Neural grafts with microenvironment regulation function and preparation method and application thereof

Abstract

The application provides a nerve graft with a microenvironment regulation function and a preparation method and application thereof, and belongs to the field of biomedical materials, wherein the nerve graft comprises an outer layer mechanical enhancement zone and an inner layer conductive regulation zone; the outer layer mechanical enhancement zone is a tubular skeleton formed by a three-dimensional weaving process of a multifilament and a monofilament; the inner layer conductive regulation zone is a conductive hydrogel inner core arranged in the inner cavity of the tubular skeleton; the conductive hydrogel inner core is formed with an inner ring axial through small-diameter pore for nerve cell migration and axon directional growth and an outer ring axial through large-diameter pore for vascular endothelial cell migration and capillary ingrowth; mesenchymal stem cells are inoculated in the inner ring axial through small-diameter pore; and the conductive hydrogel inner core comprises a photo-crosslinked hydrogel matrix and metal ion modified black phosphorus two-dimensional nanosheets dispersed in the photo-crosslinked hydrogel matrix. The application is beneficial to improving the peripheral nerve defect repair effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a neural graft with microenvironment regulation function, its preparation method, and its application. Background Technology

[0002] Peripheral nerve injury is one of the most common types of neurological injury in clinical practice. Currently, autologous nerve transplantation is still considered the gold standard for repairing long-segment peripheral nerve defects. However, autologous nerve transplantation has problems such as limited donor sources, donor site functional impairment, secondary surgical trauma, nerve diameter mismatch, and limited available transplant length, making it difficult to meet the clinical needs for repairing complex nerve defects. Artificial nerve conduits, as an important alternative to autologous nerve transplantation, have become a research focus in the field of peripheral nerve tissue engineering.

[0003] Current artificial nerve conduits primarily guide proximal axons to grow distally by providing a physical bridging space and, to some extent, preventing the invasion of peripheral fibrous tissue. However, peripheral nerve regeneration involves multiple processes, including Schwann cell migration, directional axonal growth, myelin regeneration, vascular ingrowth, and environmental regulation. Therefore, an ideal nerve graft must possess multiple functions, including stable mechanical support, axial guidance, electrophysiological regulation, vascularization support, and the creation of a cellular microenvironment. To improve the repair effect of artificial nerve conduits, researchers have developed various modification strategies, but the following shortcomings still exist: Existing braided conduits mostly use single fibers or uniform braided structures, making it difficult to simultaneously achieve flexibility, radial anti-collapse, and reversible resilience, which can easily lead to tissue irritation or lumen collapse; conductive hydrogels commonly use components such as polypyrrole and carbon nanotubes, which have the problem of non-degradability or long-term residue; although black phosphorus nanosheets have degradable conductivity, they are easily oxidized and degraded in water and oxygen environments, resulting in a decrease in conductivity; the internal structure of traditional conduits is mostly a single cavity or a homogeneous porous structure, lacking a spatial partition design for central nerve guidance and peripheral vascularization support, making it difficult to simultaneously meet the needs of axonal directional extension and nutrient exchange; in addition, existing cell loading methods often directly mix stem cells into the hydrogel precursor, which is easily damaged during cross-linking and pore formation, and the spatial distribution is difficult to control precisely.

[0004] In view of this, it is necessary to design a neural graft with microenvironment regulation function, as well as its preparation method and application, to solve the above problems. Summary of the Invention

[0005] In view of the technical problems existing in the background art, this application provides a neural graft with microenvironment regulation function, its preparation method and application, aiming to solve the technical problems of existing neural grafts that are difficult to simultaneously take into account flexibility and radial support, non-degradable or easily degradable conductive components, lack of spatial partition design for neural guidance and vascularization, and easy damage and uncontrollable spatial distribution during cell loading process.

[0006] In a first aspect, this application provides a neural graft with microenvironment regulation function, including an outer mechanical enhancement region and an inner electrical conductivity regulation region; The outer mechanical reinforcement region is a tubular skeleton formed by multifilament and monofilament through a three-dimensional weaving process; the inner conductive control region is a conductive hydrogel core disposed in the inner cavity of the tubular skeleton. The conductive hydrogel core has an inner ring of axially penetrating small-diameter channels and an outer ring of axially penetrating large-diameter channels. The inner ring of axially penetrating small-diameter channels is used for nerve cell migration and axonal directional growth, while the outer ring of axially penetrating large-diameter channels is used for vascular endothelial cell migration and capillary ingrowth. Mesenchymal stem cells are seeded in the inner ring of axially penetrating small-diameter channels. The conductive hydrogel core comprises a photocrosslinked hydrogel matrix and metal ion-modified black phosphorus two-dimensional nanosheets dispersed in the photocrosslinked hydrogel matrix.

[0007] As a further improvement to this application, the multifilament is one or more of polylactic acid multifilament, polycaprolactone multifilament, polylactic acid-glycolic acid copolymer multifilament, silk fibroin multifilament, and collagen multifilament; The monofilament is one or more of the following: polydioxanone monofilament, polylactic acid monofilament, polycaprolactone monofilament, and polylactic acid-caprolactone copolymer monofilament.

[0008] As a further improvement of this application, the multifilament and the monofilament are combined and woven in a ratio of (2~5):1; The three-dimensional weaving process is one of spiral weaving, radial interlacing, or spiral-radial composite weaving.

