Modular three-dimensional nerve guide catheter system and method of construction thereof

Abstract

The application discloses a modular three-dimensional nerve guide catheter system and a construction method thereof, and belongs to the technical field of biomedical materials and tissue engineering. The system comprises a plurality of functional modules arranged in sequence along an axial direction, and the modules are spliced into a whole catheter through a physical mortise and tenon structure. Each functional module is composed of a hydrogel matrix and biodegradable microspheres or nanoparticles dispersed in the hydrogel matrix, and the microspheres or nanoparticles are loaded with bioactive substances. By setting different types, loading concentrations and release characteristics of bioactive substances in different functional modules, and utilizing the discrete combination and sequential arrangement of the modules in the axial direction, a spatial concentration gradient of various bioactive substances can be constructed in the catheter, and the spatial concentration gradient is independent of each other and the distribution directions are not coupled. Further, by regulating the degradation rate of the carrier material, sequential release of different bioactive substances in the time dimension can also be achieved. The application realizes the spatiotemporal coordinated regulation of the angiogenesis and nerve regeneration process, and can be flexibly adapted to nerve defects of different lengths and complex morphologies by adjusting the number and arrangement of the modules, and has good manufacturing repeatability and clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tissue engineering technology, and particularly relates to a modular three-dimensional neural guidance conduit system and its construction method. Background Technology

[0002] (I) Clinical background and technical requirements of peripheral nerve injury Peripheral nerve injury (PNI) is one of the most common and serious types of tissue damage in clinical practice. It can be caused by a variety of factors, including trauma, tumor resection, iatrogenic injury, or chronic compression. Severe peripheral nerve injury often leads to sensory, motor, or autonomic dysfunction, significantly reducing patients' quality of life and causing long-term socioeconomic burden.

[0003] For patients with nerve defects, especially those with large defects or thick nerve diameters, direct end-to-end suturing cannot effectively repair the nerve tissue. Currently, autologous nerve transplantation remains the "gold standard" for treating long peripheral nerve defects in clinical practice. However, this method has several drawbacks, including limited donor nerve sources, the need for secondary surgery, potential damage to donor site function, difficulties in size matching, and the risk of traumatic neuroma formation, severely limiting its application in the repair of complex nerve defects.

[0004] Therefore, the Nerve Guidance Conduit (NGC) is considered a potential alternative to autologous nerve transplantation. Although several nerve conduit products have entered clinical use, they are mainly suitable for the repair of short-distance, thin-diameter sensory nerves. For large, long, or complex peripheral nerve defects, their repair effect still falls short of clinical needs.

[0005] (II) The synergistic effect of angiogenesis and neurogenesis during nerve regeneration Peripheral nerve regeneration is a highly complex biological process involving dynamic interactions between neurons, Schwann cells, endothelial cells, and various immune cells. Increasing research indicates that nerve regeneration is not a single neurogenesis process, but rather highly coupled with angiogenesis.

[0006] In the early stages of nerve injury repair, angiogenesis provides oxygen, nutrients, and pathways for the removal of metabolic waste in the regenerating area, and also provides physical support for Schwann cell migration and axonal extension. Temporally, angiogenesis typically precedes axonal extension; spatially, angiogenesis and axonal extension exhibit different dominant directions and distribution characteristics at different stages.

[0007] Therefore, designing neural conduits solely from the perspective of neurogenesis, while ignoring the regulatory needs of angiogenesis in different time windows and spatial regions, often fails to achieve ideal neural repair results. How to achieve orderly and coordinated regulation of angiogenesis and neurogenesis within the neural conduit in both time and space has become a key technical challenge in improving the repair performance of neural conduits.

[0008] (III) The role of growth factors in neural and vascular regulation and their delivery limitations Growth factors play a crucial role in regulating nerve regeneration and angiogenesis. Among them, vascular endothelial growth factor (VEGF) plays a key role in promoting vascular endothelial cell migration, proliferation, and vascular network formation; while nerve growth factor (NGF) plays an important role in promoting axonal growth, Schwann cell migration, and maintaining nerve function.

[0009] Previous studies have shown that if VEGF can preferentially exert its pro-angiogenic effect in the early stages of nerve repair and then continuously exert its neurotrophic effect in the subsequent stages, the nerve regeneration effect can be significantly improved. However, due to the short half-life of growth factors in vivo, direct administration is prone to rapid inactivation, and high-dose administration poses potential safety risks. Therefore, there is an urgent need to achieve local, controllable, and time-matched delivery of these growth factors using biomaterials.

[0010] (iv) Shortcomings of existing gradient construction techniques To simulate the spatial gradient distribution of growth factors in vivo, various gradient construction methods have been proposed, including diffusion control, passive or active adsorption, electrospinning, micro-pressing, gradient mixing, and microfluidics. However, systematic analysis reveals that these methods generally suffer from the following insurmountable technical limitations: 1) Existing gradient construction methods are mainly limited to two-dimensional or quasi-two-dimensional membrane structures, or rely on continuous mixing and diffusion processes inside the material. It is difficult to achieve a stable spatial gradient distribution that matches the axonal growth and cell migration path inside a three-dimensional neural conduit, and it is even more difficult to achieve precise construction and maintenance of gradients in a three-dimensional neural conduit with complex and precise topological guidance structures. 2) Existing methods can usually only achieve a concentration gradient in a single direction. When two or more growth factors are loaded at the same time, it is difficult to assign different spatial distribution directions to different factors, thus failing to match the spatial heterogeneity and staged regulatory needs of angiogenesis and neurogenesis. 3) The gradient construction process is highly sensitive to operating conditions, and the gradient stability and manufacturing repeatability are poor. Moreover, existing technologies often rely on continuous gradient processing within a single scaffold, making it difficult to achieve structural adaptation to peripheral nerve defects of different lengths or complex shapes by adjusting the number and / or arrangement of the building blocks without changing the overall structural design. This limits its engineering application in clinical-scale three-dimensional nerve conduits.

