A multi-gradient nanofiber membrane, a preparation method and application thereof

Abstract

The application belongs to the technical field of biological materials, and discloses a gradient nanofiber membrane as well as a preparation method and application thereof. The application provides a multi-gradient nanofiber membrane, which comprises an inner layer, a middle layer and an outer layer. The inner layer comprises a magnesium-containing compound and polycaprolactone with a mass ratio of (0.12-0.36):1. The middle layer comprises the magnesium-containing compound and the polycaprolactone with a mass ratio of (0.1-0.3):1. The outer layer comprises the magnesium-containing compound and the polycaprolactone with a mass ratio of (0.08-0.24):1. The nanofiber membrane or the multi-gradient nanofiber membrane has excellent biocompatibility, controllable mechanical properties and degradation characteristics. When applied to peripheral nerve defect repair, the nanofiber membrane or the multi-gradient nanofiber membrane has not only significant treatment potential, but also extremely high scientific research value. The application provides an important theoretical basis for popularization and application of the magnesium-containing nerve scaffold repair.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a multi-gradient nanofiber membrane, its preparation method, and its applications. Background Technology

[0002] Peripheral nerve defects refer to damage to the peripheral nerve trunk or its branches caused by accidental direct or indirect external trauma (such as trauma, tumors, and other factors), resulting in motor, sensory, and autonomic dysfunction of the trunk and limbs. Without effective treatment, it will affect the structural development of the affected limbs, thus impacting daily life, learning, and work, and also having a certain influence on psychological development. Peripheral nerve injury mainly manifests as motor dysfunction, sensory dysfunction, and neurotrophic changes. Surgical treatment is often required; some patients can be cured, but a small number suffer lifelong disability. Furthermore, peripheral nerve defects are common in young adults, leading to loss of labor capacity and high rehabilitation costs, resulting in significant economic losses for society and families.

[0003] Nerve defects typically require nerve transplantation or bridging with various biomaterials for repair. Autologous nerve transplantation is currently the gold standard in clinical treatment, involving the surgical use of a segment of nerve tissue to bridge the gap between the damaged nerve ends. Although autologous nerve transplantation remains the preferred clinical treatment option, the source of autologous nerves is limited. Harvesting autologous nerves not only increases surgical trauma but also causes complications such as surgical scarring, painful neuroma formation, and dysfunction in the nerve innervation area. Therefore, designing and fabricating novel nerve grafts and exploring their key mechanisms for promoting nerve regeneration are of significant clinical research importance for improving the repair effect of peripheral nerves. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a multi-gradient nanofiber membrane, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a multi-gradient nanofiber membrane comprising an inner layer, a middle layer, and an outer layer; the inner layer comprising a magnesium-containing compound and polycaprolactone in a mass ratio of (0.12-0.36):1; the middle layer comprising a magnesium-containing compound and polycaprolactone in a mass ratio of (0.1-0.3):1; and the outer layer comprising a magnesium-containing compound and polycaprolactone in a mass ratio of (0.08-0.24):1.

[0007] The multi-gradient nanofiber membrane of this invention is a three-layer structure. Its design takes into account various factors, reducing contaminants, controlling variables, and focusing on Mg. 2+Changes in downstream gene expression induced by sustained release. Multilayer nanofibers were fabricated using PCL mixed with different concentrations of magnesium-containing monomers. The weight ratio of MgO and MgCO3 in each layer gradually decreased from the inner to the outer layers. The composition of Mg monomers exhibited a layer-dependent variation, with the inner layer having the most MgCO3 and the outer layer the most MgO. The differences in both the concentration and composition of magnesium monomers ultimately resulted in a multi-gradient structure of the three-layer electrospun membrane, thus affecting the expression of Mg. 2+ Its high release efficiency and biocompatibility allow for sustained and effective release during treatment, promoting nerve regeneration. Furthermore, the effective controlled-release carrier optimizes its in vivo therapeutic effect. It can be applied to peripheral nerve injury repair and is safe and effective, acting not only in the early stages of nerve regeneration but also influencing nerve repair in the middle and late stages.