[0009] As a further improvement of this application, the metal ions in the metal ion-modified black phosphorus two-dimensional nanosheets are one or more of magnesium ions, copper ions, zinc ions, strontium ions, manganese ions, lithium ions, cobalt ions, and cerium ions.

[0010] As a further improvement of this application, the photocrosslinked hydrogel matrix is ​​formed by photocrosslinking and curing of a photocrosslinked polymer matrix; the photocrosslinked polymer matrix is ​​one or more of the following: methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide silk fibroin, and methacrylamide collagen.

[0011] As a further improvement of this application, the diameter of the inner ring axially penetrating small-diameter channel is 20~80μm; the diameter of the outer ring axially penetrating large-diameter channel is 100~200μm.

[0012] As a further improvement to this application, the mesenchymal stem cells are one or more selected from bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, and umbilical cord mesenchymal stem cells; the seeding volume of the mesenchymal stem cells is 50~500μL, and the seeding density is 1×10⁻⁶. 5 ~1×10 8 cells / mL.

[0013] Secondly, this application provides a method for preparing a neural graft with microenvironment regulation function as described in the first aspect, comprising the following steps: S1. Combine multifilament and monofilament in a predetermined ratio and prepare a tubular skeleton with a hollow inner cavity through a three-dimensional weaving process; S2. Disperse black phosphorus two-dimensional nanosheets in a solvent, add a metal salt solution, and react to obtain metal ion-modified black phosphorus two-dimensional nanosheets. S3. Mix the metal ion-modified black phosphorus two-dimensional nanosheets with a photocrosslinked polymer matrix to prepare a conductive hydrogel precursor solution; S4. The conductive hydrogel precursor solution is injected into the inner cavity of the tubular skeleton and cured by photocrosslinking to form a conductive hydrogel core; S5. The conductive hydrogel core is subjected to directional freezing and freeze-drying treatment to form an inner ring axially penetrating small-diameter channel and an outer ring axially penetrating large-diameter channel. S6. After the conductive hydrogel core is porous, mesenchymal stem cells are seeded into the axially penetrating small-diameter pores in the inner ring to obtain a neural graft with microenvironment regulation function.

[0014] As a further improvement of this application, the content of the metal ion modified black phosphorus two-dimensional nanosheets in the conductive hydrogel precursor solution is 0.01~2 mg / mL.

[0015] Thirdly, this application provides the use of the neural graft with microenvironment regulation function as described in the first aspect in the preparation of implantable materials, medical devices or tissue-engineered neural substitutes for repairing peripheral nerve defects.

[0016] The beneficial effects of this application are as follows: This application provides a neural graft with microenvironment regulation function, its preparation method, and its application. The neural graft includes an outer mechanical enhancement region and an inner conductive regulation region. The outer mechanical enhancement region is a tubular framework formed by multifilaments and monofilaments through a three-dimensional weaving process. The inner conductive regulation region is a conductive hydrogel core disposed within the cavity of the tubular framework. The conductive hydrogel core has an inner ring of axially penetrating small-diameter channels and an outer ring of axially penetrating large-diameter channels. The inner ring of axially penetrating small-diameter channels is used for nerve cell migration and axonal directional growth, while the outer ring of axially penetrating large-diameter channels is used for vascular endothelial cell migration and capillary ingrowth. Mesenchymal stem cells are seeded in the inner ring of axially penetrating small-diameter channels. The conductive hydrogel core includes a photocrosslinked hydrogel matrix and metal ion-modified black phosphorus two-dimensional nanosheets dispersed in the photocrosslinked hydrogel matrix. This application constructs a gradient mechanical three-dimensional braided tubular skeleton with synergistic multifilament and monofilament structures. The multifilaments endow the catheter with flexibility, bending adaptability, and suture maneuverability, while the monofilaments provide radial support, anti-collapse properties, and reversible resilience, thus overcoming the problem that traditional homogeneous catheters cannot simultaneously achieve both flexibility and mechanical support. This gradient mechanical structure can maintain a stable and open lumen structure after implantation and adapt to the stretching, bending, and compression effects in the limb movement environment. The outer braided fabric has a porous structure, which facilitates the transport of nutrients into the catheter interior, providing sufficient nutritional support for early nerve regeneration.

[0017] This application employs metal ion modification of black phosphorus two-dimensional nanosheets to construct a degradable conductive control region. The black phosphorus two-dimensional nanosheets serve as a degradable conductive framework, avoiding the long-term residue problem of traditional non-degradable conductive materials. Metal ions bind to the wrinkled structure, edge defect sites, and / or phosphorus atom coordination sites on the surface of the black phosphorus two-dimensional nanosheets through electrostatic adsorption, coordination, or ion complexation, which can improve the dispersion stability and antioxidant stability of the black phosphorus two-dimensional nanosheets. At the same time, during the degradation process, bioactive ions such as magnesium, copper, strontium, and cerium are released, achieving synergistic regulation of conductivity and ion microenvironment.

[0018] This application constructs a multi-scale axially perforated channel that combines central neural guidance with peripheral vascularization support. The inner ring of small-diameter axially perforated channels is mainly distributed in the central region of the catheter, facilitating Schwann cell migration, mesenchymal stem cell engraftment, and axonal directional extension. The outer ring of large-diameter axially perforated channels is mainly distributed in the peripheral region near the catheter wall, promoting the entry of host blood vessels, endothelial cells, and nutrients into the catheter from the outside in. This structure solves the problem that traditional single-pore or disordered porous structures cannot simultaneously provide both neural guidance and vascularization support.