[0011] Therefore, existing technologies are unable to achieve a stable and reproducible solution for decoupling the spatial gradient directions of multiple growth factors in neural conduits that simultaneously possess complex three-dimensional topological guidance structures. Summary of the Invention

[0012] To address the aforementioned technical problems, this invention proposes a modular three-dimensional neural guidance conduit system and its construction method. By deconstructing the neural conduit into multiple functional modules and assembling them through physical interlocking, a differentiated spatial concentration gradient of various bioactive substances is constructed in three-dimensional space. Combined with carrier materials, the sequential release of bioactive substances is achieved. Simultaneously, structured guiding units are introduced within each functional module to provide directional physical guidance for cell migration, orientation alignment, and nerve axon extension, thereby synergistically regulating cell migration and axon directional growth. This overcomes the shortcomings of existing technologies and improves the repair effect of peripheral nerve injuries.

[0013] To achieve the above objectives, the present invention provides the following technical solution: A modular three-dimensional neural guidance conduit system, comprising: Outer structure module; At least two functional modules arranged sequentially along the axial direction of the conduit; The functional module and the outer structural module are connected by a physical interlocking structure to form the modular three-dimensional neural guidance conduit system; Each functional module contains a hydrogel matrix and microspheres or nanoparticles loaded with bioactive substances. Optionally, the outer structure module can be cylindrical or have a main stem and multiple branches.

[0014] The functional modules are arranged in a gradient according to the concentration of the bioactive substances they contain.

[0015] Optionally, the bioactive substances include, but are not limited to, angiogenesis-related factors, neurotrophic factors, nerve growth factors, cytokines, bioactive proteins, polypeptide molecules, and small molecule compounds with tissue regulatory effects, as well as mixtures thereof in any proportion. The raw materials for the microspheres or nanoparticles are selected from at least one or a mixture of several of the following in any proportion: polycaprolactone (PCL), poly(lactide-caprolactone) copolymer (PLCL), polyurethane (PU), polyglycerol sebacate (PGS), polydioxanone (PDS), polyglycolic acid (PGA), polylactide (PLA), poly(lactide-glycolic acid) copolymer (PLGA), polyhydroxyalkanoate (PHA), and polyethylene glycol (PEO).

[0016] Furthermore, when the outer structural module is cylindrical, the bioactive substance is angiogenesis-related factor, and the functional modules are arranged with decreasing concentrations from the middle of the nerve conduit towards the proximal and distal ends. The bioactive substance is a neurotrophic factor, and the functional modules are arranged in increasing concentrations from the proximal end to the distal end of the nerve conduit.

[0017] Furthermore, when the outer structure module has a trunk-and-multi-branch shape and the bioactive substance is angiogenesis-related factor, the functional module at the junction of the trunk and the bifurcation is loaded with a high concentration of angiogenesis-related factor. When the bioactive substance is a neurotrophic factor or nerve growth factor, the functional modules of each branch region that carry the neurotrophic factor exhibit an axial arrangement in which the concentration increases from the bifurcation region to the distal end within each branch.

[0018] Furthermore, the angiogenesis-related factor is vascular endothelial growth factor (VEGF).

[0019] Optionally, the functional module has a structured guidance unit inside, which forms an orderly guidance structure after being integrated and connected.

[0020] Furthermore, the structured guiding unit is a continuously or discretely distributed microstructure, which includes, but is not limited to, microchannels, microgrooves, or combinations thereof arranged along the axial direction of the conduit.

[0021] Optionally, the physical interlocking structure includes a mortise and tenon structure, a snap-fit ​​structure, a shape-fitting structure, or an equivalent mechanical connection structure.

[0022] A method for constructing a modular three-dimensional neural guidance conduit system as described above includes the following steps: (1) Dissolve the photosensitive bioresin in a phosphate buffer solution, add a photoinitiator and food coloring, and vortex to dissolve completely to obtain photosensitive ink; Microspheres or nanoparticles loaded with bioactive substances are added to the photosensitive ink to obtain a bio-ink; The bio-ink is molded into a functional module with structured guiding units and physical interlocking structures using a photocuring method; the photosensitive ink is molded into an outer structural module with physical interlocking structures. (2) Based on the length and shape of the target nerve defect, at least two functional modules are spliced ​​together along the axial direction in a preset order through a physical interlocking structure to form an integral nerve conduit, and then placed inside the outer structural module, so that the functional modules containing different concentrations of bioactive substances form a preset decoupling concentration gradient in space and achieve sequential release in time, thereby obtaining the modular three-dimensional nerve guidance conduit system.

[0023] Optionally, the photosensitive bioresin includes at least one or a mixture of several of the following in any proportion: methacrylamide gelatin (GELMA), polyethylene glycol diacrylate (PEGDA), polyether F127 diacrylate (F127DA), methacrylamide polyvinyl alcohol (PVAMA), methacrylamide hyaluronic acid (HAMA), acrylamide (AM), sodium alginate methacrylate (ALGMA), and silk fibroin methacrylate (SILMA).

[0024] Optionally, the photocuring method includes ultraviolet curing, photocuring in-situ 3D printing, SLA 3D printing, DLP 3D printing, or two-photon lithography 3D printing.

[0025] Compared with the prior art, the present invention has the following advantages and technical effects: 1) It realizes the directional decoupling construction of spatial gradients of various bioactive substances inside the three-dimensional neural conduit, which enables different bioactive signals to independently form preset concentration gradients in different axes or different regions, thus breaking through the technical limitation of existing technologies that can only construct gradients in a single direction or a single factor. 2) Spatial gradient construction is achieved through the discretization design and assembly of functional modules, avoiding complex continuous gradient processing within a single scaffold. Structured guidance units can be introduced collaboratively within each functional module to achieve the synergistic effect of biological signal regulation and physical guidance, significantly improving the structural stability, manufacturing repeatability, and feasibility of mass production in the gradient construction process. 3) The modular construction method allows the nerve conduit to be flexibly expanded and combined according to the length, diameter and shape of different nerve defects. By adjusting the number, arrangement and composition of functional modules, it can be adapted to complex peripheral nerve defects such as long segments, large or branched types, without the need to redesign the overall scaffold structure. 4) By leveraging the differences in degradation characteristics of carrier materials, the sequential release of various bioactive substances is achieved, and in conjunction with spatial gradient distribution, biological events such as angiogenesis, nerve regeneration, and microenvironment remodeling are orderly regulated in both temporal and spatial dimensions. 5) It provides a systematic, scalable solution with a clear engineering implementation path for tissue engineering repair of complex peripheral nerve defects, which improves the consistency and stability of regeneration effects while having good potential for translational applications. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 TEM images and average particle size of nanoparticles prepared using different biodegradable polymers in Example 1; Figure 2 The cumulative release curves of nanoparticles with different degradation properties in Example 1 are shown. Figure 3 TEM images showing the degradability of nanoparticles prepared from different polymer materials in Example 1; Figure 4 This is a schematic diagram of the microgroove and microchannel guiding unit in Example 1; Figure 5 The diagram shows the single and composite gradient nerve conduits in Example 2, along with their actual effects. Figure 6 The active molecule gradient construction process and fluorescence images in Example 2; Figure 7 A schematic diagram (A) of the spatiotemporal controlled release neural conduit system in Example 3 and bar charts (B, C) of the actual cumulative release of the two active molecules. Figure 8 A schematic diagram (A) of the modular nerve conduit applicable to different injury lengths in Example 4 and a photograph (B) of its application in a rat nerve injury model; Figure 9 This is a schematic diagram of the modular nerve conduit with one trunk and multiple branches in Example 5, along with a photograph of the actual fabrication effect; Figure 10 The histological stained section (A) and Micro-CT vascular reconstruction image (B) taken 3 months postoperatively in Example 1 are used. Detailed Implementation