[0008] Furthermore, the MgO / MgCO3 / PCL multigradient nanofiber membrane of the present invention helps to improve the mechanical properties of implants, mimics the environment of natural neural tissue, and optimizes the migration and growth of nerve cells.

[0009] In a preferred embodiment of the multi-gradient nanofiber membrane of the present invention, the magnesium-containing compound includes MgO and MgCO3.

[0010] As a further preferred embodiment of the multi-gradient nanofiber membrane of the present invention, the mass ratio of MgO to MgCO3 in the inner layer is (0.3-0.7):1; the mass ratio of MgO to MgCO3 in the middle layer is (0.8-1.4):1; and the mass ratio of MgO to MgCO3 in the outer layer is (1.5-2.5):1.

[0011] Preferably, the mass ratio of MgO to MgCO3 in the inner layer is 0.5:1; the mass ratio of MgO to MgCO3 in the middle layer is 1:1; and the mass ratio of MgO to MgCO3 in the outer layer is 2:1.

[0012] In a preferred embodiment of the multi-gradient nanofiber membrane of the present invention, the mass percentage of magnesium in the inner layer is 12.57%-24.54%; the mass percentage of magnesium in the middle layer is 11.42%-20.46%; and the mass percentage of magnesium in the outer layer is 9.43%-19.63%.

[0013] Secondly, the present invention provides a method for preparing the multi-gradient nanofiber membrane, comprising the following steps:

[0014] (1) Add the polycaprolactone to the solvent and stir to obtain a mixed solution;

[0015] (2) Add the magnesium-containing compounds to the inner, middle and outer layers respectively, stir evenly, and obtain the electrospinning solutions of the inner, middle and outer layers respectively;

[0016] (3) Electrospinning is performed using the obtained inner, middle and outer electrospinning solutions. The electrospinning is stacked layer by layer to form a three-layer composite electrospinning membrane, which is a multi-gradient nanofiber membrane.

[0017] The preparation method of this invention utilizes electrospinning technology to prepare a product with long-lasting sustained-release Mg 2+ Single-channel nerve conduits utilize multi-gradient nanofiber membranes, employing rational and effective Mg... 2+ The sustained-release system regulates the local neural regeneration microenvironment, promoting the transformation of nerve cells (SCs) into a repair phenotype to repair peripheral nerve defects. It can achieve 4-6 weeks of effective Mg treatment. 2+ Slow-release.

[0018] In a preferred embodiment of the preparation method of the present invention, in step (1), the solvent is trifluoroethanol; the mass-to-volume ratio of polycaprolactone to the solvent is 5%-15% g / mL.

[0019] As a preferred embodiment of the preparation method described in this invention, in step (3), the electrospinning adopts a high voltage electric field of 10kV-20kV.

[0020] Thirdly, the present invention provides a nerve conduit in which the multi-gradient nanofiber membrane is embedded.

[0021] In a preferred embodiment of the nerve conduit of the present invention, the inner layer of the multi-gradient nanofiber membrane faces the inner wall of the nerve conduit.

[0022] Fourthly, the present invention includes the use of the multi-gradient nanofiber membrane and the nerve conduit in any of the following fields:

[0023] i. Preparation of agents that promote nerve regeneration;

[0024] ii. Preparation of nerve conduits that promote nerve regeneration;

[0025] iii. Preparation of formulations for the repair of peripheral nerve defects;

[0026] iv. Preparation of nerve conduits for peripheral nerve defect repair;

[0027] v. Treatment of peripheral nerve defects for purposes other than diagnosis or treatment of disease.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. The nanofiber membrane or multi-gradient nanofiber membrane of this invention possesses excellent biocompatibility, controllable mechanical properties, and degradation characteristics. Its application in peripheral nerve defect repair not only demonstrates significant therapeutic potential but also exhibits extremely high scientific research value. It provides important theoretical basis for the widespread application of magnesium-containing nerve scaffold repair.