[0019] This application employs a precise cell seeding strategy after pore formation. Compared to directly mixing cells into the hydrogel precursor and then crosslinking or freeze-drying to form pores, this application seeds mesenchymal stem cells into the inner ring of small-diameter axially interconnected pores after the conductive hydrogel core is formed and axially interconnected channels are created. This avoids the adverse effects of material mixing, photocrosslinking, nanomaterial dispersion, and freeze-drying processes on cells, improves cell viability retention, and enables spatial positioning of cells in neural regeneration channels.

[0020] This application constructs a dynamic neural regeneration microenvironment through the synergistic effects of multiple factors, including mechanics, conductivity, ion release, pore guidance, and cell loading. The outer tubular framework provides structural support and mechanical adaptation, while the inner conductive hydrogel core provides electroactive and bioactive ion release. The central-peripheral zoned pores support axon guidance and vascularization ingrowth, respectively. Subsequent inoculation with mesenchymal stem cells provides neurotrophic and paracrine regulatory effects, thereby promoting axon regeneration, myelination, vascularization reconstruction, and functional recovery after peripheral nerve loss.

[0021] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in this application will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0023] Figure 1 This is a schematic diagram of the structure of a neural graft with microenvironment regulation function provided in the embodiments of this application; Figure 2 This is a transmission electron microscope (TEM) image of magnesium ion-modified black phosphorus two-dimensional nanosheets in Example 1 of this application. Figure 3 This is the XPS energy spectrum of the magnesium ion-modified black phosphorus two-dimensional nanosheets in Example 1 of this application; Figure 4 The degradability of magnesium ion-modified black phosphorus two-dimensional nanosheets in Example 1 of this application; Figure 5 Conductivity test of the conductive hydrogel core in Example 1 of this application; Figure 6 This is a staining image showing the growth morphology of intermediate mesenchymal stem cells seeded on the surface of a conductive hydrogel in Example 1 of this application. Figure 7This is a diagram illustrating how a nerve graft in Embodiment 1 of this application promotes axonal extension and tissue regeneration during peripheral nerve defect repair. Figure labeling: 1. Outer mechanical enhancement region; 2. Inner conductivity regulation region; 3. Metal ion modified black phosphorus two-dimensional nanosheets; 4. Inner ring axially penetrating small-diameter channels; 5. Outer ring axially penetrating large-diameter channels; 6. Mesenchymal stem cells. Detailed Implementation

[0024] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0026] In the description of the embodiments of this application, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, "multiple" means two or more, unless otherwise explicitly defined.

[0027] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0028] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0029] In the description of the embodiments of this application, the term "multiple" refers to two or more (including two), similarly, "multiple sets" refers to two or more (including two sets), and "multiple pieces" refers to two or more (including two pieces).

[0030] Existing artificial nerve conduits suffer from the following shortcomings: the outer support structure is mostly homogeneous, making it difficult to simultaneously achieve flexibility, radial support, anti-collapse, and rebound buffering; conductive materials often suffer from non-degradability, long-term residue, or single-function issues; while black phosphorus two-dimensional nanosheets have the advantage of being degradable and conductive, their stability still needs improvement; the porous structure inside the conduit lacks spatial partitioning design for central nerve guidance and peripheral vascularization support; and cell loading methods are mostly direct embedding, which easily causes cell damage and is not precise in spatial positioning. Therefore, there is an urgent need to develop a novel electroactive nerve graft that can simultaneously achieve gradient mechanical support, degradable conductive regulation, release of bioactive ions, central axial nerve guidance, peripheral vascularization support, and precise cell loading after pore formation, in order to improve the repair effect of peripheral nerve defects.

[0031] To address the technical challenges of existing neural grafts, such as difficulty in simultaneously achieving flexibility and radial support, non-degradable or easily degradable conductive components, lack of spatial partitioning design for neural guidance and vascularization, and susceptibility to damage and uncontrollable spatial distribution during cell loading, this application provides a neural graft with microenvironment regulation function, its preparation method, and its application. Specifically, by constructing a peripheral nerve repair system that combines mechanical support, axial guidance, conductivity regulation, and cell delivery functions, the repair effect of peripheral nerve defects is improved.

[0032] Please see Figure 1 In a first aspect, embodiments of this application provide a neural graft with microenvironment regulation function, including an outer mechanical enhancement region 1 and an inner conductive regulation region 2; The outer mechanical reinforcement zone 1 is a tubular skeleton formed by multifilaments and monofilaments through a three-dimensional braiding process. The multifilaments are used to impart flexibility, bending adaptability and suturing operability to the catheter, while the monofilaments are used to provide radial support, anti-collapse performance and reversible elasticity. The inner conductive control region 2 is a conductive hydrogel core disposed within the lumen of the tubular skeleton. The conductive hydrogel core has an inner ring of axially penetrating small-diameter channels 4 and an outer ring of axially penetrating large-diameter channels 5. The inner ring of axially penetrating small-diameter channels 4 is used for nerve cell migration and axonal directional growth, while the outer ring of axially penetrating large-diameter channels 5 is used for vascular endothelial cell migration and capillary ingrowth. Mesenchymal stem cells 6 are seeded in the inner ring of axially penetrating small-diameter channels 4. The inner ring of axially penetrating small-diameter channels 4 is distributed in the central region of the catheter, while the outer ring of axially penetrating large-diameter channels 5 is distributed in the peripheral region near the catheter wall. The conductive hydrogel core comprises a photocrosslinked hydrogel matrix and metal ion-modified black phosphorus two-dimensional nanosheets dispersed in the photocrosslinked hydrogel matrix.