[0027] 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.

[0028] 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 each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] 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. 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. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] The present invention provides a modular three-dimensional nerve conduit system, comprising at least one outer structural module and at least two functional modules arranged along the conduit axis; The functional modules are interconnected through mortise and tenon joints, snap-fit ​​structures, or other physical interlocking structures to form an integrated neural conduit. By regulating the release functions of modules at different locations, a differentiated spatiotemporal concentration gradient of active substances can be constructed. At the same time, the functional modules contain structured guiding units, which form an orderly guiding structure after being integrated and connected.

[0033] In some optional embodiments, the functional module, by weight, comprises 1 part of a photocurable hydrogel matrix and 0.0001-0.5 parts of biodegradable microspheres or nanoparticles uniformly dispersed in the hydrogel matrix. The release function of a single module can be controlled by adding microspheres or nanoparticles loaded with different active substances and possessing different degradation properties.

[0034] Furthermore, the photocurable hydrogel matrix, by mass, consists of 1 part phosphate buffer solution and 0.01-1 part biocompatible photosensitive resin.

[0035] Furthermore, the biocompatible photosensitive resin is a mixture of at least one or more of the following in any proportion: methacrylamide gelatin (GELMA), polyethylene glycol diacrylate (PEGDA), polyether F127 diacrylate (F127DA), methacrylamide polyvinyl alcohol (PVAMA), sodium alginate methacrylate (ALGMA), silk fibroin methacrylate (SILMA), hyaluronic acid methacrylate (HAMA), and acrylamide (AM).

[0036] In some alternative embodiments, the nanoparticles, by weight, include 1 part of biodegradable polymer material and 0.001-0.1 g of bioactive substances for regulating angiogenesis, nerve regeneration, or tissue reconstruction processes.

[0037] Furthermore, the biodegradable polymer material is any mixture of at least one or more of the following: polycaprolactone (PCL), poly(lactide-caprolactone) copolymer (PLCL), polyurethane (PU), polyglycerol sebacate (PGS), polydioxanone (PDS), polyglycolic acid (PGA), polylactide (PLA), poly(lactide-glycolic acid) copolymer (PLGA), polyhydroxyalkanoate (PHA), and polyethylene glycol (PEO).

[0038] Furthermore, bioactive substances include, but are not limited to, angiogenic factors, neurotrophic factors, growth factors, cytokines, bioactive proteins, polypeptides, and small molecule compounds with tissue regulatory effects, as well as mixtures thereof in any proportion.

[0039] In some alternative embodiments, the structured guiding unit is a continuously or discretely distributed microstructure, the structural form of which includes, but is not limited to, microchannels, microgrooves or combinations thereof arranged along the axial direction of the conduit, and its scale can be in the range of micrometers to hundreds of micrometers, to simulate the structural features in natural nerve bundles.

[0040] Furthermore, this invention also discloses a method for constructing the aforementioned modular three-dimensional neural conduit system, comprising the following steps: Step 1, Preparation: Mix the prescribed amount of biocompatible photosensitive resin with a solvent, then add the prescribed amount of polymer nanoparticles and disperse them evenly to prepare a photosensitive ink solution; Step 2, Module preparation: The photosensitive ink solution is cured by light to obtain the outer structural module and the inner functional module respectively; Step 3, Catheter Assembly: Assemble the outer structural module and the inner functional module to obtain a modular nerve conduit.

[0041] In some alternative embodiments, the photopolymerization method includes ultraviolet curing, photopolymerization in situ 3D printing, SLA 3D printing, DLP 3D printing, and two-photon lithography 3D printing.

[0042] Furthermore, in the photocuring and shaping method, when ultraviolet light is used for curing, the obtained photosensitive ink solution is added into the mold, and the degree of gelation is controlled by parameters such as irradiation intensity, irradiation time, and irradiation angle. After demolding, the photocurable scaffold material of the desired shape is obtained.

[0043] Furthermore, in the photopolymerization shaping method, during in-situ photopolymerization 3D printing, the obtained photosensitive ink solution is loaded into a barrel, which is then mounted on the 3D printer. The diameter and degree of gelation of the extruded gel are controlled by adjusting parameters such as the barrel's three-dimensional (x, y, z-axis) movement trajectory, the barrel's piston speed, the needle diameter, the receiver rod's rotational speed and lateral movement speed, the irradiation intensity, and the irradiation time. This allows for the production of a photopolymerized scaffold material of the desired shape.

[0044] Furthermore, when using SLA 3D printing or DLP 3D printing, the photocurable shaping method involves loading the obtained photosensitive ink solution into a material tank, installing the deposition platform on the 3D printer, and adjusting parameters such as slice layer height, single-layer irradiation light intensity and irradiation time, peeling speed and scraping speed to ensure good gel formation, thereby obtaining the photocurable scaffold material of the desired shape.

