cell adhesion and reduces the incidence of tissue adhesion after catheter implantation.

[0020] (3) Enhanced electroactivity: The conductive components are uniformly dispersed in the hydrogel, which gives the material good electrical conductivity and can provide weak electrical signal stimulation, which helps to activate nerve cell function.

[0021] (4) Controlled release of biological factors: Nerve growth factors are embedded in the hydrogel network to achieve good sustained release capacity and provide continuous biological stimulation for nerve repair.

[0022] (5) Good biocompatibility and molding performance: The materials used in this invention have good biodegradability and compatibility, and the process is simple and easy to prepare on a large scale.

[0023] (6) The overall catheter performance is synergistically matched with the neural regeneration microenvironment, which can effectively improve the efficiency of functional reconstruction of neural tissue and has broad clinical translation prospects. Attached Figure Description

[0024] Figure 1 A macroscopic image of the nerve regeneration guiding conduit of the present invention is shown.

[0025] Figure 2The chemical composition and hydrophilicity characterization of the nanofiber surface are shown, where A represents the chemical composition of each group of nanofiber surfaces, and B represents the change in water contact angle of PLLA and PDLA nanofiber membranes before and after modification.

[0026] Figure 3 The morphology characterization and piezoelectric property analysis of the nanofibers are shown, where A represents the morphology of each group of nanofibers and B represents the piezoelectric properties of each group of nanofibers.

[0027] Figure 4 The adhesion behavior of L929 cells on the nanofiber surface is shown. A and C are the results of confocal laser scanning microscopy analysis of cell adhesion behavior, and B and D are the quantitative statistical analysis results of A and C, respectively.

[0028] Figure 5 The results of the evaluation of the induced differentiation behavior of PC12 cells on the nanofiber surface are shown.

[0029] Figure 6 The results of optimization and screening of electrical conductivity based on conductive hydrogels are shown.

[0030] Figure 7 The results of repairing sciatic nerve injury in mice using a nerve-guided conduit are shown. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0034] Nerve regeneration guiding catheter In one aspect, the present invention provides a nerve regeneration guiding conduit for promoting nerve cell differentiation, comprising a conduit body and a conductive gel located inside the conduit. The nerve regeneration guiding conduit of the present invention can promote nerve cell differentiation, thereby effectively promoting axonal growth and functional recovery of nerves (e.g., but not limited to the sciatic nerve), and has advantages such as excellent conductivity, biocompatibility, mechanical adaptability, reduced risk of tissue adhesion, and stable release of bioactive factors.

[0035] In some implementations, "neural cell differentiation" refers to the process by which nerve cells or their precursor cells mature, grow axons, and establish connections under the influence of physical, chemical, and / or electrical signals provided by a nerve regeneration guiding conduit.

[0036] In a preferred embodiment, the catheter body is made of polylactic acid (e.g., but not limited to poly-L-lactic acid, poly-D-lactic acid, etc.), and the wall of the catheter body is modified to include an anti-adhesion layer. The anti-adhesion layer comprises a hydrophilic polymer, which is not particularly limited, but examples include, but are not limited to, polyethylene glycol, hyperbranched polyglycerol, poly(2-hydroxyethylacrylamide), polyvinyl alcohol, polyvinylpyrrolidone, poly(N-acryloylmorpholine), etc.

[0037] In a preferred embodiment, the anti-adhesion layer is connected to the pipe wall via polydopamine.

[0038] The nerve regeneration guiding conduit of the present invention, after being modified to include an anti-adhesion layer, can achieve a superhydrophilic state with a contact angle of 0-10°, preferably 0-9°, even more preferably 0-8°, further preferably 0-7°, more preferably 0-6°, and even more preferably 0-5°, such as 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5°, etc.