[0033] This application utilizes a three-dimensional weaving technique involving multifilaments and monofilaments to form a tubular framework with gradient mechanical properties. The inner cavity is filled with a conductive hydrogel core composed of metal ion-modified black phosphorus two-dimensional nanosheets and a photocrosslinked hydrogel matrix. A directional cryopreservation technique is used to construct inner-ring small-diameter axially permeable channels and outer-ring large-diameter axially permeable channels within the core. Mesenchymal stem cells are then seeded into the inner-ring small-diameter channels. This achieves a synergistic effect between the outer layer's mechanical support and the inner layer's conductive microenvironment, a synergistic combination of axonal directional guidance in the central region and vascularization support in the peripheral region, and precise cell loading after pore formation to maintain high cell viability.

[0034] Further, in some embodiments, the multifilament is one or more of polylactic acid multifilament, polycaprolactone multifilament, polylactic acid-glycolic acid copolymer multifilament, silk fibroin multifilament, and collagen multifilament; the monofilament is one or more of poly(p-dioxanone) monofilament, polylactic acid monofilament, polycaprolactone monofilament, and polylactic acid-caprolactone copolymer monofilament. The multifilament and the monofilament are combined and woven in a ratio of (2~5):1; the three-dimensional weaving process is one of spiral weaving, radial interlacing, and spiral-radial composite weaving.

[0035] In the technical solution of this application embodiment, by controlling the ratio of multifilaments to monofilaments and adopting a composite braided structure, a gradient mechanical distribution can be formed in the axial and circumferential directions of the catheter. The area dominated by multifilaments provides compliant deformation capability, while the area dominated by monofilaments provides compressive support. The two work together to enable the nerve graft to adapt to the dynamic mechanical environment of the limb after implantation and to maintain an open lumen for a long time. At the same time, the inherent porous structure of the braid allows tissue fluid and nutrients to permeate from the outside to the inside in the early stage, providing sufficient nutritional support for the cells and regenerating nerves in the inner conductive hydrogel. In addition, the interfacial mechanical constraint between the braided skeleton and the inner conductive hydrogel can further enhance the mechanical-electrical coupling effect between the two when the catheter undergoes slight compression / stretch deformation, thereby improving the efficiency of electrical signal transmission.

[0036] Furthermore, in some embodiments, the metal ions in the metal ion-modified black phosphorus two-dimensional nanosheets 3 are one or more of magnesium ions, copper ions, zinc ions, strontium ions, manganese ions, lithium ions, cobalt ions, and cerium ions.

[0037] In the technical solution of this application embodiment, metal ions are bound to the surface wrinkled structure, edge defect sites or phosphorus atom coordination sites of black phosphorus two-dimensional nanosheets through electrostatic adsorption, coordination or ion complexation, thereby improving the antioxidant stability of black phosphorus two-dimensional nanosheets in hydrogel matrix, and realizing the controllable release of bioactive ions as the black phosphorus two-dimensional nanosheets degrade after implantation.

[0038] Furthermore, in some embodiments, the photocrosslinked hydrogel matrix is ​​formed by photocrosslinking and curing of a photocrosslinked polymer matrix; the photocrosslinked polymer matrix is ​​one or more of methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide silk fibroin, and methacrylamide collagen.

[0039] In the technical solution of this application embodiment, by selecting one or more of the above-mentioned matrix combinations, the mechanical strength, degradation rate, pore structure, and bioactivity of the conductive hydrogel core can be flexibly controlled according to the specific type of nerve defect and repair needs. This allows for synergistic matching with the gradient mechanical properties of the woven shell and the conductive / ion-releasing function of the black phosphorus nanosheets, jointly constructing an ideal microenvironment for nerve regeneration. The conductive hydrogel core is formed through cross-linking under ultraviolet or visible light.

[0040] Furthermore, in some embodiments, the diameter of the inner ring axially penetrating small-diameter channel 4 is 20~80μm; the diameter of the outer ring axially penetrating large-diameter channel 5 is 100~200μm.

[0041] In the technical solution of this application embodiment, by constructing a partitioned structure of small-diameter guiding channels in the central region and large-diameter vascularized channels in the peripheral region within the same hydrogel core, the neural graft can simultaneously support axonal directional regeneration and vascularization reconstruction in space, resolving the contradiction that traditional single-pore or random porous structures cannot simultaneously address nerve guidance and nutrient exchange. Furthermore, these multi-scale channels are all axially connected along the catheter, significantly reducing the resistance to axial migration of endogenous cells and tissue fluid, accelerating the integration of the host-graft interface, and forming an interconnected material exchange network with the porous structure of the outer woven skeleton, providing continuous nutritional support and metabolic pathways for the repair of long-segment nerve defects. Specifically, the number of axially connected small-diameter channels 4 in the inner ring is 1-5, and the number of axially connected large-diameter channels 5 in the outer ring is 3-12. Through this center-periphery partitioned channel structure, an axial nerve regeneration channel can be formed in the central region of the catheter, and a vascularization and nutrient exchange channel can be formed in the peripheral region, thereby constructing a regenerative microenvironment where central nerve guidance and peripheral vascularization support work synergistically.