[0045] Furthermore, in the photopolymerization and shaping method, when using two-photon lithography 3D printing, the obtained photosensitive ink solution is loaded into the material tank, the deposition platform is installed on the 3D printer, and parameters such as laser power, laser focus offset speed and calibration platform horizontal position are adjusted to make the gel precisely formed at the specified position, thereby obtaining the photopolymerization scaffold material of the required shape.

[0046] Furthermore, the different functional modules differ in at least one of the following aspects, and / or both: 1) The types of bioactive substances loaded are different. Bioactive substances are used to regulate angiogenesis, nerve regeneration, cell migration, axonal extension or tissue microenvironment remodeling. 2) Different loading concentrations of the same bioactive substance are used to create differentiated local release intensities in different modules; 3) The degradation rates of microspheres or nanoparticles loaded with bioactive substances are different, and the degradation rate can be controlled by adjusting the molecular weight, monomer ratio, crystallinity or structural morphology of the carrier material.

[0047] Furthermore, by sequentially assembling multiple functional modules along the axial direction of the nerve conduit, a predetermined concentration gradient distribution of different bioactive substances is achieved in three-dimensional space: 1) The first type of bioactive substances form a spatially decreasing concentration distribution from the middle of the nerve conduit towards the proximal and distal ends, which is used to preferentially regulate the tissue remodeling process in the middle and bifurcation or key areas of the nerve conduit; 2) The second type of bioactive substances form a spatially increasing concentration distribution from the proximal end to the distal end of the nerve conduit, which is used to guide the regenerated axons or related cells to migrate directionally to the distal target area; 3) The spatial gradients of the different bioactive substances mentioned above are set independently, without interdependence or mutual coupling, thus allowing for differentiated spatial regulation of multiple bioactive signals in different directions within the same neural conduit.

[0048] The aforementioned spatial gradient is not formed through continuous mixing, diffusion, or gradient blending within a single support, but rather through the splicing and arrangement of multiple functional modules with discrete concentration units in space, forming an approximately continuous three-dimensional concentration gradient on a macroscopic scale.

[0049] Meanwhile, the spatial gradient and the structured guidance units inside each functional module are spatially coordinated to form a matching relationship between the bioactive signal gradient and the physical guidance direction in different regions, thereby enhancing the precision of regulation of cell behavior and axonal growth pathways.

[0050] Furthermore, by selecting biodegradable materials with different degradation or release characteristics as carriers of bioactive substances, the bioactive substances in different functional modules exhibit differentiated release behaviors over time, thereby achieving sequential release regulation: 1) Class I bioactive substances are loaded onto carriers with a fast degradation rate to achieve rapid or preferential release in the early stages of implantation, thereby regulating early angiogenesis, immune response, or microenvironment establishment. 2) The second type of bioactive substances are loaded into carriers with a slower degradation rate, so as to achieve continuous or slow release over a longer time scale to maintain the process of nerve regeneration, axonal extension or functional maturation. This allows for coordinated regulation with the aforementioned spatial gradient distribution in the temporal dimension, enabling the spatiotemporal orderly guidance of vascularization and neurotransmission events during peripheral nerve regeneration.

[0051] Unless otherwise specified, the term "parts" in this invention refers to parts by weight.

[0052] All raw materials used in this invention were purchased commercially. The specific sources of the raw materials used in the following embodiments of this invention are as follows: Phosphate-buffered saline (PBS): Shanghai Aladdin Biochemical Technology Co., Ltd.; Photoinitiator LAP: Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; Food coloring: Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; Bovine serum albumin (BSA): Sigma aldrich (St. Louis, MO, USA); Recombinant human vascular endothelial growth factor (VEGF): Suzhou Nearshore Protein Technology Co., Ltd.; Recombinant human nerve growth factor (NGF): Suzhou Nearshore Protein Technology Co., Ltd.; Rhodamine-modified recombinant human vascular endothelial growth factor (Rhodamine-VEGF): Sigma aldrich (St. Louis, MO, USA); Photosensitive hydrogel green fluorescent dye: Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; Sodium docusate: Sigma aldrich (St. Louis, MO, USA); Polyvinyl alcohol (PVA): Type 1788, Sigma aldrich (St. Louis, MO, USA); Trichloromethane: Shanghai McLean Biochemical Technology Co., Ltd.; Meloxicam: Sigma aldrich (St. Louis, MO, USA); Micro-fil contrast agent: Flow Tech, Inc. (Carver, MA, USA); Isoflurane: Shenzhen Ruiwode Life Science & Technology Co., Ltd. 3-0 and 9-0 nylon sutures: Ningbo Medical Suture Co., Ltd. Hematoxylin-Einstein (H&E) staining kit: Beijing Regen Biotechnology Co., Ltd.; Sterile saline solution: Sichuan Kelun Pharmaceutical Co., Ltd.

[0053] Photosensitive bioresin: Polyethylene glycol diacrylate (PEGDA): Sigma aldrich (St. Louis, MO, USA); Polyether Prönnicke diacrylate (F127DA): Sigma aldrich (St. Louis, MO, USA); Sodium methacrylated alginate (ALGMA): Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; Methacrylated silk fibroin (SILMA): Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; Methacrylated hyaluronic acid (HAMA): Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; Methacrylated gelatin (GELMA): Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; Methacrylamide polyvinyl alcohol (PVAMA): Shanghai Lingjiu Medical Technology Co., Ltd.; Biodegradable polymer materials: Poly(L-lactide-caprolactone) (PLCL): Viscosity: 2.6-2.8, ratio 50:50, Jinan Daigang Bioengineering Co., Ltd. Polycaprolactone (PCL): Molecular weight: 40,000, Sigma Aldrich (St. Louis, MO, USA); Polylactic acid (PLA): Molecular weight: 10,000, Sigma aldrich (St. Louis, MO, USA); Polyglycolic acid (PGA): Molecular weight: 200,000, Sigma aldrich (St. Louis, MO, USA); Acid-terminated poly(lactide-glycolic acid) copolymer (PLGA-acid): molecular weight: 16,000, molar ratio of lactide to glycolic acid 50:50, Jinan Daigang Bioengineering Co., Ltd. Ester-terminated poly(lactide-glycolic acid) copolymer (PLGA-ester): molecular weight: 300,000, molar ratio of lactide to glycolic acid 80:20, Jinan Daigang Bioengineering Co., Ltd.

