Bionic nerve conduit and preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, in particular to a bionic nerve conduit and a preparation method and application thereof. Through layered functional design, the multifunctional nerve conduit which sequentially comprises an inner layer, a middle layer and an outer layer from inside to outside is prepared. Wherein the inner layer is used for constructing parallel oriented nanofibers through an electrostatic spinning technology, and is connected with divalent metal ions after being modified by connecting molecules, so as to regulate and control the behaviors of endothelial cells and blood vigor cells; the middle layer adopts a melt spinning technology to form micron fibers which are arranged in a crossed manner, so that sufficient radial and circumferential mechanical support is provided to enhance the deformation resistance; the outer layer utilizes an electrostatic spinning technology to construct compact and randomly arranged nanofibers, so that on one hand, fibrosis infiltration can be prevented; on the other hand, the structural integrity can be effectively ensured. The bionic nerve conduit provides a new material and a new strategy with clinical transformation potential for peripheral nerve defect repair in a complex environment.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a biomimetic nerve conduit, its preparation method, and its application. Background Technology

[0002] Peripheral nerve defects caused by severe trauma, disease, or surgery often lead to sensory loss and limb motor dysfunction. Current clinical treatment primarily relies on autologous or allogeneic nerve transplantation. However, autografting faces limitations due to donor site damage and availability, while allogeneic transplantation presents challenges such as immune rejection and size mismatch. Neuro-guided catheters (NGCs) are medical devices used to repair damaged nerves, designed to provide controlled pathways between damaged nerve ends to promote axonal regeneration. They are gradually replacing autologous or allogeneic nerve transplantation techniques, becoming an indispensable instrument in neurosurgery and neurointerventional procedures. However, mainstream NGC systems used clinically or in extensive research still face problems such as insufficient mechanical stability leading to lumen collapse, lack of effective topological guidance limiting axonal directional extension, and delayed vascularization and insufficient neurotrophic factor support, all of which restrict their repair efficacy. Therefore, developing a multifunctional NGC system integrating high mechanical strength, biomimetic topological guidance, and dual regulation of vascularization and neurotrophic factors is a key strategy to enhance nerve regeneration performance and functional recovery.

[0003] Mechanical stability and biomimetic topological guidance are the core foundations for effective neural regeneration in neurogenic cells (NGCs). Mechanical stability is the cornerstone of NGC function. However, according to clinical feedback, currently FDA-approved commercial NGCs often face issues such as lumen collapse, effective volume reduction, and suture displacement / extrusion after implantation, thus limiting their clinical efficacy. The fundamental reason is that the mechanical properties of the materials are difficult to match the complex dynamic loading environment in vivo, especially the radial or circumferential compressive stresses applied by surrounding tissues. Based on this, researchers have developed various anti-deformation strategies, such as using corrugated, coiled, or spiral reinforced tube designs to endow NGCs with flexibility, enhancing their resistance to compression and bending in muscle or joint areas. However, while these designs ensure long-term lumen stability to some extent, the lack of necessary topological guidance cues or bioactive modifications limits their regenerative effects. In fact, multi-channel or oriented nanofibers constructed through electrospinning can mimic the arrangement structure of natural nerve perimysium, providing physical guidance for the directional migration of endogenous cells and axonal extension. By adjusting its fiber density, scar infiltration can be effectively limited, and the material can be given excellent mechanical sutureability. However, NGCs prepared using a single process generally suffer from insufficient mechanical properties. To address this, Sun et al. prepared multi-scale NGCs through a multi-process fusion approach. The core formed by stacked and rolled micro / nano fibers possesses both cell-directed guidance capability and radial compressive strength, but the mesh-like microfiber structure still cannot provide effective anti-kinking properties. Therefore, developing flexible NGCs that integrate anti-compression, anti-kinking, and directional guidance functions has become a key direction for overcoming current bottlenecks and improving the effectiveness of nerve regeneration.

[0004] The physical properties of neural cells (NGCs) are crucial in regulating endogenous cell behavior and maintaining structural stability. However, relying solely on physical regulation strategies is insufficient to meet the multiple needs of neural regeneration, making activity modification a key step in enhancing regeneration. Peripheral nerve regeneration is essentially a biological process involving the collaborative participation of multiple cells, including vascular endothelial cells, Schwann cells, and neural stem cells. Neurovascularization, as a prerequisite for neurogenesis, provides necessary directional guidance for Schwann cell migration and axonal elongation; while neurotransmission, as the core of functional recovery, directly regulates axonal growth and myelin formation. Existing research has confirmed that in vitro pre-vascularization or loading with angiogenic factors can promote revascularization after NGC transplantation; and that delivery of neurotrophic factors or seeding of Schwann cells can effectively improve regeneration and repair. However, given the close spatiotemporal coupling and interdependence of vascularization and neurotransmission in neural regeneration, and the fact that neovascularization supports Schwann cell migration and axonal extension, and that neural signals can also feedback-regulate angiogenesis, the regeneration process remains complex.

[0005] Therefore, developing functional NGCs that can simultaneously coordinate and promote these two key processes is particularly important. However, no NGC designs with dual regulatory potential for angiogenesis and neural cell regeneration have been reported to date. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in the related art. Therefore, the first objective of the present invention is to provide a biomimetic nerve conduit; the second objective of the present invention is to provide a method for preparing a biomimetic nerve conduit; and the third objective of the present invention is to provide an application of a biomimetic nerve conduit.

[0007] To achieve the first objective, the technical solution adopted by this invention is as follows: A biomimetic nerve conduit, comprising an inner layer, a middle layer, and an outer layer from the inside out; The inner layer is formed by parallel-oriented nanofibers I, and the main raw material of the nanofibers I is selected from polymer material I; The surface of the inner layer is modified with connecting molecules, and the connecting molecules are coupled with divalent metal ions; The intermediate layer is formed by cross-arranged microfibers, and the main raw material of the microfibers is selected from polymer material II; The outer layer is formed of randomly oriented nanofibers II, and the main raw material of the nanofibers II is selected from polymer material III.

[0008] Furthermore, the diameter of the nanofiber I ranges from 545 nm to 885 nm; The diameter of the microfibers ranges from 74 μm to 84 μm, and the crossing angle between the microfibers is from 60° to 69°. The diameter of the nanofiber II ranges from 472 nm to 853 nm.

[0009] Furthermore, the linker molecule is selected from epigallocatechin gallate, and the divalent metal ion is selected from Cu. 2+ .

[0010] Furthermore, Cu 2+ The concentration range is 100µg / mL to 400µg / mL.

[0011] Furthermore, Cu 2+ The concentration was 200 µg / mL.

[0012] Furthermore, each of the polymer materials I, II, and III is independently selected from polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid-lactic acid copolymer (PGLA), polyglycerol sebacate (PGS), polyglycolic acid (PGA), and polylactic acid-caprolactone copolymer (PLCL).

[0013] Furthermore, the nanofiber I is loaded with nerve growth factor.

[0014] To achieve the second objective, the technical solution adopted by this invention is as follows: A method for preparing a biomimetic nerve conduit, used to prepare any of the biomimetic nerve conduits described above, comprising the following steps: S100, Inner layer prepared using vein spinning technology; S200. Immerse the inner layer in a modification solution to obtain an inner layer whose surface is modified with connecting molecules and whose connecting molecules are coupled with divalent metal ions. S300: The inner layer, whose surface has been modified with connecting molecules and whose connecting molecules are coupled with divalent metal ions, is wound onto the collecting rod of a melt spinning machine, and then the intermediate layer is prepared using melt spinning technology. S500. The intermediate layer is removed from the melt spinning machine and coaxially mounted on the receiver of the electrospinning machine. The outer layer is formed by spinning on the intermediate layer using electrospinning technology to obtain a biomimetic nerve conduit.