[0042] Furthermore, in some embodiments, the mesenchymal stem cells 6 are one or more of bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, and umbilical cord mesenchymal stem cells; the seeding volume of the mesenchymal stem cells 6 is 50~500μL, and the seeding density is 1×10⁶. 5 ~1×10 8 cells / mL.

[0043] In the technical solution of this application embodiment, mesenchymal stem cells 6 are seeded into the inner ring axially penetrating small-diameter channels 4 by dripping, perfusion, micro-injection, or negative pressure aspiration, rather than being pre-mixed into the conductive hydrogel precursor. This method allows cells to be introduced after the conductive hydrogel molding and pore formation are completed, thereby reducing the adverse effects on cells during material preparation, photocrosslinking, and cryopreservation, and improving cell viability retention, spatial distribution uniformity, and subsequent adhesion and extension capabilities. Through synergistic optimization of cell type, seeding volume, and density, a stable, efficient, and controllable source of bioactivity is provided for the nerve graft, significantly enhancing its repair capacity for long-segment peripheral nerve defects. Preferably, the seeding volume of mesenchymal stem cells 6 is 100~300 μL, and the seeding density is 1×10⁶. 6 ~1×10 7 cells / mL.

[0044] Secondly, embodiments of this application provide a method for preparing a neural graft with microenvironment regulation function as described in the first aspect, comprising the following steps: S1. Combine multifilament and monofilament in a predetermined ratio and prepare a tubular skeleton with a hollow inner cavity through a three-dimensional weaving process; Specifically, the tubular skeleton has an inner diameter of 1-5 mm, preferably 1.5-3 mm; a length of 5-50 mm, preferably 10-20 mm; and a wall thickness of 0.1-1.5 mm, preferably 0.2-0.8 mm. The tubular skeleton has a porous woven structure, and its pores are conducive to nutrient exchange, tissue fluid infiltration, and peripheral vascularization support. S2. Disperse black phosphorus two-dimensional nanosheets in a solvent, add a metal salt solution, and react to obtain metal ion-modified black phosphorus two-dimensional nanosheets 3; Specifically, the solvent is deionized water, phosphate buffer, or weakly acidic buffer. Black phosphorus nanosheets are dispersed by ultrasonication to form a dispersion, wherein the concentration of the two-dimensional black phosphorus nanosheets is 0.05–2 mg / mL, preferably 0.1–1 mg / mL. The metal salt can be one or more of magnesium chloride, magnesium sulfate, magnesium acetate, copper chloride, copper sulfate, strontium chloride, strontium nitrate, cerium chloride, and cerium nitrate. The metal salt solution is added to achieve a final concentration of 0.1–10 mmol / L, preferably 0.5–5 mmol / L. The reaction is carried out under light-protected, inert gas-protected, or low-oxygen conditions with stirring for 0.5–12 h, preferably 2–6 h. After the reaction is complete, unbound metal ions are removed by centrifugation and washing, and the precipitate is redispersed in deionized water or buffer for later use. In one specific embodiment, cerium ions can be used alone or in combination with magnesium ions, copper ions, and strontium ions to modify black phosphorus two-dimensional nanosheets, so as to further improve the antioxidant stability of black phosphorus two-dimensional nanosheets and endow them with the ability to regulate the redox microenvironment. S3. Metal ion-modified black phosphorus two-dimensional nanosheets 3 were mixed with a photocrosslinked polymer matrix to prepare a conductive hydrogel precursor solution; Specifically, a photocrosslinked polymer matrix is ​​dissolved in phosphate buffer to prepare a photocrosslinked polymer solution with a concentration of 5-15% w / v. A photoinitiator is then added to the solution to obtain a basic hydrogel precursor solution. The photoinitiator can be LAP, Irgacure 2959, riboflavin system, or other biocompatible photoinitiating systems. Preferably, LAP is used as the photoinitiator, with a concentration of 0.01-0.5% w / v, and more preferably 0.05-0.25% w / v. Metal ion-modified black phosphorus two-dimensional nanosheets 3 are added to the basic hydrogel precursor solution and dispersed uniformly by gentle vortexing, low-power ultrasound, or mechanical stirring to obtain a conductive hydrogel precursor solution. This conductive hydrogel precursor solution can rapidly crosslink under light irradiation to form a conductive hydrogel core. The metal ion-modified black phosphorus two-dimensional nanosheets 3 are dispersed in the hydrogel network, which can impart conductivity to the hydrogel.