[0054] Cell source: Neural stem cells were obtained from the Beike Biotechnology Integrated Cell Bank; then, they were formed into neural stem cell spheres using ultra-low adsorption 96-well plates (Corning, NY, USA) for subsequent experiments. The vascular organ is derived from the team's self-developed technology "A Vascularized Heart Organoid with a Cavity Structure and Its Preparation Method", with the relevant patent number CN 202211277761.2.

[0055] Animal source: All laboratory animals used were... Rattus norvegicus (Sprague-Dawley), i.e., SD rats, all male, 8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0056] The technical solution of the present invention will be further illustrated by the following embodiments.

[0057] Example 1: Preparation of functional modules with different release properties based on different nanoparticles (1) First, nanoparticles with different release properties were prepared: The active molecule VEGF to be loaded was dissolved at a concentration of 0.1 mg / mL in a protective solution containing 1% (w / v) bovine serum albumin (BSA) and 10% (w / v) sodium docusate as the inner aqueous phase (W1). Simultaneously, an organic phase (O) was prepared by dissolving the aforementioned polymer materials (poly(L-lactide-caprolactone) (PLCL), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), acid-terminated poly(lactide-glycolic acid) copolymer (PLGA-acid), and ester-terminated poly(lactide-glycolic acid) copolymer (PLGA-ester)) in the volatile organic solvent chloroform at a concentration of 2% (w / v). 0.2 ml of the aqueous phase was slowly added dropwise to 3 ml of the organic phase, and the first ultrasonic emulsification was performed using an ultrasonic cell disruptor in an ice bath. The power was set to 45 W, the ultrasonic time to 60 s, and the ultrasonic treatment was repeated for 4 s with a 4-s interval to prevent overheating, forming a primary emulsion (W1 / O). The colostrum was quickly added to 4 ml of external aqueous phase, which was an aqueous solution containing the stabilizer polyvinyl alcohol (PVA) at a concentration of 3% (w / v). The colostrum was then subjected to a second ultrasonic emulsification in an ice bath with a power of 30 W and an ultrasonic time of 240 s to form a stable double emulsion (W1 / O / W2). The prepared double emulsion was placed on a magnetic stirrer and stirred in a dialysis cabinet at 4 °C for 8 h to allow the organic solvent to evaporate completely and the polymer material to gradually solidify into nanoparticles. After the solvent had completely evaporated, the resulting nanoparticle suspension was transferred to centrifuge tubes and centrifuged at 18,000 mL / min at 4 °C. g Centrifuge at 15 min to precipitate the nanoparticles, discard the supernatant to remove free active molecules and excess PVA solution residue; resuspend the precipitate in deionized water or PBS buffer, and repeat centrifugation and washing 2-3 times under the same conditions to obtain high-purity nanoparticles loaded with active molecules. Lyophilize the nanoparticles into powder and store in a -80 ℃ freezer protected from light.

[0058] (2) Nanoparticles were incorporated into bio-ink and prepared into functional modules with different release properties: By weight, 0.005 parts of LAPI photoinitiator and 0.001 parts of food coloring were simultaneously dissolved in 1 part of phosphate buffer solution, and 0.2 parts of the aforementioned photosensitive bio-resin PEGDA were added. The solution was vortexed at 37 °C until completely dissolved. Then, 0.01 parts of nanoparticles were added and vortexed until homogeneous to obtain a bio-ink containing nanoparticles. The printer parameters were then adjusted, and the printing light intensity was set to 15 mW / cm². 2The layer height was set to 25-50 μm. The functional module model was sliced ​​and imported into the printer. The obtained photosensitive ink solution (bio-ink) was added to the feed tank, and printing was performed. After printing, the model was peeled off from the deposition platform, yielding a module with structured guiding units and controlled release of active molecules.

[0059] Figure 1 For the nanoparticles prepared using different biodegradable polymers in Example 1, transmission electron microscopy (TEM) images were used, and their particle size was characterized using a nanoparticle size analyzer. The images show that the nanoparticles prepared using the six polymers are all uniformly spherical, and their particle sizes are uniformly distributed within the range of 130 nm to 150 nm, demonstrating the effectiveness and stability of the nanoparticle preparation method.

[0060] Figure 2 The cumulative release curves of nanoparticles with different degradation properties in Example 1 are shown. VEGF was used as the active ingredient, encapsulated within the nanoparticles, and functional modules of the same material and shape were fabricated. The release behavior of different modules was then detected using a VEGF kit. As shown in the figure, the PLA, PLGA-acid, and PGA groups tended towards burst release, while the PCL, PLGA-ester, and PLCL groups tended towards sustained release. The release rates of the six groups decreased sequentially, demonstrating that the release behavior of encapsulated molecules can be accurately controlled by regulating the degradation properties of the polymers. The release cycles of the PLGA-acid and PLGA-ester groups are close to the window periods of vascular remodeling and neurogenesis during peripheral nerve regeneration, respectively. Therefore, when considering the regenerative regulation of these two processes, PLGA-acid and PLGA-ester were preferentially selected to prepare the controlled-release modules.

[0061] Figure 3 These are photographs depicting the degradation properties of nanoparticles prepared using different polymeric materials (PLGA-acid and PLGA-ester) in Example 1. Following the method described in Example 1, nanoparticles were prepared using PLGA-acid and PLGA-ester, respectively, and dispersed in sterile PBS solution. Samples were taken on day 0 and day 28, and their morphology was characterized by transmission electron microscopy. The images show that the PLGA-acid and PLGA-ester groups had similar morphologies on day 0, exhibiting uniform spherical shapes. However, on day 28, the PLGA-acid group showed almost complete degradation and collapse, while the PLGA-ester group retained some of its morphology. This further demonstrates the feasibility of controlled release through regulating polymer degradation.

[0062] Figure 4This is a schematic diagram of different structured guiding units in Example 1. The figure shows a microgroove structure with a spacing of 50 μm and a microchannel structure with a diameter of 300 μm. After being assembled with the outer tube through a tenon and mortise structure, different modules of the same configuration can form a continuous guiding structure, such as a through microchannel and a continuous microgroove, thereby guiding and supporting the directional growth of nerves.