[0039] The nerve regeneration guiding catheter of the present invention, after being modified to include an anti-adhesion layer, can inhibit the adhesion of non-specific cells (such as, but not limited to, fibroblasts, inflammatory cells, vascular endothelial cells, etc.), reduce the incidence of tissue adhesion after catheter implantation, reduce inflammatory response and avoid scar formation.

[0040] In a preferred embodiment, the nerve conduit has a length of 10-15 mm (e.g., 10, 10.2, 10.4, 10.6, 10.8, 11, 11.2, 11.4, 11.6, 11.8, 12, 12.2, 12.4, 12.6, 12.8, 13, 13.2, 13.4, 13.6, 13.8, 14, 14.2, 14.4, 14.6, 14.8, 15 mm), an inner diameter of 1.5-2.5 mm (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 mm), and a wall thickness of 0.4-0.5 mm. mm (e.g., 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5 mm).

[0041] In some embodiments, the conductivity of the conductive gel is 10. -2 -10 2 The conductive gel of the present invention comprises poly(3,4-ethylenedioxythiophene):dextran sulfate and a gel matrix, wherein the gel matrix is ​​not particularly limited, and examples include, but are not limited to, alginate or derivatives thereof, chondroitin sulfate or derivatives thereof, dextran or derivatives thereof, chitosan or derivatives thereof, hyaluronic acid or derivatives thereof, etc. "Nerve growth factor precursor" refers to a compound that can generate and release nerve growth factor under operating conditions, and such substances are generally stable in vitro. The nerve growth factor includes, but is not limited to, NGF, GDNF, FGF, etc. In a preferred embodiment, the gel matrix is ​​methacrylamide hyaluronic acid, and the nerve growth factor is NGF.

[0042] Preparation method In one aspect, the present invention provides a method for preparing a neural regeneration guiding conduit for promoting neural cell differentiation.