[0015] Furthermore, in step S100, the spinning solution in the electrospinning technology contains one or more of the following components: polycaprolactone, polylactic acid, polyglycolic acid-lactic acid copolymer, polyglycerol sebacate, polyglycolic acid, and polylactic acid-caprolactone copolymer.

[0016] Furthermore, the spinning solution also contains nerve growth factor and hyaluronic acid, with a mass ratio of nerve growth factor to hyaluronic acid of 1:100-150.

[0017] Furthermore, the modified solution is a buffer solution containing epigallocatechin gallate and CuCl2; The concentration of epigallocatechin gallate was 1.5–2.5 mM, and the concentration of CuCl2 was 100 µg / mL–400 µg / mL.

[0018] To achieve the third objective, the technical solution adopted by this invention is as follows: An application of a bionic nerve conduit, wherein a medical device for repairing nerve damage is prepared using any of the above-described bionic nerve conduits.

[0019] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The biomimetic nerve conduit and its preparation method provided by this invention, through a layered functional design, fabricate a multifunctional nerve conduit comprising an inner layer, a middle layer, and an outer layer, from the inside out. The inner layer utilizes electrospinning technology to construct parallel-oriented nanofibers, which are then modified with connecting molecules and linked with divalent metal ions to regulate the behavior of endothelial cells and hematopoietic cells. The middle layer employs melt spinning technology to form cross-arranged microfibers, providing sufficient radial and circumferential mechanical support to enhance deformation resistance. The outer layer utilizes electrospinning technology to construct dense and randomly arranged nanofibers, which on the one hand prevent fibrotic infiltration (avoiding scar tissue formation) and on the other hand effectively ensure the integrity of the structure (giving it suture-like properties).

[0020] In vitro experimental results show that the biomimetic nerve conduit provided by the present invention exhibits excellent anti-compression and anti-kink properties, and the active ingredients released by it can synergistically regulate the spreading, migration and angiogenesis gene expression of endothelial cells, and significantly promote the pro-repair phenotype transformation of blood cells and the expression of neurotrophic factor genes.

[0021] In in vivo experiments using a rat model of sciatic nerve defect, the bionic nerve conduit provided by this invention exhibited high structural stability after implantation in rats, maintaining patency of the lumen without any observed collapse. Furthermore, this bionic nerve conduit significantly promoted the functional regeneration of the defective nerve and effectively reduced the degree of target muscle atrophy by synergistically driving the targeted recruitment of endogenous cells, promoting the formation of a functional vascular network, guiding orderly axonal regeneration and myelination processes, and remodeling the immune microenvironment with pro-regenerative functions.

[0022] Therefore, the bionic nerve conduit provided by this invention offers a new material and strategy with clinical translational potential for repairing peripheral nerve defects under complex environments such as mechanical compression or joint areas, and is expected to be applied to the preparation of medical devices for repairing nerve damage.

[0023] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the fabrication process of the bionic nerve conduit provided by the present invention.

[0025] Figure 2These are the results of the structural characterization and functional performance study of the bionic nerve conduit provided in Embodiment 1 of the present invention; Figure A is a scanning electron microscope (SEM) image; Figure B is the overall appearance of the Ta-PCL and Sr-PCL conduits after bending and radial compression mechanical tests; Figure C is the stress distribution during the bending process simulated by finite element analysis (FEA); Figure D is the load-strain curve (n=3 independent samples) of the Ta-PCL group and the Sr-PCL group after 100 radial compression cycles; Figure E is the quantitative analysis of the maximum compressive load retained after 100 complete cycles; Figure F is the SEM image and fiber diameter frequency distribution histogram of the PCL and PCL-Cu inner layers; Figure G is the two-dimensional and three-dimensional atomic force microscope (AFM) image; Figure H is the Cu... 2+ Statistical diagrams of surface roughness of fibers before and after modification. Figure I shows the Fourier transform infrared (FTIR) spectra of PCL, PCL-Cu, PCL@NGF, and PCL-Cu@NGF; Figure J shows the X-ray photoelectron spectroscopy (XPS) spectra of PCL, PCL-Cu, PCL@NGF, and PCL-Cu@NGF; Figure K shows the particle size distribution of microgel particles in organic solvents via dynamic light scattering (DLS); Figure L shows the laser confocal scanning microscope image of PCL@NGF; and Figure M shows the surface roughness of Cu... 2+ The in vitro release curves of ions are shown in Figure N, which represents the in vitro release curve of nerve growth factor (NGF).

[0026] Figure 3 The following figures illustrate the results of the functionalized PCL-Cu@NGF fiber membrane regulating HUVEC behavior provided in Example 2 of this invention: Figure A is an F-actin immunofluorescence image (morphology of human umbilical vein endothelial cells cultured on different membrane materials for 1 and 3 days); Figure B is a quantitative statistical chart of the adhesion area of ​​HUVECs on different membranes for 1 and 3 days; Figure C is an immunofluorescence image; Figure D is a quantitative VEGF fluorescence intensity of HUVECs; Figure E is a schematic diagram of cell morphology in each group; Figure F is a HUVEC migration image after 1 day of scratch assay; Figure G is a statistical chart of the quantitative results of scratch assay; Figure H is an F-actin immunostaining image (HUVEC tube formation after 4 hours of incubation in different culture media); Figure I is a quantitative statistical chart of the number of tube formation nodes; Figures J, K, and L are bar charts showing the mRNA expression of HIF-1α, vWF, and CD31 after treatment with different culture media; Figure M is a representative image stained with crystal violet (the effect of different material conditions and culture media treatment for 24 hours on the migration of HUVEC cells); and Figure N is a bar chart of quantitative analysis of the number of migrating cells.

[0027] Figure 4This diagram illustrates the regulation of neural cell behavior by PCL-Cu@NGF provided in Example 3 of this invention. Figure A shows representative confocal and bright-field images; Figure B is a bar chart of quantitative analysis of Schwann cells phagocytizing myelin fragments; Figure C is a schematic diagram of PCL-Cu@NGF-induced Schwann cell differentiation changes; and Figures D, E, F, G, H, I, and J are bar charts showing the qRT-PCR analysis results of mRNA expression levels of neural regeneration-related genes in Schwann cells after treatment with different materials.

[0028] Figure 5 This document describes the in vivo compression resistance and enhanced cellularization of the bionic nerve conduit provided in Embodiment 4 of the present invention. Figure A shows the hematoxylin-eosin (H&E) staining results of a longitudinal section of the regenerated nerve tissue 14 days after implantation; Figure B shows the hematoxylin-eosin (H&E) staining results of a longitudinal section of the regenerated nerve tissue 90 days after implantation; Figure C shows the DAPI nuclear staining results of cell infiltration in the bionic nerve conduit at 14 days; Figure D shows the DAPI nuclear staining results of cell infiltration in the bionic nerve conduit at 90 days; Figure E shows a bar chart of quantitative analysis of cell number in the bionic nerve conduit at 14 and 90 days after implantation; and Figure F shows a bar chart of analysis of lumen area deformation in the bionic nerve conduit at 14 and 90 days after implantation.