[0063] Example 2: Multifunctional modular neural conduit based on modular assembly and its spatial gradient construction method (1) By weight, dissolve 0.005 parts of LAPT photoinitiator and 0.001 parts of food coloring simultaneously in 1 part of phosphate buffer solution, and add 0.2 parts of the aforementioned photosensitive bioresin PEGDA. Vortex at 37°C until completely dissolved. Then, adjust the printer parameters and set the printing light intensity to 15 mW / cm². 2 The layer height is set to 50-100 μm. The outer layer structure module model is sliced ​​and imported into the printer. The resulting photosensitive ink solution is added to the ink tank, and printing is performed.

[0064] (2) Using the method described in Example 1, and specifying the polymer material as PLGA-acid in Example 1, several cylindrical functional modules with the same axial dimensions were prepared. Each functional module was made of a photocurable hydrogel, in which biodegradable microparticles or nanoparticles loaded with bioactive substances (specifically VEGF and / or NGF in Example 2) were uniformly dispersed. The functional modules were identical in structure and morphology, but differed in the types and / or concentrations of the loaded bioactive substances.

[0065] (3) The above-mentioned different functional modules are spliced ​​and assembled along the axial direction in a preset order through a mortise and tenon structure with physical interlocking function to form an overall neural conduit structure (including single and multiple composite gradient neural conduits). The spatial gradient is not formed by continuous mixing or diffusion within a single scaffold, but by the spatial arrangement and combination of multiple discrete functional modules along the axial direction to form a continuously changing concentration gradient of bioactive substances on a macroscopic scale.

[0066] Among them, single composite gradient (heterogeneous gradient) neural conduits: a. In functional modules located in the middle of the nerve conduit axis, nanoparticles are loaded with a high concentration of angiogenesis-related bioactive substances; in functional modules located far from the middle, the loading concentration of angiogenesis-related bioactive substances is relatively lower. b. In the functional modules near the distal nerve end, the nanoparticles are further loaded with a higher concentration of neurotrophic bioactive substances; Multiple composite gradient neural conduits: In the functional module located in the middle of the nerve conduit axis, the nanoparticles are loaded with a high concentration of angiogenesis-related bioactive substances; in the functional module located far from the middle, the loading concentration of angiogenesis-related bioactive substances is relatively lower; and in the functional module closer to the distal nerve, the nanoparticles are further loaded with a high concentration of neurotrophic bioactive substances.

[0067] After assembly, angiogenesis-related bioactive substances show a spatial distribution trend of decreasing from the middle to both ends of the nerve conduit, while neurotrophic bioactive substances show a spatial distribution trend of increasing from the proximal end to the distal end, thus providing differentiated spatial guidance signals for angiogenesis and axonal directional extension.

[0068] Figure 5 This diagram illustrates the single and multiple composite gradient neural conduits used in Example 2, along with actual fabrication results, showcasing the heterogeneous spatial distribution of active molecules within the conduit. The method in Example 2 not only achieves the "axially increasing" and "high in the middle and low on both sides" gradient distribution patterns of single active molecules, but also integrates these two patterns to achieve a heterogeneous distribution of active molecule A (neurotrophic bioactive substance, NGF) with "axially increasing" and active molecule B (angiogenic bioactive substance, VEGF) with "high in the middle and low on both sides." This aligns with the different spatial distribution trends of angiogenic bioactive substances and neurotrophic bioactive substances described in Example 2, providing differentiated spatial guidance signals for angiogenesis and axonal regeneration.

[0069] Figure 6 This is a fluorescence image of the active molecule gradient in Example 2. Rhodamine-VEGF was selected as the encapsulating active ingredient (VEGF is used here, Rhodamine is only for ease of observation). Nanoparticles were prepared using the method in Example 1, and then mixed with FITC-labeled photosensitive ink in different proportions to sequentially process functional modules with different drug loadings. These modules were then assembled into an "axially increasing" gradient conduit, and the fluorescence signals between adjacent modules were observed using a confocal microscope. The image shows that the FITC fluorescence signals of adjacent modules are basically consistent, while the Rhodamine-VEGF fluorescence signals show significant differences, demonstrating the successful construction of the active molecule concentration gradient.

[0070] Example 3: Modular neural conduit for sequential release based on functional modules with different release characteristics Using PLGA-acid and PLGA-ester polymers, several functional modules with identical structures but different release characteristics were prepared using the method described in Example 1. Each functional module consisted of a photocurable hydrogel, in which biodegradable nanoparticles were dispersed and loaded. By adjusting the material composition, molecular weight, monomer ratio, or crystallinity of the nanoparticles, different in vivo degradation rates of the carrier materials in the different functional modules were achieved.

[0071] In the functional module used to load angiogenesis-related bioactive substances, the nanoparticles use a carrier material with a relatively fast degradation rate, which is limited to PLGA-acid in this embodiment 3; in the functional module used to load neurotrophic bioactive substances, the nanoparticles use a carrier material with a relatively slow degradation rate, which is limited to PLGA-ester in this embodiment 3.

[0072] The functional modules (G1-G5) with different release properties are assembled axially in a predetermined order to form an integral neural conduit structure. The specific predetermined order is as follows: the concentration of molecule A, human endothelial growth factor (VEGF), decreases from the intermediate functional module G3 towards both ends (G1 or G5 functional modules), i.e., the encapsulation amount is 2 × 10⁻⁶. 3 ng→0; The concentration of molecule B human nerve growth factor (NGF) increases axially according to the G1→G5 functional modules, i.e., the encapsulation amount 0→2×10 3 ng.

[0073] After implantation, angiogenesis-related bioactive substances in the early release module are preferentially released to promote early angiogenesis within the neural conduit; while neurotrophic bioactive substances in the slow release module are continuously released over a longer timescale to support subsequent axonal extension and neural maturation.

[0074] Through the coordinated design of functional modules in terms of spatial location and release characteristics, the sequential release regulation of bioactive substances in the time dimension was realized, and it formed a synergistic effect with the spatial gradient distribution.