[0043] In a preferred embodiment, the method for preparing the nerve regeneration guiding conduit of the present invention includes the following steps: (1) Electrospinning is performed using polylactic acid (PLA) in an electrospinning solution obtained in an organic solvent to obtain a directionally aligned PLA film. The obtained PLA film is then subjected to two annealing treatments. The mass ratio of PLA to organic solvent is 8-12% (e.g., 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12%). The electrospinning conditions include: applying a positive and negative high voltage of 10-15 kV (preferably 11-14 kV, more preferably 12-14 kV, even more preferably 13-14 kV, e.g., 13, 13.2, 13.4, 13.6, 13.8, 14 kV), and a feed pump speed of 0.05-0.15 mm / min (preferably 0.06-0.14 mm / min, even more preferably 0.07-0.13 mm / min, even more preferably 0.08-0.12 mm / min, and more preferably 0.09-0.11 mm / min). The receiving speed is 15-25 cm (preferably 16-24 cm, even more preferably 17-23 cm, more preferably 18-22 cm, such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22 cm), and the receiving shaft speed is 3000-5000 rpm (preferably 3100-4900 rpm, even more preferably 3200-4800 rpm, further preferably 3300-4700 rpm, more preferably 3400-4600 rpm, even more preferably 3500-4500 rpm, such as 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500). The first annealing is carried out at 90-120℃ (preferably 92-118℃, more preferably 94-116℃, further preferably 96-114℃, more preferably 98-112℃, and even more preferably 100-110℃, such as 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110℃) for 5-15 h (preferably 6-14 h, more preferably 7-13 h, such as 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13 rpm); The second annealing is carried out at 150-170℃ (preferably 151-169℃, even more preferably 152-168℃, further preferably 153-167℃, more preferably 154-166℃, and even more preferably 155-165℃, for example 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165℃) for 5-15 h (preferably 6-14 h, even more preferably 7-13 h, for example 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13 h). (2) The annealed polylactic acid film is co-incubated with a dopamine solution at a concentration of 1-10 mg / ml (preferably 2-9 mg / ml, even more preferably 3-8 mg / ml, for example 3, 4, 5, 6, 7, 8 mg / ml) for 20-40 min (preferably 21-39 min, even more preferably 22-38 min, further preferably 23-37 min, more preferably 24-36 min, even more preferably 25-35 min, for example 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 min). Then the film is co-incubated with a polyethylene glycol solution at a concentration of 1-5 mg / ml (for example 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg / ml) for 24-48 h (24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48). h), the obtained polylactic acid film is rolled into a nerve conduit with a diameter of 1-3 mm (preferably 1.1-2.9 mm, more preferably 1.2-2.8 mm, further preferably 1.3-2.7 mm, more preferably 1.4-2.6 mm, even more preferably 1.5-2.5 mm, for example 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5 mm), preferably, at 60-80°C (preferably 61-79°C, more preferably 62-78°C, further preferably 63-77°C, more preferably 64-76°C, even more preferably 65-75°C, for example 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75°C) for 10-30 min (preferably 11-29 min, more preferably 12-28 min, even more preferably 13-27 min). 14-26 min, more preferably 15-25 min, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 min; (3) The conductive gel precursor was infused into the interior of the nerve conduit at a concentration of 10-30 mW / cm². 2 (Preferred value: 11-29mW / cm) 2 Furthermore, a value of 12-28 mW / cm is preferred. 2 Further optimization of 13-27 mW / cm 2 More preferably 14-26 mW / cm 2 Even better is 15-25 mW / cm 2 For example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mW / cm 2Curing time is 60-180 s under light intensity (preferably 65-175 s, more preferably 70-170 s, further preferably 75-165 s, more preferably 80-160 s, and even more preferably 90-150 s, for example 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150). s), to obtain the neural regeneration guiding conduit for promoting nerve cell differentiation, wherein the conductive gel precursor comprises poly(3,4-ethylenedioxythiophene):dextran sulfate, nerve growth factor or its precursor, a curing agent, and methacrylamide hyaluronic acid, wherein the mass ratio of poly(3,4-ethylenedioxythiophene):dextran sulfate is 0.5-2% (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2%), the mass ratio of methacrylamide hyaluronic acid is 2-3% (e.g., 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3%), and the NGF concentration is 1-10 μg / conduit (preferably 2-9 μg / conduit, more preferably 3-8 μg / conduit). μg / catheter, for example 3, 4, 5, 6, 7, 8 μg / catheter), LAP concentration 0.1-0.5% (e.g. 0.1, 0.2, 0.3, 0.4, 0.5%), preferably, the nerve regeneration guiding catheter is stable at 0-10℃ (preferably 1-9℃, more preferably 2-8℃, further preferably 2-7℃, more preferably 2-6℃, for example 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6℃) for 8-12 hours (e.g. 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 hours).

[0044] In this invention, the organic solvents include, but are not limited to, trifluoroethanol, dichloromethane, N,N-dimethylformamide, chloroform, ethanol, isopropanol, 2-pyrrolidone, glycerol, propylene glycol, polyethylene glycol, tetraethylene glycol, acetone, etc.

[0045] In a preferred embodiment, the conductive gel precursor comprises poly(3,4-ethylenedioxythiophene):dextran sulfate, nerve growth factor or a precursor thereof, a curing agent, and methacrylamide hyaluronic acid. The curing agent includes, but is not limited to, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, and tris(2,2'-bipyridine)ruthenium(II) dichloride.

[0046] In vitro nerve regeneration methods In one aspect, the present invention provides an in vitro neural regeneration method for promoting nerve cell differentiation, comprising the step of contacting the neural regeneration guiding conduit described herein with nerve cells in vitro.

[0047] In a preferred embodiment, the in vitro nerve regeneration method of the present invention includes: culturing nerve cells; bringing the nerve regeneration guiding conduit described in the present invention into contact with nerve cells (e.g., but not limited to PC12 cells) in vitro and applying ultrasound treatment; and detecting the differentiation of nerve cells. The method for detecting nerve cell differentiation can be performed using methods and apparatus known in the art, and is not particularly limited thereto. In some embodiments, the method is a therapeutic method, i.e., for therapeutic purposes. In other embodiments, the method is a non-therapeutic method, such as for commercial experimental purposes.