[0029] Figure 6 The images show the effects of the bionic nerve conduit provided in Embodiment 5 of this invention on angiogenesis on days 14 and 90 after implantation. Figure A shows the angiogenesis of the proximal, middle, and distal segments of the regenerated nerve on day 14 after implantation of different nerve conduits; Figure B shows the angiogenesis of the proximal, middle, and distal segments of the regenerated nerve on day 90 after implantation of different nerve conduits; Figure C shows representative VEGF staining images of the proximal, middle, and distal segments of the nerve stump on day 14 after implantation of different nerve conduits; and Figure D shows representative VEGF staining images of the proximal, middle, and distal segments of the nerve stump on day 90 after implantation of different nerve conduits. Figure E shows representative immunofluorescence images of α-smooth muscle actin (α-SMA) expression in the proximal, middle, and distal segments of the nerve stump on day 14 after implantation of different nerve conduits. Figure F shows representative immunofluorescence images of α-SMA expression in the proximal, middle, and distal segments of the nerve stump on day 90 after implantation of different nerve conduits. Figure G is a quantitative analysis of VEGF fluorescence intensity in Figure C. Figure H is a quantitative analysis of VEGF fluorescence intensity in Figure D. Figure I is a quantitative analysis of the number of α-SMA-positive capillaries in Figure E. Figure J is a quantitative analysis of the number of α-SMA-positive capillaries in Figure F.

[0030] Figure 7This invention, in Example 6, describes the effects of different nerve conduit implantations on functional regeneration of damaged nerves 90 days after implantation. Figure A shows the hindfoot gait pattern of rats; Figure B is a bar chart of quantitative analysis of the sciatic nerve function index (SFI); Figure C is a schematic diagram of target muscle renervation detection; Figure D shows representative waveforms of compound muscle action potentials (CMAP); Figure E shows gross images of the gastrocnemius muscle on the experimental side (ES) and the normal side (NS); Figure F shows cross-sections of the gastrocnemius muscle in each group after HE staining; Figure G shows collagen deposition in each group after Masson staining; and Figure H... Figure 1 shows a schematic diagram of the distal nerve stump (DS) region and the neuromuscular junction (NMJ) region. Figure 2 shows the immunofluorescence staining results of the distal nerve stump (DS) region and the neuromuscular junction (NMJ) region. Figure 3 shows a bar chart of quantitative analysis of CMAP latency in Figure 4. Figure 4 shows a bar chart of quantitative analysis of CMAP amplitude in Figure 4. Figure 5 shows a bar chart of quantitative analysis of gastrocnemius muscle wet weight ratio (experimental side / normal side). Figure 6 shows a bar chart of quantitative analysis of collagen deposition density in Figure 5. Figure 7 shows a bar chart of quantitative analysis of fluorescence intensity in different regions in Figure 4.

[0031] Figure 8 This document describes the role of the biomimetic nerve conduit provided in Embodiment 7 of the present invention in nerve regeneration and myelin formation. Figure A shows a representative immunofluorescence image of the regenerated nerve; Figure B shows a bar chart of quantitative analysis of NF-09 fluorescence intensity in different nerve conduits; Figure C shows a bar chart of quantitative analysis of S100 Schwann cell fluorescence intensity in different nerve conduits; Figure D shows an immunofluorescence image of mature neurons (β-tubulin, green) in the regenerated nerve; Figure E shows the expression of myelin protein MPZ; Figure F shows the results of toluidine blue (TB) staining and Ponceau S-Lite blue (LFB) staining of regenerated myelinated axons; Figure G shows a bar chart of quantitative analysis of β-tubulin fluorescence intensity in Figure D; Figure H shows a bar chart of quantitative analysis of MPZ fluorescence intensity in Figure E; Figure I shows a bar chart of quantitative results of myelin-positive area in LFB staining; and Figure J shows a nightingale rose diagram of the arrangement angles of neurites in different nerve conduits.

[0032] Figure 9 This document describes the regulatory effects of different neural conduits on the inflammatory microenvironment and macrophage phenotypic polarization provided in Example 8 of the present invention. Figure A shows a representative immunofluorescence image of TNF-α, Figure B is a bar chart of quantitative analysis of TNF-α fluorescence intensity, Figure C shows immunofluorescence images of pro-inflammatory macrophages in different neural conduits, Figure D is a bar chart of quantitative results of iNOS-positive cells in different neural conduits, Figure E shows immunofluorescence images of anti-inflammatory macrophages in different neural conduits, and Figure F is a bar chart of quantitative results of CD206-positive macrophages. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0034] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0035] Example 1 The process of fabricating a biomimetic nerve conduit is as follows: I. Preparation of the main solutions.

[0036] To prepare a 10% (w / v) PCL spinning solution: Weigh 2g of PCL particles and dissolve them in 20mL of hexafluoroisopropanol. Place the solution on a magnetic stirrer and stir for 6-8 hours until fully dissolved.

[0037] Preparation of a microsol-spinning solution loaded with nerve growth factor (NGF): First, hyaluronic acid (HA) (0.06 g) was dissolved in deionized water (5 mL) and stirred at room temperature until completely dissolved to obtain an HA aqueous solution. Then, rat-derived NGF was resuspended in a 0.1% (w / v) bovine serum albumin (BSA) aqueous solution to achieve a final concentration of 100 μg / mL. Next, the resuspended NGF solution (30 μL) was mixed with the HA aqueous solution (600 μL) to obtain an HA-NGF aqueous solution. Span 80 (10 mg) and dichloromethane (DCM) (10 mL) were then added to the mixture. The mixture was ultrasonically treated using an ultrasonic emulsifier to obtain a homogeneous and stable water-in-oil (WO) emulsion with a power setting of 10% and a time setting of 2 min. Finally, PCL (1 g) and dimethylformamide (DMF) (2 mL) were added to the emulsion and dissolved completely to obtain the NGF-loaded microsol-spinning solution.

[0038] Preparation of copper ion modified solution: Epigallocatechingallate (EGCG) and CuCl2 were dissolved together in Tris-HCl buffer (10 mM, pH 8.5) to prepare a 2 mM EGCG solution. 2 +Copper ion modified solutions (EGCG / Cu) with concentrations of 0 µg / mL, 100 µg / mL, 200 µg / mL, 400 µg / mL, and 800 µg / mL were used. 2+ (solution).

[0039] II. Preparation of biomimetic nerve conduits.

[0040] Using PCL as raw material, electrospinning, melt spinning, and drug delivery technologies were employed to prepare an inner, middle, and outer layer in three steps, forming a functionalized anti-kink biomimetic nerve conduit. The inner layer consists of parallel-oriented nanofibers prepared by electrospinning; the middle layer uses melt spinning to prepare cross-arranged microfibers that are circumferentially wound to provide sufficient mechanical support and exert anti-kink function; the outermost layer is encapsulated by dense, random nanofibers to restrict fibroblast infiltration and prevent adhesion. A schematic diagram of the preparation process is shown below. Figure 1 As shown, the details are as follows: Step 1: Prepare the inner layer of the biomimetic nerve conduit using electrospinning technology, as follows: Load a syringe (10mL) with a 10% (w / v) PCL spinning solution or microsol spinning solution prepared as described above, adjust the distance between the needle and the receiver to 12cm, and after spinning, obtain an inner layer formed of PCL nanofibers (thickness 0.1-0.15mm) or an inner layer formed of PCL@NGF nanofibers (thickness 0.1-0.15mm). The electrospinning machine has the following spinning parameters set during the spinning process: 21G needle, 15kV voltage, 2mL / h spinning flow rate, 6cm diameter stainless steel receiver, and 2500rpm rotation speed.

[0041] Furthermore, taking advantage of the strong bonding properties of metal-phenolic networks on various material surfaces, Cu was coupled onto the surfaces of the PCL nanofibers and PCL@NGF nanofibers prepared above. 2+ (Preferred concentration: 200 µg / mL) The specific procedure is as follows: Immerse the nanofiber membrane in the aforementioned copper ion modification solution, gently shake at 100 rpm for 6 hours at room temperature, then remove the treated membrane and repeatedly wash it with deionized water to obtain the Cu-modified membrane. 2+ The modified PCL nanofiber layer or PCL@NGF nanofiber layer is denoted as PCL-Cu and PCL-Cu@NGF, respectively.