[0030] 2. Addressing the limitations and complications of autologous nerves and magnesium-containing nerve scaffolds, this invention solves the following technical problems: 1) Source limitations: The preparation method of this invention is simple, low-cost, uses abundant raw materials, is highly efficient, mass-producible, widely adaptable, and highly compatible; 2) Surgical trauma and complications: This invention eliminates the need for additional surgical trauma, postoperative pain, donor site scarring, painful neuroma formation, and dysfunction in the nerve innervation area; 3) Mg 2+ Long-lasting sustained release: This invention can achieve effective Mg release for 4-6 weeks depending on the magnesium concentration. 2+ Slow-release.

[0031] 3. This invention applied this nerve conduit to repair a 10mm sciatic nerve defect in rats, and high expression of the SCs phenotypic conversion characteristic gene C-Jun was detected. Compared with autologous nerve repair, it significantly promoted axonal myelination and muscle tissue re-innervation. In vivo and in vitro studies elucidated the role of Mg... 2+ This invention investigates the role of Schwann cell phenotypic transformation during nerve regeneration. By comprehensively utilizing transcriptome sequencing and single-cell sequencing to detect key upstream and downstream genes, it screens specific cell types and key signaling pathways that respond to magnesium-containing scaffold materials during peripheral nerve regeneration, revealing the molecular mechanism by which magnesium-containing scaffold materials promote peripheral nerve repair. This invention is methodologically innovative, fully exploring the role of magnesium in Schwann cell phenotypic transformation. 2+ The regulation of various key cells during nerve regeneration provides an important theoretical basis for the clinical translation of magnesium-containing biomaterials for repair. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the technical route of the present invention.

[0033] Figure 2 shows the scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDXPS) analyses of the MgO / MgCO3 / PCL film; in the figure, Figure 2A SEM images showing the vertical structural features of the MgO / MgCO3 / PCL film; Figure 2B C and D represent the EDS analysis and mapping of the inner, middle and outer layers of the MgO / MgCO3 / PCL multi-gradient nanofiber membranes in Examples 1-3, respectively, confirming the existence of the multi-gradient structure and showing a trend of gradually decreasing Mg element ratio from the inner layer to the outer layer.

[0034] Figure 3 shows the fabrication and characterization of the MgO / MgCO3 / PCL multi-gradient nanofiber membrane; in the figure, Figure 3A A schematic diagram of the fabrication process of MgO / MgCO3 / PCL multigradient nanofiber membrane; Figure 3B The cumulative release of Mg from the MgO / MgCO3 / PCL multi-gradient nanofiber membrane over 6 weeks 2+ ; Figure 3C SEM characterization of the MgO / MgCO3 / PCL multigradient nanofiber membrane; Figure 3D To calculate the diameter of the nanofibers; Figure 3E To measure the contact angle of MgO / MgCO3 / PCL multigradient nanofiber membrane.

[0035] Figure 4 shows the H&E analysis and remyelination of regenerated nerves; in the figure, Figure 4A This is a diagram illustrating animal surgery. Figure 4B For the harvested regenerated nerves; Figure 4C H&E staining for autologous transplantation, PCL nerve conduit, 10% MgO / MgCO3 / PCL, 20% MgO / MgCO3 / PCL and 30% MgO / MgCO3 / PCL groups; Figure 4D The diameter (μm) of the regenerated nerve (n=4); Figure 4E Toluidine blue staining and TEM analysis of transverse sections of the graft harvested 12 weeks post-surgery and remyelinated axons; Figure 4F The diameter of the myelinated axon (μm) (n=5); Figure 4G The number of myelinated axons (n=5); Figure 4H The thickness of the newly formed myelin sheath (μm) (n=5).