[0048] application In one aspect, the present invention provides the use of the nerve regeneration guiding catheter described herein in the preparation of medical products that promote nerve cell differentiation and / or nerve regeneration. The medical products include, but are not limited to, nerve repair products, nerve repair scaffolds, nerve repair systems, and nerve repair devices.

[0049] Preparation Example 1 This preparation example illustrates a method for preparing a nerve regeneration guiding conduit with anti-adhesion and electrical conductivity functions.

[0050] (1) Weigh 1.0 g of polylactic acid (PLLA) and dissolve it in 10 mL of trifluoroethanol (mass ratio of 10%). Stir overnight at room temperature using a magnetic stirrer to fully dissolve it and obtain a uniform and transparent electrospinning solution.

[0051] (2) The spinning solution obtained in step (1) is loaded into a syringe, and the electrospinning parameters are set as follows: high voltage 13 kV, injection pump speed 0.10 mm / min, collection distance 20 cm, and receiving roller speed 4000 rpm. Electrospinning is then performed to obtain a polylactic acid electrospun nanofiber membrane with longitudinal orientation.

[0052] (3) The PLLA film obtained in step (2) is annealed at 105°C for 10 hours and then annealed at 160°C for 10 hours to improve the mechanical stability and structural compactness of the film.

[0053] (4) The annealed PLLA membrane was incubated in 5 mg / mL dopamine Tris buffer (pH=8.5) for 30 minutes to allow the surface to self-assemble and form a dopamine coating. It was then rinsed 2-3 times with deionized water to remove unbound dopamine. The membrane was then incubated in 2 mg / mL polyethylene glycol (PEG) aqueous solution for another 24 hours to form a stable hydrophilic, anti-adhesion hydration layer. After treatment, it was washed 2-3 times again and air-dried.

[0054] (5) The modified electrospun film is wound onto the surface of a stainless steel rod with a diameter of 2 mm and a length of 100 mm, and then wrapped and fixed with a polytetrafluoroethylene (PTFE) film. The rod is then heated and kept at 70°C for 20 minutes to set the shape. After cooling, the PTFE film is removed and the steel rod is taken out to obtain a cylindrical PLLA conduit with an inner diameter of about 2 mm and a wall thickness of 0.4-0.5 mm.

[0055] (6) Prepare 1% PEDOT:DSS solution and 2.5% HAMA solution (both w / v concentration) separately, and stir or sonicate them at room temperature for 2 hours. Mix them at a 1:1 volume ratio, add nerve growth factor (NGF) solution with a final concentration of 5 μg / branch, and stir magnetically at 4°C for 1 hour. Finally, add photoinitiator LAP solution with a final concentration of 0.25% (w / v), mix thoroughly, and obtain a homogeneous mixture.

[0056] (7) Inject the PEDOT-HAMA-NGF mixed solution obtained in step (6) into the PLLA catheter prepared in step (5), ensuring uniform and complete perfusion. Use an ultraviolet light source (wavelength 365 nm, intensity 20 mW / cm²). 2 Irradiate the mixture for about 120 seconds to complete the rapid photocrosslinking reaction and solidify the gel.

[0057] (8) The photocured catheter sample was placed in a 4°C environment and allowed to stand for 12 hours to enhance the internal network stability of the gel and the encapsulation efficiency of NGF. Finally, an electroactive nerve regeneration guiding catheter with anti-adhesion function and high conductivity was obtained.

[0058] Preparation Example 2 This preparation example illustrates a method for preparing a nerve regeneration guiding conduit with anti-adhesion and electrical conductivity functions.