[0075] Figure 7This diagram illustrates the construction of the spatiotemporally controlled release neural conduit system in Example 3, along with the actual release histogram. Following the method in Example 1, nanoparticles were prepared using PLGA-acid and PLGA-ester to encapsulate molecules A (VEGF) and B (NGF), respectively. Heterogeneous gradient functional modules for the two molecules were constructed according to Example 2. Subsequently, the release behavior of the G1-G5 functional modules for the two molecules was measured using a kit. The release histogram shows that the release of both molecules A and B conforms to the gradient design. Furthermore, molecule A, encapsulated with PLGA-acid, is released more rapidly within 14 days, while molecule B, encapsulated with PLGA-ester, is released more slowly within 28 days. This demonstrates that the sequential release of bioactive substances over time can synergistically interact with the spatial gradient distribution, proving the effectiveness of the spatiotemporally controlled release system design.

[0076] Example 4: Scalable modular neural conduit suitable for different defect lengths To address the repair needs of peripheral nerve defects of varying lengths, this embodiment provides a scalable, modular neural conduit construction method. The neural conduit is assembled sequentially along the axial direction from multiple functional modules of identical axial dimensions. While the functional modules are structurally identical, they differ in the types, concentrations, and / or release characteristics of the bioactive substances they contain.

[0077] In practice, the overall length of the nerve conduit is adjusted by increasing or decreasing the number of functional modules according to the length of the nerve defect to be repaired, while maintaining the axial arrangement of the functional modules and the distribution pattern of their corresponding bioactive substances. This method allows for the creation of a consistent spatial gradient pattern in nerve conduits of different lengths without requiring continuous gradient fabrication or redesign of a single scaffold.

[0078] Modular design allows the construction of spatial gradients to rely on the number and arrangement of discrete functional modules, rather than on complex material mixing or processing parameter control, thereby significantly improving the structural adaptability of nerve conduits to different defect lengths, manufacturing repeatability, and batch production stability.

[0079] Figure 8This diagram illustrates the modular nerve conduit applicable to different defect lengths in Example 4, along with application examples. By adjusting the length of the outer shell, scaffolds can be fabricated for different nerve defect models. As shown in the figure, when performing small-gap closure surgery, a 5mm outer shell is used, and a microgroove functional module is applied to assemble it into a small-gap nerve cannula, reserving more space for nerve regeneration. When dealing with longer nerve defects, a 13mm outer shell is used, and a microchannel functional module is applied to assemble it into a nerve conduit, accelerating the guidance of nerve regeneration. This demonstrates the personalized design and flexible adaptability of this modular conduit, enabling it to address various injury scenarios through simple assembly.

[0080] Example 5: Modular nerve conduit with one trunk, two branches, or multiple branches suitable for complex peripheral nerve defects. For common clinical peripheral nerve defect models with one trunk, two branches, or multiple branches, this embodiment provides a method for constructing a nerve conduit with an integrally molded outer shell and modular internal functional assembly.

[0081] The neural conduit comprises a monolithically photopolymerized printed trunk-branch shell and multiple replaceable functional modules disposed within the shell cavity. The shell forms a proximal trunk channel and at least two distal branch channels. The overall structure is formed in a single step during the printing process to match the macroscopic morphology of complex bifurcated nerves.

[0082] The functional modules are independently molded photocurable hydrogel modules or hydrogel-containing composite modules. Biodegradable microspheres or nanoparticles loaded with bioactive substances are uniformly dispersed within each module. Different functional modules may have similar or identical structural morphologies, but the types, concentrations, and release characteristics of the bioactive substances they contain differ. The functional modules are assembled into corresponding positions within the outer shell cavity through physical interlocking structures, shape-fitting structures, or positioning and limiting structures, thereby creating a pre-defined spatially heterogeneous distribution of bioactive substances in the main region, branching regions, and various sub-regions.

[0083] In the specific spatial design, the functional modules located at the junction of the main trunk and the bifurcation are loaded with a high concentration of angiogenesis-related bioactive substances to preferentially promote blood revascularization; the functional modules located in each branch area are loaded with neurotrophic bioactive substances, and their distribution in each branch shows an increasing trend from the bifurcation area to the distal end along the axial direction, thereby guiding the regenerating axons to extend directionally to the corresponding branch target area.

[0084] By changing the number of functional modules or adjusting the formulation parameters of microspheres or nanoparticles in each module, it is possible to flexibly adapt to different branch lengths, diameters, and target organ requirements. At the same time, by selecting carrier materials with different release characteristics, sequential delivery of angiogenesis-related bioactive substances and neurotrophic bioactive substances with sustained or delayed release can be achieved in the time dimension.

[0085] The construction method, which combines the one-piece molding of the outer shell with the modular assembly of internal functions, avoids the technical difficulties of directly processing multi-directional and multi-factor continuous gradients in a single three-dimensional bifurcated scaffold. This significantly improves the feasibility, manufacturing repeatability, and structural adaptability of spatial gradient construction in complex peripheral nerve defect models.

[0086] Figure 9 This is a design diagram and fabrication effect diagram of the modular nerve conduit with one trunk and multiple branches in Example 5. By adopting the spatiotemporal controlled release method in Example 2, it is possible to achieve spatial gradient distribution and "window period" release of angiogenesis-related active substances (VEGF) and nerve growth-related active substances (NGF), thereby guiding vascular remodeling from both ends to the middle and the directional extension of regenerated axons, respectively.

[0087] Application Example 1: Repairing sciatic nerve defects in rats using neural conduits with different spatiotemporal release patterns of growth factors. To demonstrate the in vivo repair effects of different spatiotemporal release patterns of growth factors and to verify the superior repair performance of dual gradients, the method described in Example 3 was used to prepare heterogeneous VEGF / NGF concentration gradient conduits, uniform VEGF / NGF concentration gradient conduits, and blank factor-free conduits, respectively. Healthy adult male SD rats (weighing 200-250g) were then used to create a 1.1cm sciatic nerve defect model and implant the nerve conduit. Before the experiment, rats were acclimatized for one week in standard stainless steel cages, with free access to a complete nutritional diet sterilized by 60Co irradiation and sterile drinking water. The surgery was performed in a sterile operating room. After being anesthetized with isoflurane inhalation, the rats were fixed in a prone position. The surgical area was prepared and disinfected with iodine and alcohol. A longitudinal incision was made on the posterolateral aspect of the right hind limb femur. The gluteal and biceps femoris muscles were bluntly dissected to expose the main trunk of the sciatic nerve. The nerve was sharply transected 5 mm distal to the ischial tuberosity using microsurgical scissors. A segment of the nerve trunk was then removed, creating a precise 1.1 cm defect. The previously prepared nerve conduit was infiltrated with sterile saline. Then, the proximal and distal ends of the nerve conduit were sutured end-to-end to the two severed ends of the sciatic nerve using 9-0 sutures, ensuring alignment and tension-free nerve bundles. Subsequently, the muscle fascia and skin incision were sutured layer by layer using 3-0 sutures. Immediately postoperatively, a subcutaneous injection of meloxicam was administered, and the rats continued to be fed. Three months post-surgery, final sampling was performed. The samples were perfused intravascularly with contrast agent via micro-fil, and micro-CT scans were used to assess the three-dimensional growth of blood vessels. Further histological staining was then performed to comprehensively evaluate the regenerative effects of blood vessels and nerves.