[0036] Figure 5 shows the functional recovery analysis, assessed using SFI values, electrophysiological evaluation, and Masson staining of cross-sectional samples of the triceps surae muscle; in the figure, Figure 5A A schematic diagram for calculating SFI values; Figure 5B The SFI values ​​of all groups gradually increased over time, indicating partial functional recovery at 6 and 12 weeks postoperatively (n=5). Figure 5C The CAMP level for the five groups 12 weeks post-surgery; Figure 5D CAMP amplitude (unit: mV) (n=4); Figure 5E Masson staining of a calf muscle sample from the 3rd cephalic version 12 years post-surgery; Figure 5F This represents the cross-sectional area of ​​the muscle fiber. Figure 5G This represents the cross-sectional area of ​​the collagen fiber. Figure 5H This represents the percentage of collagen fiber area (n=4); Figure 5I The value is the G ratio; data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA and Tukey's test. Detailed Implementation

[0037] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0038] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. Data were analyzed using one-way ANOVA and Tukey post-test, and are expressed as mean ± SD. *P<0.05; **P<0.01.

[0039] Example 1: Long-lasting sustained-release Mg 2+ Preparation of electrospun membranes

[0040] Multilayer nanofiber ducts were fabricated using electrospinning technology with polycaprolactone (PCL) (catalog number 178301000, Thermofisher) mixed with different concentrations of magnesium-containing monomers (MgO (catalog number 529699, Sigma) and MgCO3 (catalog number 63032, Sigma)). The specific steps are as follows:

[0041] (1) Add 3g of polycaprolactone to 30mL of trifluoroethanol solvent and stir to obtain a mixed solution with a mass-volume ratio of 10%.

[0042] (2) Add magnesium-containing monomers accounting for 10% of the mass percentage of polycaprolactone, stir evenly, and obtain the middle layer of polycaprolactone magnesium-containing monomer electrospinning solution.

[0043] (3) Prepare electrospinning solutions of magnesium-containing polycaprolactone monomers for the inner and outer layers respectively;

[0044] Compared to the intermediate layer, the magnesium monomer concentration in the inner and outer layers increases by 20% (magnesium monomer accounts for 12% of the mass of polycaprolactone) and decreases by 20% (magnesium monomer accounts for 8% of the mass of polycaprolactone), respectively, resulting in a gradual decrease in the weight ratio of MgO and MgCO3 to PCL from the inner to the outer layers. Specifically, the mass ratio of MgO to MgCO3 changes layer by layer as follows: 1:2 in the inner layer, 1:1 in the intermediate layer, and 2:1 in the outer layer.

[0045] (4) Using a 15kV high voltage electric field, electrospinning was performed on the inner, middle and outer three-layer polycaprolactone magnesium monomer electrospinning solution (TL-01, Tongli Technologies Co., China). The electrospinning was stacked layer by layer to form a three-layer composite electrospinning membrane, which was denoted as 10% MgO / MgCO3 / PCL multigradient nanofiber membrane.

[0046] Example 2: Long-lasting sustained-release Mg 2+ Preparation of electrospun membranes

[0047] Multilayer nanofiber ducts were fabricated using electrospinning technology with polycaprolactone PCL (178301000, Thermofisher) mixed with different concentrations of magnesium-containing monomers (MgO (529699, Sigma) and MgCO3 (63032, Sigma)). The specific steps are as follows:

[0048] (1) Add 3g of polycaprolactone to 30mL of trifluoroethanol solvent and stir to obtain a mixed solution with a mass-volume ratio of 10%.

[0049] (2) Add magnesium-containing monomers accounting for 20% of the mass percentage of polycaprolactone, stir evenly, and obtain the middle layer of polycaprolactone magnesium-containing monomer electrospinning solution.

[0050] (3) Prepare electrospinning solutions of magnesium-containing polycaprolactone monomers for the inner and outer layers respectively;

[0051] Compared to the intermediate layer, the magnesium monomer concentration in the inner and outer layers increases by 20% (magnesium monomer accounts for 24% of the mass of polycaprolactone) and decreases by 20% (magnesium monomer accounts for 16% of the mass of polycaprolactone), respectively, resulting in a gradual decrease in the weight ratio of MgO and MgCO3 to PCL from the inner to the outer layers. Specifically, the mass ratio of MgO to MgCO3 changes layer by layer as follows: 1:2 in the inner layer, 1:1 in the intermediate layer, and 2:1 in the outer layer.