[0059] (1) Weigh 1.0 g of polylactic acid (PLLA) and dissolve it in 10 mL of trifluoroethanol (mass ratio of 10%). Stir overnight at room temperature using a magnetic stirrer to fully dissolve it and obtain a uniform and transparent electrospinning solution.

[0060] (2) The spinning solution obtained in step (1) is loaded into a syringe, and the electrospinning parameters are set as follows: high voltage 13 kV, injection pump speed 0.10 mm / min, collection distance 20 cm, and receiving roller speed 4000 rpm. Electrospinning is then performed to obtain a polylactic acid electrospun nanofiber membrane with longitudinal orientation.

[0061] (3) The PLLA film obtained in step (2) is annealed at 105°C for 10 hours and then annealed at 160°C for 10 hours to improve the mechanical stability and structural compactness of the film.

[0062] (4) The annealed PLLA membrane was incubated in 5 mg / mL dopamine Tris buffer (pH=8.5) for 30 minutes to allow the surface to self-assemble and form a dopamine coating. It was then rinsed 2-3 times with deionized water to remove unbound dopamine. The membrane was then incubated in 2 mg / mL polyethylene glycol (PEG) aqueous solution for another 48 hours to form a stable hydrophilic, anti-adhesion hydration layer. After treatment, it was washed 2-3 times again and air-dried.

[0063] (5) The modified electrospun film is wound onto the surface of a stainless steel rod with a diameter of 2 mm and a length of 100 mm, and then wrapped and fixed with a polytetrafluoroethylene (PTFE) film. The rod is then heated and kept at 70°C for 20 minutes to set the shape. After cooling, the PTFE film is removed and the steel rod is taken out to obtain a cylindrical PLLA conduit with an inner diameter of about 2 mm and a wall thickness of 0.4-0.5 mm.

[0064] (6) Prepare 1.5% PEDOT:DSS solution and 2.5% HAMA solution, stir or sonicate separately and then mix, add 5 μg NGF, then add 0.25% LAP initiator, and stir evenly at low temperature (4℃).

[0065] (7) Inject the PEDOT-HAMA-NGF mixed solution obtained in step (6) into the PLLA catheter prepared in step (5) to ensure uniform and full perfusion. Irradiate the mixture with an ultraviolet light source (wavelength 365 nm, intensity 20 mW / cm²) for about 120 seconds to complete the rapid photocrosslinking reaction and gel solidification.

[0066] (8) The photocured catheter sample was placed in a 4°C environment and allowed to stand for 12 hours to enhance the internal network stability of the gel and the encapsulation efficiency of NGF. Finally, an electroactive nerve regeneration guiding catheter with anti-adhesion function and high conductivity was obtained.

[0067] Preparation Example 3 This preparation example illustrates a method for preparing a nerve regeneration guiding conduit with anti-adhesion and electrical conductivity functions.

[0068] (1) Weigh 1.0 g of polylactic acid (PLLA) and dissolve it in 10 mL of trifluoroethanol (mass ratio of 10%). Stir overnight at room temperature using a magnetic stirrer to fully dissolve it and obtain a uniform and transparent electrospinning solution.

[0069] (2) The spinning solution obtained in step (1) is loaded into a syringe, and the electrospinning parameters are set as follows: high voltage 13 kV, injection pump speed 0.10 mm / min, collection distance 20 cm, and receiving roller speed 4000 rpm. Electrospinning is then performed to obtain a polylactic acid electrospun nanofiber membrane with longitudinal orientation.

[0070] (3) The PLLA film obtained in step (2) is annealed at 105°C for 10 hours and then annealed at 160°C for 10 hours to improve the mechanical stability and structural compactness of the film.