[0042] Step 2: Prepare the intermediate layer of the biomimetic nerve conduit using melt spinning technology, as follows: Place PCL particles (Mn=80,000Da) into a 30mL stainless steel syringe with a 19-gauge needle in a melt spinning machine. The distance between the needle and the collecting rod (1.6mm in diameter tungsten rod) is 1cm. Before melt spinning, heat the stainless steel syringe at 120℃ for 1 hour to ensure uniform melting of the PCL particles and complete removal of air bubbles from the syringe. The extrusion speed of the melt spinning device is controlled by an air pump. The collecting rod is connected to a stepper motor to provide rotation and lateral reciprocating translation to prepare the PCL intermediate layer (manufacturing parameters: rotation speed 390rpm, lateral translation speed 180Hz). Before melt spinning, the prepared inner layer (PCL-Cu or PCL-Cu@NGF) needs to be fixed in the coaxial position of the collecting rod (tungsten rod) to ensure that the melt-spun PCL fibers are directly deposited on the surface of the inner layer, achieving gapless composite.

[0043] Step 3: Prepare the outer layer of the biomimetic nerve conduit using electrospinning technology, as follows: Coaxially mount the previously prepared PCL intermediate layer onto the receiver; then, use a syringe (10 mL) to load the 10% (w / v) PCL spinning solution prepared according to the above method, adjust the distance between the needle and the receiver to 12 cm, and spin on the PCL intermediate layer to form the outer layer of the biomimetic nerve conduit. After spinning is completed, the biomimetic nerve conduit (prepared with PCL-Cu as the inner layer) is obtained. The electrospinning machine has the following spinning parameters set during the spinning process: needle is 21G, spinning flow rate is 2.0mL / h, applied voltage is 11kV, receiving distance is 12cm, receiver rotation speed is 200rpm, and spinning time is 2-3h.

[0044] The structural characterization and functional performance research of biomimetic nerve conduits, such as... Figure 2 As shown; Figure A shows a scanning electron microscope (SEM) image. From this image, it can be seen that the biomimetic nerve conduit (denoted as Ta-PCL with PCL-Cu as the inner layer, and Fta-PCL with PCL-Cu@NGF as the inner layer) has a clear three-layer structure: the inner layer consists of parallel-oriented nanofibers (716.1±168.4 nm in diameter) that can guide cell orientation; the middle layer consists of cross-arranged microfibers with a diameter of 78.9±4.5 μm and a fiber crossing angle of 64.9°±4.0°; and the outer layer consists of randomly oriented nanofibers (662.3±190.1 nm in diameter). The control example, a monolayer PCL nerve conduit (denoted as Sr-PCL, prepared using the electrospinning technique in step three, with a 10% PCL spinning solution), only exhibits a uniform random fiber structure, without obvious layering, and lacks cell orientation guidance function. Figure B shows the overall appearance of Ta-PCL and Sr-PCL catheters after bending and radial compression mechanical tests. From the figure, it can be seen that the Ta-PCL catheter can maintain its structural integrity after circumferential bending, showing excellent bending resistance; while the Sr-PCL catheter kinks in the middle section under the same conditions, resulting in lumen occlusion; the Ta-PCL group shows good elastic recovery ability and can return to its original shape after compression, while the Sr-PCL group suffers irreversible structural collapse. To gain a deeper understanding of its mechanical behavior mechanism, this invention reconstructed 3D models of two types of catheters using SolidWorks software and performed finite element analysis (FEA) on the stress changes when the catheters were bent.

[0045] Figure C shows the stress distribution during bending simulation using finite element analysis (FEA). From this figure, we can see that the Ta-PCL sandwich structure effectively disperses stress and avoids localized stress concentration during bending. In contrast, the Sr-PCL structure is prone to significant stress concentration under load, leading to structural deformation. Figure D shows the load-strain curves (n=3 independent samples) of the Ta-PCL group and the Sr-PCL group after 100 radial compression cycles. Figure E shows the quantitative analysis of the maximum compressive load (Newtons) retained after 100 complete cycles; From Figures D and E, we can see that after 100 cycles of compression, the Ta-PCL group can still maintain a maximum compressive load of 1.86±0.04N, which is significantly higher than the Sr-PCL group's 0.17±0.05N. This further confirms that the Ta-PCL group has better pressure resistance and mechanical stability. Figure F shows the scanning electron microscope images of the inner layers of PCL and PCL-Cu and the histogram of fiber diameter frequency distribution. From this figure, it can be seen that both PCL and PCL-Cu are uniform nanofibers. After Cu doping, the fiber diameter is slightly reduced and the distribution is more concentrated. Figure G shows two-dimensional and three-dimensional atomic force microscopy (AFM) images. From this figure, we can see that the 2D topology shows that the PCL-Cu surface is rougher, while the 3D topology image intuitively shows that the surface protrusions / depressions are more significant. H diagram is Cu 2+ The surface roughness statistics of the fibers before and after modification show that the roughness (Ra) of PCL-Cu is significantly higher than that of pure PCL. The increased surface roughness can enhance the adhesion of nerve conduits to nerve cells. Figure I shows the Fourier transform infrared (FTIR) spectra of PCL, PCL-Cu, PCL@NGF, and PCL-Cu@NGF. From this figure, it can be seen that PCL-Cu@NGF exhibits the characteristic absorption peak of NGF, confirming the successful loading of NGF. Figure J shows the X-ray photoelectron spectroscopy (XPS) spectra of PCL, PCL-Cu, PCL@NGF, and PCL-Cu@NGF. From the figure, it can be seen that PCL-Cu@NGF has a characteristic peak at 932.6 eV, confirming that Cu exists in the form of divalent ions (which have better biocompatibility and can promote nerve regeneration). The K-figure shows the particle size distribution of microgel particles in organic solvents by dynamic light scattering (DLS). The figure shows that the NGF particles are concentrated around 300 nm in size and are uniform in size, indicating that NGF is well dispersed on the surface of the nerve conduit and does not aggregate. Figure L shows a PCL@NGF laser confocal scanning microscope image. From this figure, we can see that NGF and Cu... 2+ Co-positioning on the surface / inside of nanofibers results in uniform loading. M is Cu 2+ The in vitro release curve of ions shows that Cu within 14 days... 2+ It exhibits a continuous and slow release, with a release rate approaching 90% after 14 days, exhibiting no burst release effect and avoiding high concentrations of Cu. 2+ Cytotoxicity; The N-figure shows the in vitro release curve of nerve growth factor (NGF). From the figure, it can be seen that NGF exhibits long-term sustained release within 28 days, with the release rate gradually increasing to 80%, which can meet the long-term nutritional needs of nerve regeneration.

[0046] Example 2 This invention evaluates Cu 2+ The effects of modified PCL fibrous membranes on the behavior of human umbilical vein endothelial cells (HUVECs), such as Figure 3 As shown.

[0047] After 1 and 3 days of co-culture, F-actin staining showed that cells in all groups were oriented and extended along the fiber direction, as indicated by Cu. 2+ The modified groups (PCL-Cu and PCL-Cu@NGF groups) showed fuller cell morphology and significantly improved spreading, with a spreading area greater than that without Cu. 2+ The modification groups (PCL and PCL@NGF groups) are shown in Figure A. Figure B shows the quantitative statistical chart of adhesion area on different HUVEC films after 1 and 3 days. From this figure, it can be seen that: although Cu 2+ There was no significant difference in cell spreading area between the modified group and day 3, but both were significantly better than the group without Cu. 2+ Modified groups. Furthermore, vascular endothelial growth factor (VEGF) fluorescence staining showed strong red fluorescence signals in the PCL-Cu, PCL@NGF, and PCL-Cu@NGF groups, while the PCL group showed a weaker fluorescence signal. Figure C shows the immunofluorescence pattern; Figure D shows the quantitative analysis of VEGF fluorescence intensity in HUVECs; From Figures C and D, we can see that the VEGF fluorescence intensity in the PCL-Cu group and the PCL-Cu@NGF group is significantly higher than that in the PCL group, but there is no statistically significant difference between the PCL@NGF group and the PCL group. Figure E is a schematic diagram of the cell morphology of each group. From this figure, we can see that: Cu 2+ Modification, alone or in combination with NGF, can promote endothelial cell adhesion, puzhan1, and VEGF expression.