[0045] S4. Inject the conductive hydrogel precursor solution into the inner cavity of the tubular skeleton, and solidify it through photocrosslinking to form a conductive hydrogel core; Specifically, ultraviolet or visible light irradiation is used to cause the conductive hydrogel precursor solution to crosslink and solidify in situ within the tubular framework cavity, forming a conductive hydrogel core; the irradiation wavelength is 365~450nm, preferably 405nm; the irradiation intensity is 1~50mW / cm². 2 Preferably, it is 5~20mW / cm 2 The illumination time is 5~300s, preferably 20~120s; S5. The conductive hydrogel core is subjected to directional freezing and freeze-drying treatment to form an inner ring axially penetrating small-diameter channel 4 and an outer ring axially penetrating large-diameter channel 5. Specifically, the directional freezing device includes a substrate with a metal cold head, a liquid nitrogen cold source, a low-temperature metal plate or a programmed cooling device. By transferring cold energy along the axial direction of the conduit, ice crystals are induced to grow axially in the conductive hydrogel core. Subsequently, freeze-drying is performed for 24-72 hours to remove the ice crystal template, forming multi-scale through-holes extending along the axial direction of the conduit. By controlling the freezing direction, cooling rate, shape, number, diameter, arrangement, solid content of hydrogel and content of nanosheets, the inner ring axially through-holes with small diameter 4 are mainly formed in the central region of the conduit, and the outer ring axially through-holes with large diameter 5 are mainly formed in the outer peripheral region near the conduit wall, forming a multi-scale channel structure with a center-periphery partition. S6. After forming pores in the conductive hydrogel core, mesenchymal stem cells 6 are seeded into the inner ring axially penetrating small-diameter channels 4 to obtain a neural graft with microenvironment regulation function.

[0046] Specifically, mesenchymal stem cells 6 were expanded to passages 3-5. After the cells reached a suitable degree of fusion, they were digested with trypsin or other cell digestion solutions, collected by centrifugation, and resuspended in culture medium, physiological saline, PBS (phosphate buffered saline), or a cell suspension containing a small amount of hydrogel matrix. The cell suspension was seeded into the axially penetrating small-diameter channels 4 of the inner ring of the conductive hydrogel core by dripping, perfusion, microinjection, or negative pressure aspiration. The cells were then statically cultured for 2-6 hours under suitable conditions to allow the cells to fully adhere to the inner wall of the channels. Subsequently, culture medium was added and cultured for a further period of time to obtain electrically active neural grafts loaded with mesenchymal stem cells 6.

[0047] Compared with the method of directly mixing cells into the hydrogel precursor and then performing photocrosslinking and cryopreservation, this application adopts a precise cell seeding method after pore formation, which can avoid the adverse effects of nanomaterial dispersion, photocrosslinking and low temperature pore formation on cells, improve cell viability retention rate, and enable cells to be mainly located in the central nerve guide channel of the duct, forming a spatial partitioning synergy with the peripheral vascularized support channel.

[0048] Furthermore, in some embodiments, the content of metal ion modified black phosphorus two-dimensional nanosheets 3 in the conductive hydrogel precursor solution is 0.01~2 mg / mL.

[0049] In the technical solution of this application embodiment, controlling the content of metal ion-modified black phosphorus two-dimensional nanosheets 3 within a suitable range can ensure that the conductive hydrogel core has sufficient conductivity and controllable release capability of bioactive ions, while maintaining good photocrosslinking molding properties, suitable degradation rate, uniform porous structure, and excellent cell compatibility, providing a stable, safe, and efficient electroactive microenvironment for the repair of long-segment peripheral nerve defects. Preferably, the content of metal ion-modified black phosphorus two-dimensional nanosheets 3 in the conductive hydrogel precursor solution is 0.05~1 mg / mL.

[0050] Thirdly, embodiments of this application provide the application of a neural graft with microenvironment regulation function in the preparation of implantable materials, medical devices, or tissue-engineered neural substitutes for repairing peripheral nerve defects.