[0071] (4) The annealed PLLA membrane was incubated in 5 mg / mL dopamine Tris buffer (pH=8.5) for 30 minutes to allow the surface to self-assemble and form a dopamine coating. It was then rinsed 2-3 times with deionized water to remove unbound dopamine. The membrane was then incubated in 2 mg / mL polyethylene glycol (PEG) aqueous solution for another 24 hours to form a stable hydrophilic, anti-adhesion hydration layer. After treatment, it was washed 2-3 times again and air-dried.

[0072] (5) The modified electrospun film is wound onto the surface of a stainless steel rod with a diameter of 2 mm and a length of 100 mm, and then wrapped and fixed with a polytetrafluoroethylene (PTFE) film. The rod is then heated and kept at 70°C for 20 minutes to set the shape. After cooling, the PTFE film is removed and the steel rod is taken out to obtain a cylindrical PLLA conduit with an inner diameter of about 2 mm and a wall thickness of 0.4-0.5 mm.

[0073] (6) Prepare 2% PEDOT:DSS and 3% HAMA solutions, disperse them separately and then mix them. Add 5 μg NGF and 0.25% LAP and stir at low temperature until homogeneous.

[0074] (7) Inject the PEDOT-HAMA-NGF mixed solution obtained in step (6) into the PLLA catheter prepared in step (5) to ensure uniform and full perfusion. Irradiate the mixture with an ultraviolet light source (wavelength 365 nm, intensity 20 mW / cm²) for about 120 seconds to complete the rapid photocrosslinking reaction and gel solidification.

[0075] (8) The photocured catheter sample was placed in a 4°C environment and allowed to stand for 12 hours to enhance the internal network stability of the gel and the encapsulation efficiency of NGF. Finally, an electroactive nerve regeneration guiding catheter with anti-adhesion function and high conductivity was obtained.

[0076] Example This embodiment illustrates the characterization, optimization, and application of a nerve regeneration guiding catheter.

[0077] 1. Macroscopic Form The macroscopic morphology of the nerve regeneration guiding conduit was observed using a microscope, and the results are as follows: Figure 1 As shown, the conduit has a regular hollow tubular structure, designed to mimic the natural epineurium and provide physical support space for the directional regeneration of damaged axons.

[0078] The main material of the nerve regeneration guiding conduit in this embodiment is piezoelectric polylactic acid. The conduit wall is combined with an anti-adhesion layer, and the inside of the conduit contains a high-conductivity hydrogel, which can synergistically regulate the physical, chemical and electrical microenvironment to construct an ideal nerve regeneration auxiliary platform.

[0079] 2. Characterization of the chemical composition and hydrophilicity of nanofiber surfaces The chemical composition of the nanofiber surface was analyzed using XPS full-spectrum and C 1s high-resolution energy dispersive spectroscopy, and the results are as follows: Figure 2 As shown in Figure A, XPS full spectrum reveals a distinct N 1s characteristic peak in the modified sample, confirming the successful grafting of dopamine and PEG onto the fiber surface. High-resolution C 1s peak analysis shows that the signal intensities of CN (derived from dopamine) and CO (derived from PEG) functional groups significantly increased with the progress of the modification process, validating the effectiveness of the surface chemical modification at the molecular level.

[0080] The changes in water contact angle of PLLA and PDLA nanofiber membranes before and after modification were tested, and the results are as follows: Figure 2 As shown in Figure B, the original polylactic acid substrate exhibits strong hydrophobicity; after modification with dopamine and PEG, the contact angle is significantly reduced, demonstrating excellent hydrophilicity. Furthermore, the degree of improvement in surface wettability is positively correlated with the modification time; as the reaction time increases from 1 h to 48 h, the droplets spread more fully on the fiber surface, ultimately achieving a superhydrophilic state.

[0081] 3. Morphological characterization and piezoelectric property analysis of polylactic acid nanofibers The morphology of polylactic acid nanofibers was analyzed using scanning electron microscopy, and the results are as follows: Figure 3 As shown in Figure A, all groups of fibers exhibited a highly consistent orientation, with smooth fiber surfaces and no obvious defects. The results indicate that surface PEGylation modification did not disrupt the morphological integrity or arrangement regularity of the nanofibers.