[0048] The effects of different bioactive materials on HUVEC migration, tube formation, and angiogenesis gene expression were investigated using a transwell co-culture system.

[0049] Figure F shows the HUVEC migration images after 1 day of scratch assay. From this figure, it can be seen that after 1 day of co-culture, the number of endothelial cells that migrated to the lower chamber in the PCL-Cu group, PCL@NGF group and PCL-Cu@NGF group was greater than that in the PCL group, and the PCL-Cu@NGF group showed the highest migration efficiency. Figure G is a statistical chart of the quantitative results of the scratch assay. From the figure, it can be seen that the number of cells migrating in the PCL-Cu group, PCL@NGF group and PCL-Cu@NGF group is significantly higher than that in the PCL group, but there is no significant difference among the three groups. Figure H shows the formation of HUVEC tubes after 4 hours of incubation in different culture media under F-actin immunostaining conditions. From this figure, it can be seen that the tubeformation assay shows that after 4 hours of co-culture, HUVECs in the PCL-Cu group, PCL@NGF group and PCL-Cu@NGF group can all connect with each other to form a vascular network structure, and the vascular network formed in the PCL-Cu@NGF group is the most abundant.

[0050] Subsequently, the present invention performs statistical analysis on the number of nodes, branches, and intersections forming blood vessels; Figure I is a quantitative statistical chart of the number of vascular nodes. From Figure I, it can be seen that the number of vascular nodes in the PCL-Cu group and the PCL-Cu@NGF group is significantly higher than that in the PCL group. After 3 days of co-culture, the expression levels of pro-angiogenic genes HIF-1α, vWF, and CD31 in the PCL-Cu, PCL@NGF, and PCL-Cu@NGF groups are shown in Figures J, K, and L. These figures reveal that the expression levels of HIF-1α, vWF, and CD31 were significantly upregulated, showing significant differences compared to the PCL group. Furthermore, the PCL-Cu@NGF group exhibited a synergistic enhancement trend in gene expression promotion, with significantly higher HIF-1α expression levels and significantly higher CD31 expression levels compared to both the PCL-Cu and PCL@NGF groups. These results indicate that the PCL-Cu@NGF group can promote endothelial cell migration, tubule formation, and the expression of pro-angiogenic genes. Figure M shows a representative image of crystal violet staining (the effect of different material conditions and culture medium treatment for 24 h on the migration of blood cells). From this figure, it can be seen that the PCL-Cu@NGF group has the most blue spots, indicating that the cells have the strongest migration ability. The N-figure is a bar chart for the quantitative analysis of the number of migrating cells. From this figure, it can be seen that the number of migrating cells in the CL-Cu@NGF group is significantly higher than that in the PCL, PCL-Cu and PCL@NGF groups.

[0051] Example 3 To investigate the regulatory effects of PCL-Cu@NGF on neuronal behavior, this invention further evaluated the effects of different modified fiber inner layers on neuronal cell (PC12) survival, proliferation, adhesion and spreading, as well as Schwann cell (RSC96) migration, myelin debris clearance, and neurotrophic factor gene expression. Figure 4 As shown.

[0052] Figure A shows representative confocal and bright-field images. From this figure, it can be seen that after 1 day of co-culture, the number of RSC96 cells that internalized MPZ (red fluorescent label) in the PCL@NGF group and the PCL-Cu@NGF group was higher than that in the PCL group and the PCL-Cu group. The PCL group showed the lowest internalization activity and the weakest MPZ fluorescence. Figure B is a bar chart of quantitative analysis of Schwann cells that phagocytose myelin fragments. From this figure, it can be seen that the proportion of cells that phagocytosed MPZ in the PCL@NGF group and the PCL-Cu@NGF group was higher than that in the PCL group and the PCL-Cu group, but there was only a significant difference with the PCL group. Figure C is a schematic diagram of the changes in Schwann cell differentiation induced by PCL-Cu@NGF. Figures D, E, F, G, H, I, and J are bar charts showing the qRT-PCR analysis results of the mRNA expression levels of neural regeneration-related genes in Schwann cells after different material treatments. Figures C, D, and E show that the active modified fiber membrane can induce the expression of c-Jun and ATF-3 genes in RSC96 cells. Specifically, the expression levels of c-Jun and ATF-3 genes in the PCL-Cu@NGF group were significantly higher than those in the PCL and PCL-Cu groups, but there was no significant difference compared to the PCL@NGF group. This result indicates that this combined modification can effectively drive the transformation of RSC96 cells to the repair phenotype. Figures F, G, and H show that the expression levels of neurotrophic factor-related genes NGF, BDNF, and GDNF were all upregulated in the PCL-Cu@NGF group. Specifically, the expression levels of NGF and BDNF genes in the PCL-Cu@NGF group were statistically significantly higher than those in the PCL group, while the expression level of GDNF gene was significantly higher than that in both the PCL and PCL-Cu groups. Figures I and J show that the PCL-Cu@NGF group exhibited the highest expression levels of PMP2 and NRG1 genes. Although there was no statistical difference compared to other groups, the overall expression trend was still better than that of other groups. In summary, PCL-Cu@NGF lays the foundation for the repair of damaged nerves after in vivo implantation by regulating neuronal morphological extension, Schwann cell migration, and upregulating the expression levels of genes related to neuroregeneration.

[0053] Example 4 The investigation into the in vivo compression resistance and enhanced cellularization effects of biomimetic nerve conduits was conducted as follows: This invention implants three different types of nerve conduits into a 12mm defect site of the sciatic nerve in rats, namely, a single-layer hollow NGC group (Sr-PCL), an NGC group without NGF modification (Ta-PCL), and a Cu... 2+ The study included a composite NGC group with double NGF modification (Fta-PCL), with autologous transplantation (Autograft) serving as a positive control. Samples were collected 14 days post-surgery for observation. Results were as follows: Figure 5 As shown; Figure A shows the hematoxylin-eosin (H&E) staining results of a longitudinal section of the regenerated nerve tissue 14 days after implantation. The figure reveals that 14 days after implantation of the bionic nerve conduit, endogenous cell infiltration was observed at both ends of each conduit, accompanied by sparse extracellular deposition. However, a large area of ​​cavity remained in the middle section, without cell or tissue filling. Figure B shows the hematoxylin-eosin (H&E) staining results of longitudinal sections of regenerated nerve tissue 90 days after implantation. This figure reveals that 90 days after implantation, the cavities of the bionic nerve conduits in all groups were completely filled with cells and tissue. The Fta-PCL group showed significantly higher nerve fiber density and extracellular matrix deposition than the Ta-PCL and Sr-PCL groups. Figure C shows the DAPI nuclear staining results of the bionic nerve conduit at 14 days. From this figure, it can be seen that 14 days after the bionic nerve conduit was implanted, the infiltrating cells were mainly concentrated at both ends of the lumen (both ends of the dotted line), while no cell infiltration was observed in the central area of ​​the graft. Figure D shows the DAPI nuclear staining results of the bionic nerve conduit at 90 days. From this figure, it can be seen that from the implantation of the bionic nerve conduit to the 90th day, the number of infiltrating cells in each group of conduits increased significantly, achieving complete cellularization of the lumen. Figure E is a bar chart showing the quantitative analysis of cell count in the bionic nerve conduit on day 14 and day 90 after implantation. The chart shows that the cell infiltration count in the Fta-PCL group was higher than that in the Sr-PCL group and the Ta-PCL group, but there was no significant difference among the three groups. Figure F is a bar chart showing the results of the luminal area deformation analysis of the bionic nerve conduit on days 14 and 90 after implantation. The chart reveals that on day 14 post-implantation, the Sr-PCL group exhibited severe deformation, with a significant reduction in luminal area. In contrast, the Ta-PCL and Fta-PCL groups maintained structural stability, with lumen deformation rates of 4.2±1.7% and 6.0±0.2% respectively, significantly lower than the 33.9±5.1% in the Sr-PCL group. On day 90 post-implantation, the luminal area of ​​the Sr-PCL group further narrowed, with the lumen deformation rate increasing to 40.9±3.5%. In comparison, the Ta-PCL and Fta-PCL groups maintained high structural stability, with lumen deformation rates of only 4.0±1.4% and 2.7±1.0% respectively, significantly better than the Sr-PCL group. In summary, the constructed three-layer composite biomimetic neural conduit exhibits excellent deformation resistance and good tissue integration, while the functional modification strategy further promotes the targeted infiltration of cells and the construction of a neural regeneration microenvironment.