[0051] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0052] I. Preparation Method Example 1 This embodiment provides a method for preparing a neural graft with microenvironment regulation function, including the following steps: S1. Polylactic acid multifilaments (100D / 48F) and polydioxanone monofilaments (0.15mm diameter) are mixed and braided at a ratio of 3:1. During the braiding process, the polylactic acid multifilaments are distributed radially to improve the overall flexibility and bending adaptability of the catheter; the polydioxanone monofilaments are distributed helically to improve the radial support strength and anti-collapse performance of the catheter. The resulting tubular skeleton has an inner diameter of 2mm, a length of 15mm, and a wall thickness of 0.5mm. S2. Disperse black phosphorus two-dimensional nanosheets in deionized water to prepare a black phosphorus nanosheet dispersion with a concentration of 0.5 mg / mL. Add magnesium chloride solution to the dispersion to make the final magnesium ion concentration 3 mmol / L. Stir and react for 4 h under light protection and nitrogen protection. After the reaction is completed, remove unbound metal ions by centrifugation and wash, and redisperse the precipitate in deionized water to obtain a magnesium ion modified black phosphorus two-dimensional nanosheet dispersion. Magnesium ion-modified black phosphorus two-dimensional nanosheets were characterized using transmission electron microscopy. Figure 2 (a) shows the microstructure of unmodified black phosphorus two-dimensional nanosheets, and (b) shows the microstructure of magnesium ion-modified black phosphorus two-dimensional nanosheets. The two-dimensional sheet morphology of the nanosheets can be seen from the figure. Figure 3 Unmodified black phosphorus two-dimensional nanosheets (BP) and magnesium ion-modified black phosphorus two-dimensional nanosheets (BP@Mg) 2+ XPS spectra, combined with Figure 3 Energy dispersive spectroscopy analysis confirmed that magnesium ions were successfully bound to the surface of black phosphorus two-dimensional nanosheets, realizing the effective modification of black phosphorus nanosheets by metal ions. The effect of metal ion modification on the stability of black phosphorus two-dimensional nanosheets was evaluated using stability testing. Unmodified black phosphorus two-dimensional nanosheets (BP) and magnesium ion-modified black phosphorus two-dimensional nanosheets (BP@Mg) were compared. 2+ The phosphorus was dispersed separately in deionized water and left at room temperature for different times. The degree of degradation of black phosphorus was evaluated by ultraviolet absorption testing. The results are as follows: Figure 4 As shown, unmodified black phosphorus nanosheets are prone to oxidative degradation, leading to performance degradation. However, metal ion modification effectively inhibits this process and significantly improves the dispersion stability and antioxidant stability of black phosphorus two-dimensional nanosheets. S3. Dissolve methacrylated gelatin in phosphate buffer to prepare a 10% w / v methacrylated gelatin solution. Add photoinitiator LAP to the solution to obtain the basic hydrogel precursor solution with a LAP concentration of 0.1% w / v. Magnesium ion-modified black phosphorus two-dimensional nanosheet dispersion was added to the above basic hydrogel precursor solution to make the final concentration of black phosphorus nanosheets 0.5 mg / mL. The solution was ultrasonically treated for 5 min (power 100 W) to obtain a conductive hydrogel precursor solution. S4. Fix the tubular skeleton obtained in step S1 in the mold to maintain its hollow tubular structure. Inject the conductive hydrogel precursor solution into the inner cavity of the tubular skeleton to fully fill the entire inner cavity. Irradiate with a 405nm light source for 60s to crosslink the precursor solution in situ and form a conductive hydrogel core. The conductivity, impedance spectrum, and electrical signal transmission capability of the conductive hydrogel core were tested using an electrochemical workstation. The results are as follows: Figure 5 As shown, GelMA represents methacrylamide gelatin hydrogel without added conductive materials, GelMA / BP@Mg 2+ This indicates the addition of magnesium ion-modified black phosphorus two-dimensional nanosheets (BP@Mg). 2+ The conductive hydrogel shows that metal ion-modified black phosphorus two-dimensional nanosheets can form a conductive network in the hydrogel matrix, reduce the core impedance of the hydrogel, and improve the ability to transmit conductive signals. S5. Place the tubular framework containing the conductive hydrogel core on a plastic substrate with a metal cold head, so that the conductive hydrogel core is in contact with the metal cold head; then immerse the bottom of the substrate in liquid nitrogen, and transfer the low temperature along the axial direction of the tube through the metal cold head to form an axial ice crystal template inside the hydrogel; after freezing for 30 min, place the sample in a freeze dryer and freeze dry at -50℃ for 48 h to sublimate the ice crystals and form 4 inner ring axially penetrating small diameter channels 4 with a diameter of 30 μm, and 8 outer ring axially penetrating large diameter channels 5 with a diameter of 100 μm; S6. Take third-generation bone marrow mesenchymal stem cells and resuspend them in culture medium (αMEM medium + 10% fetal bovine serum) to prepare a density of 1×10⁻⁶. 7 A cell suspension of 200 μL was injected via microinjection into the inner ring of the small-diameter channel 4 in the central region of the duct. The cells were then incubated at 37°C and 5% CO2 for 4 h to allow them to adhere fully to the inner wall of the channel. Subsequently, culture medium was added and the cells were cultured for a longer period to obtain a neural graft with microenvironment regulation function.

[0053] The staining image shows the growth morphology of mesenchymal stem cells seeded on the surface of a conductive hydrogel. Figure 6 As shown, (a) is a staining image of the growth morphology of mesenchymal stem cells seeded on the surface of GelMA hydrogel, and (b) is a staining image of mesenchymal stem cells seeded on GelMA / BP@Mg 2+The staining images of the growth morphology on the surface of the conductive hydrogel show that the mesenchymal stem cells (MSCs) using the precise seeding strategy after pore formation maintained high activity. Moreover, compared with non-conductive hydrogels, conductive hydrogels can better promote the adhesion and spread of MSCs, demonstrating that the conductive microenvironment is beneficial to cell growth and functional expression.

[0054] Comparative Example 1 Comparative Example 1 provides a method for preparing a neural graft with microenvironment regulation function. The only difference from Example 1 is that only a braided hollow catheter is used and no conductive hydrogel core is injected. Other experimental parameters and conditions are basically the same as those in Example 1, and will not be repeated here.

[0055] Comparative Example 2 Comparative Example 2 provides a method for preparing a neural graft with microenvironment regulation function. The only difference from Example 1 is that mesenchymal stem cells were not inoculated. Other experimental parameters and conditions are basically the same as those in Example 1, and will not be repeated here.

[0056] Application examples The nerve grafts obtained in Example 1 and Comparative Examples 1 and 2 were used for nerve tissue repair to establish a sciatic nerve defect model in SD rats. After anesthesia, the sciatic nerve was exposed, and a certain length of nerve segment was removed to form a peripheral nerve defect. The obtained electroactive nerve grafts were implanted into the nerve defect site, with the proximal and distal nerve stumps inserted into the two ends of the conduit, respectively, and fixed with microsutures. The length of the nerve defect was 10 mm, and the length of the electroactive nerve graft was slightly longer than the length of the nerve defect to ensure that the nerve stump could be stably inserted into both ends of the conduit. Postoperative behavioral, electrophysiological, histological, and immunofluorescence evaluations were performed at different time points.