[0082] The piezoelectric properties of nanofiber membranes with different compositions were tested, and the results are as follows: Figure 3 As shown in Figure B, under periodic loading, all samples exhibited significant voltage pulse output. Pure PLLA had the highest peak output voltage (approximately 1V), while the piezoelectric output voltage decreased to varying degrees with the introduction of PEG components and within the PDLA system.

[0083] 4. Cell adhesion behavior The adhesion behavior of L929 cells on the nanofiber surface was analyzed using confocal laser scanning microscopy. The adhesion morphology and quantitative results of L929 cells on the PLLA substrate are as follows: Figure 4 As shown in Figures A and B, the adhesion morphology and quantitative results of L929 cells on the PDLA substrate are as follows: Figure 4 Figures C and D are shown. The green fluorescence signal indicates a large number of densely distributed cells adhering to the original polylactic acid (PLLA / PDLA) surface. However, with increasing PEG modification time (1 h, 24 h, 48 h), the number of cells adhering to the surface sharply decreased, exhibiting significant protein / cell anti-adhesion properties. Quantitative statistical analysis of cell adhesion showed that the introduction of PEG led to a time-dependent decrease in cell adhesion. After 48 h of modification, the number of adherent cells on the surface reached its lowest value, confirming that the hydrophilic shielding layer formed by the PEG molecular chains effectively inhibits the interaction between cells and the substrate.

[0084] 5. Induction of PC12 cell differentiation The differentiation of PC12 cells was analyzed after 7 days of culture on PLLA, PDLA, and their PEG-modified surfaces. The results are as follows: Figure 5 As shown. Under ultrasound (US)-triggered piezoelectric stimulation, the growth of nerve axons in each group of cells was promoted to varying degrees. The PEG-modified surface was conducive to the development of nerve fibers into longer strands. Piezoelectric response and differentiation efficiency: PLLA group: After ultrasound stimulation, both PLLA and its PEG-modified group showed significant axonal elongation enhancement, confirming that the piezoelectric effect of PLLA can effectively promote nerve differentiation. PDLA group: Although PEG modification itself improved the differentiation environment, the promoting effect of the PDLA group on nerve fiber differentiation was not significant under ultrasound stimulation. In the ultrasound coupling experiment, the PEG-24 h modified group showed better overall differentiation potential. Therefore, the conditions of the PEG-24 h modified group were selected as the optimal parameters for subsequent in vivo animal experiments.

[0085] 6. Optimization of conductive hydrogels PEDOT: The effect of DSS concentration, such as Figure 6As shown in Figure A, the effect of different PEDOT:DSS contents (0.1%, 0.5%, and 1.0%) on the conductivity of the hydrogel was investigated under the condition of a fixed HAMA (2.0 wt%) concentration. The results showed that the conductivity of the system increased linearly with the increase of the conductive component concentration.

[0086] The effect of HAMA concentration, such as Figure 6 Figure B shows the effect of different HAMA mass fractions on conductivity under the condition of fixed PEDOT:DSS (1.5 wt%) content. With the increase of HAMA concentration, the gel network density increases, and the conductivity also increases slightly.

[0087] The electrical conductivity of the nerve regeneration guiding conduit prepared in this embodiment is 10. -2 -10 2 S / m can effectively promote nerve regeneration and signal transduction. Considering both the mechanical strength and electrical stability of the materials, PEDOT:DSS 1.5%-HAMA2% was ultimately selected as the optimal component ratio for subsequent biological experiments.