[0054] Example 5 Rapid revascularization is essential for graft survival and is fundamental to the restoration of neurophysiological function. Therefore, this invention investigates the pro-angiogenic capacity of different nerve conduits after implantation, and the results are as follows: Figure 6 As shown; Figure A shows the angiogenesis of the proximal, middle, and distal segments of the regenerated nerve on day 14 after implantation of different nerve conduits; Figure B shows the angiogenesis of the proximal, middle, and distal segments of the regenerated nerve on day 90 after implantation of different nerve conduits; Figures A and B show that VEGF is a key regulator of angiogenesis. Figure C shows representative VEGF staining images in the proximal, middle, and distal segments of the nerve stump on day 14 after implantation of different nerve conduits. From this figure, it can be seen that on day 14 after implantation, the VEGF fluorescence signals in the proximal (Pro), middle (Mid), and distal (Distal) segments of the FTa-PCL group were significantly stronger than those in the Sr-PCL group and the Ta-PCL group. Figure G is a quantitative analysis of the VEGF fluorescence intensity in Figure C. From this figure, it can be seen that although the intensity of each segment in the FTa-PCL group is lower than that in the autograft group, there is no significant difference between the two. Figure D shows representative VEGF staining images in the proximal, middle, and distal segments of the nerve stumps 90 days after implantation of different nerve conduits. From this figure, it can be seen that: 90 days after implantation, the VEGF fluorescence signal in the Sr-PCL group and Ta-PCL group showed an increasing trend in all locations, approaching that of the FTa-PCL group and the autograft group, while the expression of VEGF in the middle and distal segments of the FTa-PCL group showed an increasing trend. Figure H is a quantitative analysis of the VEGF fluorescence intensity in Figure D. From this figure, it can be seen that the VEGF fluorescence intensity in the proximal, distal and middle segments of the regenerated nerve is similar in the Sr-PCL group, Ta-PCL group, FTa-PCL group and the autograft group, with no significant difference. Figure E shows representative immunofluorescence images of α-smooth muscle actin (α-SMA) expression in the proximal, middle, and distal segments of the nerve stumps on day 14 after implantation of different nerve conduits. This figure indicates that α-SMA was observed in all groups 14 days post-implantation. + Blood vessels infiltrate into the material, mainly concentrated in the proximal and distal regions of the material; Figure I is a quantitative analysis of the number of α-SMA-positive capillaries in Figure E. From this figure, we can see that the FTa-PCL group has α-SMA in the proximal position. + The number of blood vessels was higher in the Sr-PCL group than in the Ta-PCL group, although the difference was not statistically significant. The number of blood vessels in the distal part was also higher than in these two groups, and there was a significant difference compared with the Ta-PCL group, but it was still lower than in the autograft group. Figure F shows representative immunofluorescence images of α-SMA expression in the proximal, middle, and distal segments of the nerve stump at 90 days after implantation of different nerve conduits. This figure indicates that at 90 days post-implantation, the expression of α-SMA in each group... + The number of blood vessels has increased significantly; Figure J is a quantitative analysis of the number of α-SMA-positive capillaries in Figure F. From this figure, it can be seen that in both the proximal and distal regions, the α-SMA in the FTa-PCL group...+ The number of blood vessels in the FTa-PCL group was higher than that in the Sr-PCL group and the Ta-PCL group, but there was no significant difference among the three groups. In the mid-segment, the number of blood vessels in the FTa-PCL group was still higher than that in the Sr-PCL group and the Ta-PCL group, and there was a significant difference between the FTa-PCL group and the Ta-PCL group, and it was close to that in the autograft group. The above results confirm that FTa-PCL nerve conduit implantation can promote the upregulation of VEGF expression and α-SMA. + The maturation of functional capillaries provides crucial blood supply support for the neural regeneration microenvironment.