[0088] Figure 10This section shows representative tissue slice images obtained 3 months post-surgery in Application Example 1, stained with hematoxylin and eosin (H&E), as well as representative Micro-CT reconstructed images obtained after Micro-fil contrast agent perfusion. Figure 10 As shown in (A), in the catheter group loaded with VEGF and NGF at heterogeneous concentration gradients (experimental group 1), the regenerated tissue was the densest and largest in volume, forming a rich and clearly oriented vascular network, with numerous deeply stained nerve fibers visible in the intervascular spaces. In contrast, the catheter group loaded with the same growth factors but at a uniform concentration distribution (experimental group 2) exhibited a relatively loose regenerated tissue structure, lower vascularization, and a significantly reduced number of accompanying nerve fibers; while in the blank catheter control group (experimental group 3) without any growth factors, only a very small amount of vascular infiltration was observed, with almost no nerve tissue regeneration. These results indicate that a heterogeneous concentration gradient of VEGF / NGF has the optimal promoting effect on the synergistic regeneration of blood vessels and nerves. Furthermore, Figure 10 Micro-CT images in (B) show that the heterogeneous concentration gradient catheters formed a large number of orderly and well-structured blood vessels, while the homogeneous concentration catheters, although having a larger number of vessels, had a disordered distribution and failed to construct a continuous and effective blood transport network. This comparison further confirms that the spatial concentration gradient of active substances (growth factors) is crucial for guiding the directional growth of blood vessels.

[0089] In summary, this invention not only verifies the superiority of the heterogeneous spatiotemporal release pattern of VEGF / NGF in the in vivo tissue regeneration microenvironment, but also reveals the synergistic effect between angiogenesis and neuroregeneration processes.

[0090] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A modular three-dimensional neural guidance conduit system, characterized in that, include: Outer structure module; At least two functional modules arranged sequentially along the axial direction of the conduit; The functional module and the outer structural module are connected by a physical interlocking structure to form the modular three-dimensional neural guidance conduit system; Each functional module contains a hydrogel matrix and microspheres or nanoparticles loaded with bioactive substances. The functional modules are arranged in a gradient according to the concentration of the bioactive substances they contain.

2. The modular three-dimensional neural guidance conduit system according to claim 1, characterized in that, The bioactive substances include at least one of angiogenesis-related factors, neurotrophic factors, nerve growth factors, cytokines, bioactive proteins, polypeptides, and small molecule compounds with tissue regulatory effects. The raw materials for the microspheres or nanoparticles are selected from at least one of polycaprolactone, poly(lactide-caprolactone) copolymer, polyurethane, polyglycerol sebacate, polydioxane, polyglycolic acid, polylactide, poly(lactide-glycolic acid) copolymer, polyhydroxy fatty acid ester, and polyethylene glycol.

3. The modular three-dimensional neural guidance conduit system according to claim 2, characterized in that, The bioactive substance is angiogenesis-related factor, and the functional modules are arranged with concentrations decreasing from the middle of the neural conduit towards the proximal and distal ends; and / or, The bioactive substance is a neurotrophic factor or nerve growth factor, and the functional modules are arranged in increasing concentrations from the proximal end to the distal end of the nerve conduit.

4. A modular three-dimensional neural guidance conduit system according to claim 3, characterized in that, The angiogenesis-related factor is vascular endothelial growth factor.

5. A modular three-dimensional neural guidance conduit system according to claim 1, characterized in that, The functional module has a structured guidance unit inside, which forms an orderly guidance structure after being integrated and connected.

6. A modular three-dimensional neural guidance conduit system according to claim 5, characterized in that, The structured guiding unit is a continuously or discretely distributed microstructure, which includes microchannels, microgrooves, or combinations thereof arranged along the axial direction of the conduit.

7. A modular three-dimensional neural guidance conduit system according to claim 1, characterized in that, The physical interlocking structure includes a mortise and tenon structure or a snap-fit ​​structure.

8. A method for constructing a modular three-dimensional neural guidance conduit system as described in any one of claims 1-7, characterized in that, Includes the following steps: (1) Dissolve the photosensitive bioresin in a phosphate buffer solution, add a photoinitiator and food coloring, and vortex to dissolve completely to obtain photosensitive ink; Microspheres or nanoparticles loaded with bioactive substances are added to the photosensitive ink to obtain a bio-ink; The bio-ink is molded into a functional module with structured guiding units and physical interlocking structures using a photocuring method; the photosensitive ink is molded into an outer structural module with physical interlocking structures. (2) Based on the length and shape of the target nerve defect, at least two functional modules are spliced ​​together along the axial direction in a preset order through a physical interlocking structure to form an integral nerve conduit, and then placed inside the outer structural module, so that the functional modules containing different concentrations of bioactive substances form a preset decoupling concentration gradient in space and achieve sequential release in time, thereby obtaining the modular three-dimensional nerve guidance conduit system.

9. The method for constructing a modular three-dimensional neural guidance conduit system according to claim 8, characterized in that, The photosensitive bioresin includes at least one of methacrylamide gelatin, polyethylene glycol diacrylate, polyether F127 diacrylate, methacrylamide polyvinyl alcohol, methacrylamide hyaluronic acid, and acrylamide.

10. The method for constructing a modular three-dimensional neural guidance conduit system according to claim 8, characterized in that, The photocuring and shaping methods include ultraviolet light curing, photocuring in-situ 3D printing, SLA 3D printing, DLP 3D printing, or two-photon lithography 3D printing.

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