[0057] Immunofluorescence staining was performed on regenerated nerve tissue to detect neuroregeneration and myelin-related markers such as NF200 and MBP. Results are as follows: Figure 7 As shown, Comparative Example 1 (braided hollow catheter only) lacked conductive hydrogel support, resulting in lumen collapse and disordered axon growth; Comparative Example 2 (GelMA / BP@Mg 2+ Although the lumen structure was intact, the rate of nerve regeneration and the degree of myelination were lower than in Example 1 (GelMA / BP@Mg). 2+ +MSC), while the electroactive nerve graft provided in Example 1 can better maintain the opening of the catheter lumen, promote the orderly growth of axons along the axial direction of the catheter, promote the ingrowth of peripheral blood vessels into the catheter, and significantly improve the maturity and functional recovery level of regenerated nerve tissue.

[0058] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A neural graft with microenvironment regulation function, characterized in that, This includes an outer mechanical enhancement region and an inner conductivity control region; The outer mechanical reinforcement region is a tubular skeleton formed by multifilament and monofilament through a three-dimensional weaving process; the inner conductive control region is a conductive hydrogel core disposed in the inner cavity of the tubular skeleton. The conductive hydrogel core has an inner ring of axially penetrating small-diameter channels and an outer ring of axially penetrating large-diameter channels. The inner ring of axially penetrating small-diameter channels is used for nerve cell migration and axonal directional growth, while the outer ring of axially penetrating large-diameter channels is used for vascular endothelial cell migration and capillary ingrowth. Mesenchymal stem cells are seeded in the inner ring of axially penetrating small-diameter channels. The diameter of the inner ring of axially penetrating small-diameter channels is 20-80 μm, and the diameter of the outer ring of axially penetrating large-diameter channels is 100-200 μm. The conductive hydrogel core comprises a photocrosslinked hydrogel matrix and metal ion-modified black phosphorus two-dimensional nanosheets dispersed in the photocrosslinked hydrogel matrix; The method for preparing the neural graft with microenvironment regulation function includes the following steps: S1. Combine multifilaments and monofilaments in a ratio of (2~5):1 and prepare a tubular skeleton with a hollow inner cavity through a three-dimensional weaving process; S2. Disperse black phosphorus two-dimensional nanosheets in a solvent, add a metal salt solution, and react to obtain metal ion-modified black phosphorus two-dimensional nanosheets. S3. Mix the metal ion-modified black phosphorus two-dimensional nanosheets with a photocrosslinked polymer matrix to prepare a conductive hydrogel precursor solution; S4. The conductive hydrogel precursor solution is injected into the inner cavity of the tubular skeleton and cured by photocrosslinking to form a conductive hydrogel core; S5. Place the tubular skeleton containing the conductive hydrogel core on a plastic substrate with a metal cooling head, so that the conductive hydrogel core is in contact with the metal cooling head, and perform directional freezing and freeze-drying treatment on the conductive hydrogel core. By controlling the freezing direction, cooling rate, cooling head shape, number of cooling heads, cooling head diameter, cooling head arrangement, hydrogel solid content and nanosheet content, an inner ring axially penetrating small-diameter channel is formed in the central region of the conduit, and an outer ring axially penetrating large-diameter channel is formed in the outer peripheral region near the conduit wall. S6. After the conductive hydrogel core is porous, mesenchymal stem cells are seeded into the axially penetrating small-diameter pores in the inner ring to obtain a neural graft with microenvironment regulation function.

2. The neural graft with microenvironment regulation function according to claim 1, characterized in that, The multifilament is one or more of polylactic acid multifilament, polycaprolactone multifilament, polylactic acid-glycolic acid copolymer multifilament, silk fibroin multifilament, and collagen multifilament; The monofilament is one or more of the following: polydioxanone monofilament, polylactic acid monofilament, polycaprolactone monofilament, and polylactic acid-caprolactone copolymer monofilament.

3. The neural graft with microenvironment regulation function according to claim 1, characterized in that, The three-dimensional weaving process is one of spiral weaving, radial interlacing, or spiral-radial composite weaving.

4. The neural graft with microenvironment regulation function according to claim 1, characterized in that, The metal ions in the metal ion-modified black phosphorus two-dimensional nanosheets are one or more of magnesium ions, copper ions, zinc ions, strontium ions, manganese ions, lithium ions, cobalt ions, and cerium ions.

5. The neural graft with microenvironment regulation function according to claim 1, characterized in that, The photocrosslinked hydrogel matrix is ​​formed by photocrosslinking and curing a photocrosslinked polymer matrix; the photocrosslinked polymer matrix is ​​one or more of the following: methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide silk fibroin, and methacrylamide collagen.

6. The neural graft with microenvironment regulation function according to claim 1, characterized in that, The mesenchymal stem cells are one or more of bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, and umbilical cord mesenchymal stem cells; the seeding volume of the mesenchymal stem cells is 50-500 μL, and the seeding density is 1×10⁻⁶. 5 ~1×10 8 cells / mL.

7. The neural graft with microenvironment regulation function according to claim 1, characterized in that, The content of the metal ion modified black phosphorus two-dimensional nanosheets in the conductive hydrogel precursor solution is 0.01~2 mg / mL.

8. The use of the neural graft with microenvironment regulation function according to any one of claims 1 to 7 in the preparation of medical devices for repairing peripheral nerve defects.

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