[0055] Example 6 This invention systematically investigated the effects of different neural conduit implantations on functional regeneration of damaged nerves 90 days after implantation using a functional assessment system including rat motor function index, electrophysiological signal transduction, and target organ histological staining. Figure 7 As shown; Figure A shows the gait pattern of the rat hind feet. Macroscopic observation of the rat hind limbs reveals varying degrees of atrophy on the operated side in all groups. Specifically, the toe abduction width of the hind limbs in the FTa-PCL group was significantly greater than that in the Sr-PCL and Ta-PCL groups. Figure B is a bar chart of quantitative analysis of the sciatic nerve function index (SFI). From the figure, it can be seen that the FTa-PCL group can effectively promote the functional regeneration of the damaged nerve in rats, with an SFI index of -53.9±11.8, which is better than the Sr-PCL group (-78.7±12.4) and the Ta-PCL group (-71.9±8.7), and close to the autograft group (-42.0±8.6). Figure C is a schematic diagram of the detection of target muscle renervation. From this figure, it can be seen that sciatic nerve electrophysiological testing is an important means of evaluating functional recovery after sciatic nerve injury. Figure D shows a representative waveform of compound muscle action potential (CMAP). From this figure, it can be seen that the CMAP waveform induced in the Fta-PCL group is clear with sharp peaks, and its distribution pattern is closer to that of the autograft group. In contrast, the waveforms in the Sr-PCL and Ta-PCL groups are disordered and have attenuated amplitudes. Figure J is a bar chart of quantitative analysis of CMAP latency in Figure D. From this figure, it can be seen that the maximum amplitude of CMAP of regenerated nerves in the Fta-PCL group is significantly higher than that in the Sr-PCL group and the Ta-PCL group, and there is no significant difference from the autograft group. The K-plot is a bar chart for the quantitative analysis of CMAP amplitude in the D-plot. From this plot, it can be seen that the CMAP latency in the Fta-PCL group is significantly shorter than that in the Sr-PCL group and the Ta-PCL group, and is close to that in the autograft group. Figure E shows gross images of the gastrocnemius muscle on the experimental side (ES) and the normal side (NS). From this figure, it can be seen that the gastrocnemius muscle on the surgical side of each group showed a reduction in volume and atrophy. However, the muscle fiber morphology and volume recovery of the Fta-PCL group were significantly better than those of the Sr-PCL group and the Ta-PCL group, but still did not reach the level of normal muscle. Figure L is a bar chart showing the quantitative analysis of the gastrocnemius muscle wet weight ratio (experimental side / normal side). The chart reveals that the ES / NS ratio in the Fta-PCL group was 42.7±7.0%, which was not significantly different from the 60.0±4.1% in the autograft group. However, the wet weight ratios in the Sr-PCL and Ta-PCL groups were only 25.0±7.1% and 28.7±6.9%, respectively, lower than those in the Fta-PCL and autograft groups, and significantly different from those in the autograft group. Figure F shows the cross-sections of the gastrocnemius muscle in each group after HE staining. From this figure, it can be seen that the muscle fiber size in the Sr-PCL group is significantly reduced and accompanied by structural disorder, exhibiting typical atrophy characteristics. In contrast, the Fta-PCL group shows a more uniform cross-sectional area with increased size, indicating a lower degree of muscle fiber atrophy. Figure G shows the collagen deposition in each group as revealed by Masson staining. This figure indicates that collagen fiber deposition was present in the Sr-PCL, Ta-PCL, and Fta-PCL groups. Among them, the Fta-PCL group showed the lowest collagen deposition density with tightly packed muscle fibers, with only a small amount of light blue collagen fibers observed at the perimysium; its deposition level was similar to that of the autograft group. In contrast, the Sr-PCL group exhibited significant muscle fiber atrophy, with a large number of collagen fibers filling the interfibrillary spaces, demonstrating significant fibrosis characteristics. Figure M is a bar chart for quantitative analysis of collagen deposition density in Figure G. From this figure, we can see that the collagen deposition density in the Fta-PCL group is 3.1±0.9%, which is significantly lower than that in the Sr-PCL group (19.8±2.8%) and the Ta-PCL group (7.3±1.5%), and close to that in the autograft group (2.0±0.8%). Figure H is a schematic diagram of the distal nerve stump (DS) region and the neuromuscular junction (NMJ) region; Figure 1 shows the immunofluorescence staining results of the distal nerve stump (DS) region and the neuromuscular junction (NMJ) region. From Figures H and I, it can be seen that NF-09 staining of the target muscle was used to assess nerve regeneration and re-innervation in the distal sciatic nerve region (DS region) and the neuromuscular junction region (NMJ region). Immunofluorescence results showed that in the DS region, the Pta-PCL group exhibited dense, punctate NF-09+ signals, significantly stronger than the Sr-PCL and Ta-PCL groups. The Sr-PCL group showed nerve fiber bundle atrophy in the DS region, while the autograft group showed the strongest fluorescence signal. In the NMJ region, the Fta-PCL group showed intact "salt and pepper"-like NF-09+ fiber terminals, but their distribution area was still smaller than that of the autograft group. In contrast, only sparse, punctate NF-09+ signals were observed in the Sr-PCL and Ta-PCL groups. Figure N is a bar chart of quantitative analysis of fluorescence intensity in different regions of Figure I. From this figure, it can be seen that in the DS region and NMJ region, the fluorescence intensity of NF-09 in the Pta-PCL group is significantly higher than that in the Sr-PCL group and the Ta-PCL group, but still does not reach the level of the autograft group. The above results confirm that the Fta-PCL nerve conduit can effectively promote the functional regeneration of damaged nerves and the reconstruction of the function of nerve-innervated muscles.

[0056] Example 7 This invention evaluated the role of functionalized composite biomimetic nerve conduits in nerve regeneration and myelination, and the results are as follows: Figure 8 As shown; Figure A shows a representative immunofluorescence image of the regenerated nerve. From this image, we can see that histological analysis of the mid-segment longitudinal section of the nerve conduit 90 days after implantation shows that NF09 in the Ta-PCL group and the FTa-PCL group... + Nerve fibers and S100 + Schwann cells all showed a highly oriented arrangement, and their morphological characteristics were similar to those of the autograft group, while the Sr-PCL group showed a random and disordered distribution. Figure B is a bar chart showing the quantitative analysis of NF-09 fluorescence intensity in different nerve conduits; Figure C is a bar chart showing the quantitative analysis of fluorescence intensity of S100 Schwann cells in different neural conduits; Figures B and C show that the density of NF09+ nerve fibers and S100+ Schwann cells in the FTa-PCL group was significantly higher than that in the Sr-PCL group and the Ta-PCL group, and the FTa-PCL group showed the highest fluorescence intensity. Figure D shows an immunofluorescence image of mature neurons (β-tubulin, green) in regenerated nerves; Figure G is a bar chart showing the quantitative analysis of the fluorescence intensity of β-tubulin in Figure D; From graphs D and G, it can be seen that a significant enhancement of β-tublin can be observed in the Fta-PCL group. + The fluorescence signal was significantly higher than that of the Ta-PCL group and the Sr-PCL group, indicating that Fta-PCL can effectively promote neuronal maturation and axonal regeneration. Myelin zero (MPZ) is the most abundant structural transmembrane glycoprotein in myelin, participating in myelin maturation and the recovery of neurotransmission function. Figure E shows the expression of myelin protein MPZ. From this figure, it can be seen that in the FTa-PCL group, oriented and widely covered MPZ+ red fluorescence signals can be observed. In contrast, the Sr-PCL and Ta-PCL groups only show weak and discretely distributed MPZ+ fluorescence signals. Figure H is a bar chart of quantitative analysis of MPZ fluorescence intensity in Figure E. From this figure, it can be seen that the MPZ fluorescence intensity of the FTa-PCL group is significantly higher than that of the Sr-PCL group and the Ta-PCL group, but still does not reach the level of autograft. Toluidine blue (TB) dye can label newly formed Schwann cells within the myelin sheath, and quick blue (LFB) dye can also bind to the myelin sheath and appear bright blue. Figure F shows the results of toluidine blue (TB) staining and Ponceau S quick blue (LFB) staining of regenerated myelinated axons. From this figure, it can be seen that compared with the Sr-PCL group and the Ta-PCL group, the FTa-PCL group has higher myelin sheath axon density and thickness. Figure I is a bar chart showing the quantitative results of the myelin-positive area in LFB staining. From this figure, we can see that the myelination density in the FTa-PCL group is as high as 39.9±4.3%, which is significantly higher than that in the Ta-PCL group (29.3±6.8%) and the Sr-PCL group (23.7±6.4%), but still significantly lower than that in the autologous transplantation group (49.4±5.5%). This invention quantifies the orientation of regenerated myelin sheath in LFB staining. Figure J is a nightingale rose diagram showing the arrangement angle of neurites in different nerve conduits. As shown in the figure, in the Ta-PCL group and FTa-PCL group containing the oriented nanofiber inner layer, the myelin sheath extends from the proximal end to the distal end, and the angle with the horizontal direction is concentrated between 75 degrees and 90 degrees, showing an orientation arrangement pattern similar to that of the autograft group. The Sr-PCL group, on the other hand, shows a random and disordered distribution, which also confirms the effective guiding role of the oriented nanofiber inner layer in the directional regeneration of myelin sheath. The above results indicate that the FTa-PCL neural conduit can significantly promote axonal regeneration, Schwann cell migration, and orderly myelin formation, demonstrating excellent neural structure repair and regeneration guidance capabilities.