[0088] 7. In vivo experiments Twelve weeks after sciatic nerve transection, the physical morphology and weight of the recipient-side gastrocnemius muscle in mice of each group were as follows: Figure 7 As shown in Figures A and C, compared to the pure PLLA or PDLA groups, the gastrocnemius muscle volume was significantly increased and the weight was higher in the groups implanted with PEDOT-modified catheters (PLLA-PEDOT and PDLA-PEDOT). Among them, the PDLA-PEDOT group showed the best muscle mass maintenance ability, indicating that the catheter can effectively promote the recovery of nerve function and reduce muscle atrophy caused by denervation.

[0089] Masson staining and collagen fiber quantification results are as follows: Figure 7 As shown in Figures B and D, red represents muscle fibers and blue represents collagen fibers. The results indicate that the PDLA-PEDOT group had the lowest proportion of collagen fibers and the most regular arrangement of muscle fibers, confirming that this conduit significantly inhibits muscle tissue fibrosis by accelerating nerve regeneration.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A neural regeneration guiding conduit for promoting nerve cell differentiation, characterized in that, Includes a catheter body and a conductive gel located inside the catheter, wherein: The catheter body is made from polylactic acid-containing raw materials, and the wall of the catheter body is modified to include an anti-adhesion layer; The conductivity of the conductive gel is 10. -2 -10 2 S / m, and contains nerve growth factor or its precursor; Preferably, the nerve conduit has a length of 10-15 mm, an inner diameter of 1.5-2.5 mm, and a wall thickness of 0.4-0.5 mm.

2. The neural regeneration guiding conduit for promoting nerve cell differentiation according to claim 1, characterized in that, The anti-adhesion layer comprises a hydrophilic polymer and has a contact angle of 0-10°.

3. The neural regeneration guiding conduit for promoting nerve cell differentiation according to claim 1, characterized in that, The anti-adhesion layer is connected to the pipe wall via polydopamine.

4. The neural regeneration guiding conduit for promoting nerve cell differentiation according to claim 1, characterized in that, The conductive gel is a methacrylamide hyaluronic acid hydrogel containing poly(3,4-ethylenedioxythiophene):dextran sulfate.

5. A method for preparing a neural regeneration guiding conduit for promoting neural cell differentiation, characterized in that, Includes the following steps: (1) Electrospinning is performed using polylactic acid in an organic solvent to obtain a polylactic acid film with oriented arrangement, and the polylactic acid film is annealed. (2) The annealed polylactic acid film is treated with dopamine, and then polyethylene glycol is grafted onto the treated polylactic acid film to obtain a hydration layer. The polylactic acid film is then rolled up to form a nerve conduit. (3) The conductive gel precursor is infused into the interior of the nerve conduit to obtain the nerve regeneration guiding conduit for promoting nerve cell differentiation.

6. The method for preparing a neural regeneration guiding conduit for promoting neural cell differentiation according to claim 5, characterized in that, The organic solvent includes at least one of trifluoroethanol, dichloromethane, N,N-dimethylformamide, chloroform, ethanol, isopropanol, 2-pyrrolidone, glycerol, propylene glycol, polyethylene glycol, tetraethylene glycol, and acetone.

7. The method for preparing a neural regeneration guiding conduit for promoting neural cell differentiation according to claim 5, characterized in that, The conductive gel precursor comprises poly(3,4-ethylenedioxythiophene):dextran sulfate, nerve growth factor or its precursor, a curing agent, and methacrylamide hyaluronic acid.

8. The method for preparing a neural regeneration guiding conduit for promoting neural cell differentiation according to claim 5, characterized in that, The procedure further includes a step of promoting the curing of the conductive gel precursor after infusing the conductive gel precursor into the interior of the neural conduit.

9. An in vitro neural regeneration method for promoting neural cell differentiation, characterized in that, Includes the step of bringing the nerve regeneration guiding conduit according to any one of claims 1-4 into contact with nerve cells in vitro; Preferably, the step further includes applying ultrasonic treatment.

10. The use of the nerve regeneration guiding conduit according to any one of claims 1-4 in the preparation of medical products that promote nerve cell differentiation and / or nerve regeneration.

Images