[0057] Example 8 Through in vivo experiments, this invention evaluated the regulatory effects of the composite biomimetic neural conduit on the inflammatory microenvironment and macrophage phenotypic polarization, such as... Figure 9 As shown; Figure A shows a representative immunofluorescence image of TNF-α. From this image, we can see that on day 14 post-implantation, strong diffuse TNF-α+ signals were observed in the damaged areas of all groups, indicating activation of the acute inflammatory response. The signal intensity in the Sr-PCL group was significantly higher than that in the Ta-PCL group, Fta-PCL group, and autograft group. By day 90 post-implantation, the TNF-α+ signal generally decreased in all groups, but the Sr-PCL group still showed persistent TNF-α+ expression, while the Ta-PCL group, Fta-PCL group, and autograft group showed virtually no positive signal. Figure B is a bar chart of quantitative analysis of TNF-α fluorescence intensity. From the figure, it can be seen that: on day 14 after implantation, the TNF-α fluorescence intensity of the Fta-PCL group was significantly lower than that of the Sr-PCL group and the Ta-PCL group; by day 90 after implantation, the fluorescence intensity of the Fta-PCL group was greatly reduced, and there was no significant difference between it and the Ta-PCL group and the autologous transplantation group, but it was significantly lower than that of the Sr-PCL group. Inducible nitric oxide synthase (iNOS) is a key marker of macrophage M1 polarization, and its expression level directly reflects the activation state of pro-inflammatory macrophages. Figure C shows the immunofluorescence images of pro-inflammatory macrophages in different neural conduits. As shown in the figure, on day 14 after implantation, the Fta-PCL group and the autologous transplantation group showed less infiltration of iNOS+ cells in the neoplasm, while the Sr-PCL group and the Ta-PCL group showed a large number of iNOS+ cell infiltrations. By day 90 after implantation, the number of iNOS+ cells in all groups showed a decreasing trend. In the Fta-PCL group, iNOS+ signal was basically not observed, while strong iNOS+ signal was still observed in the Sr-PCL group and the Ta-PCL group. Figure D is a bar chart showing the quantitative results of iNOS-positive cells in different neural conduits. As shown in the figure, on day 14, the number of iNOS+ cells in the Fta-PCL group was significantly lower than that in the Sr-PCL group and the Ta-PCL group. Although it was still higher than that in the autologous transplantation group, there was no significant difference. By day 90 after implantation, the number of iNOS+ cells in the Fta-PCL group further decreased and was still significantly lower than that in the Sr-PCL group and the Ta-PCL group, and approached the level of the autologous transplantation group. Figure E shows immunofluorescence images of anti-inflammatory macrophages in different neural conduits. The figure reveals that 14 days post-implantation, CD206+ cell infiltration was observed in all groups, with higher cell counts in the Fta-PCL and autologous transplantation groups compared to the Sr-PCL and Ta-PCL groups. By day 90 post-implantation, the number of CD206+ cells in the Fta-PCL and autologous transplantation groups showed a similar decreasing trend, while the Sr-PCL and Ta-PCL groups showed the opposite increasing trend. Figure F is a bar chart showing the quantitative results of CD206-positive macrophages. As shown in the figure, on day 14 after implantation, the number of CD206+ cells in the Fta-PCL group was significantly higher than that in the Sr-PCL group and the Ta-PCL group, and there was no significant difference between the Fta-PCL group and the autologous transplantation group. By day 90 after implantation, the number of CD206+ cells in the Fta-PCL group was significantly lower than that in the Sr-PCL group and the Ta-PCL group, and was close to that in the autologous transplantation group.

[0058] The above results confirm that Fta-PCL can effectively inhibit TNF-α expression and M1 macrophage infiltration in the early stage of injury, and promote M2 phenotypic polarization, thereby maintaining immune microenvironment homeostasis and creating favorable conditions for nerve repair.

[0059] In summary, this invention, through innovative hierarchical functional design, constructs a multifunctional neural conduit (Fta-PCL) that combines mechanical stability, directional topological guidance, and temporal biochemical regulation, effectively overcoming the problems of traditional conduits being prone to collapse and having limited regenerative functions. In vitro and in vivo results confirm that this conduit possesses excellent resistance to compression and bending, maintaining long-term structural stability and providing space for endogenous cell infiltration and tissue ingrowth. Simultaneously, through Cu... 2+ The time-dependent release of NGF precisely regulates the behavior of vascular endothelial cells and Schwann cells, and induces M2 macrophage polarization to improve the immune microenvironment. This, in turn, synergistically promotes revascularization, axonal extension, and myelination during nerve regeneration, ultimately achieving structural and functional regeneration of the damaged nerve. The biomimetic nerve conduit provided by this invention offers a systematic regulatory strategy integrating material mechanics, structural guidance, and the release of active factors, providing a new technical solution for the clinical repair of nerve defects across joints / compression areas.

[0060] 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; and 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 biomimetic nerve conduit, characterized in that, From the inside out, it consists of an inner layer, a middle layer, and an outer layer; The inner layer is formed by parallel-oriented nanofibers I, and the main raw material of the nanofibers I is selected from polymer material I; The surface of the inner layer is modified with connecting molecules, and the connecting molecules are coupled with divalent metal ions; The intermediate layer is formed by cross-arranged microfibers, and the main raw material of the microfibers is selected from polymer material II; The outer layer is formed of randomly oriented nanofibers II, and the main raw material of the nanofibers II is selected from polymer material III.

2. The bionic nerve conduit as described in claim 1, characterized in that, The diameter of the nanofiber I ranges from 545 nm to 885 nm; The diameter of the microfibers ranges from 74 μm to 84 μm, and the crossing angle between the microfibers is from 60° to 69°. The diameter of the nanofiber II ranges from 472 nm to 853 nm.

3. The bionic nerve conduit as described in claim 1, characterized in that, The linker molecule is selected from epigallocatechin gallate; And / or the divalent metal ions are selected from Cu 2+ Cu 2+ The concentration range is 100µg / mL to 400µg / mL.

4. The bionic nerve conduit as described in claim 1, characterized in that, The polymer material I, the polymer material II, and the polymer material III are each independently selected from polycaprolactone, polylactic acid, polyglycolic acid-lactic acid copolymer, polyglycerol sebacate, polyglycolic acid, and polylactic acid-caprolactone copolymer.

5. The bionic nerve conduit as described in claim 1, characterized in that, The surface of nanofiber I is loaded with nerve growth factor.

6. A method for preparing a biomimetic nerve conduit, characterized in that, The method for preparing the biomimetic nerve conduit as described in any one of claims 1 to 5 comprises the following steps: S100, Inner layer prepared using vein spinning technology; S200. The inner layer is immersed in a modification solution to obtain an inner layer whose surface is modified with connecting molecules and whose connecting molecules are coupled with divalent metal ions. S300: The inner layer, whose surface has been modified with connecting molecules and whose connecting molecules are coupled with divalent metal ions, is wound onto the collecting rod of a melt spinning machine, and then the intermediate layer is prepared using melt spinning technology. S500. The intermediate layer is removed from the melt spinning machine and coaxially mounted on the receiver of the electrospinning machine. The outer layer is formed by spinning on the intermediate layer using electrospinning technology to obtain a biomimetic nerve conduit.

7. The method for preparing the bionic nerve conduit as described in claim 6, characterized in that, In step S100, the spinning solution in the electrospinning technology contains one or more of the following components: polycaprolactone, polylactic acid, polyglycolic acid-lactic acid copolymer, polyglycerol sebacate, polyglycolic acid, and polylactic acid-caprolactone copolymer.

8. The method for preparing the bionic nerve conduit as described in claim 7, characterized in that, The spinning solution also contains nerve growth factor and hyaluronic acid, with a mass ratio of nerve growth factor to hyaluronic acid of 1:100-150.

9. The method for preparing the bionic nerve conduit as described in claim 6, characterized in that, The modified solution is a buffer solution containing epigallocatechin gallate and CuCl2; The concentration of epigallocatechin gallate was 1.5–2.5 mM, and the concentration of CuCl2 was 100 µg / mL–400 µg / mL.

10. An application of a bionic nerve conduit, characterized in that, A medical device for repairing nerve damage was prepared using the bionic nerve conduit as described in any one of claims 1 to 5.

Images