[0052] (4) Using a 15kV high voltage electric field, electrospinning was performed on the inner, middle and outer three-layer polycaprolactone magnesium monomer electrospinning solution (TL-01, Tongli Technologies Co., China). The electrospinning was stacked layer by layer to form a three-layer composite electrospinned membrane, which was denoted as 20% MgO / MgCO3 / PCL multigradient nanofiber membrane.

[0053] Example 3: Long-lasting sustained-release Mg 2+ Preparation of electrospun membranes

[0054] Multilayer nanofiber ducts were fabricated using electrospinning technology with polycaprolactone PCL (178301000, Thermofisher) mixed with different concentrations of magnesium-containing monomers (MgO (529699, Sigma) and MgCO3 (63032, Sigma)). The specific steps are as follows:

[0055] (1) Add 3g of polycaprolactone to 30mL of trifluoroethanol solvent and stir to obtain a mixed solution with a mass-volume ratio of 10%.

[0056] (2) Add magnesium-containing monomers accounting for 30% of the mass percentage of polycaprolactone, stir evenly, and obtain the middle layer of polycaprolactone magnesium-containing monomer electrospinning solution.

[0057] (3) Prepare electrospinning solutions of magnesium-containing polycaprolactone monomers for the inner and outer layers respectively;

[0058] Compared to the intermediate layer, the magnesium monomer concentration in the inner and outer layers increases by 20% (magnesium monomer accounts for 36% of the mass of polycaprolactone) and decreases by 20% (magnesium monomer accounts for 24% of the mass of polycaprolactone), respectively, resulting in a gradual decrease in the weight ratio of MgO and MgCO3 to PCL from the inner to the outer layers. Specifically, the mass ratio of MgO to MgCO3 changes layer by layer as follows: 1:2 in the inner layer, 1:1 in the intermediate layer, and 2:1 in the outer layer.

[0059] (4) Using a 15kV high voltage electric field, electrospinning was performed on the inner, middle and outer three-layer polycaprolactone magnesium monomer electrospinning solution (TL-01, Tongli Technologies Co., China). The electrospinning was stacked layer by layer to form a three-layer composite electrospinned membrane, which was denoted as 30% MgO / MgCO3 / PCL multigradient nanofiber membrane.

[0060] like Figure 2A As shown, multilayer nanofiber membranes were synthesized using PCL and magnesium monomers (MgO and MgCO3) of varying concentrations. A series of membranes composed of MgO / MgCO3 / PCL were created using electrospinning technology. Each membrane consisted of three layers, with the MgO / MgCO3 ratio decreasing progressively from the inner to the outer layer, and the Mg monomer concentration gradually decreasing from the inside to the outside. Notably, the three layers had different Mg monomer ratios, with the inner layer containing more MgCO3 and the outer layer containing more MgO, thus forming a multi-gradient structure. Figure 3A ).

[0061] Experimental Example 1: Evaluation of the performance of MgO / MgCO3 / PCL multigradient nanofiber membranes

[0062] The following groups were set up: PCL electrospun membrane as control group; 10% MgO / MgCO3 / PCL multigradient nanofiber membrane of Example 1 as 10% MgO / MgCO3 / PCL group; 20% MgO / MgCO3 / PCL multigradient nanofiber membrane of Example 2 as 20% MgO / MgCO3 / PCL group; and 30% MgO / MgCO3 / PCL multigradient nanofiber membrane of Example 3 as 30% MgO / MgCO3 / PCL group.

[0063] (1) Scanning electron microscopy and energy dispersive X-ray spectroscopy analysis of MgO / MgCO3 / PCL film.

[0064] Scanning electron microscopy (SEM) analysis revealed the morphology of an interconnected network composed of nanofibers, in which MgO / MgCO3 nanoparticles with multi-gradient concentrations were well distributed in a three-layer structure. Cross-sectional SEM images showed the tight integration of the three-layer structure. Figure 2A ).

[0065] Energy dispersive X-ray spectroscopy (EDS) analysis of different layers of each MgO / MgCO3 / PCL film confirmed the existence of a multi-gradient structure, showing a trend of gradually decreasing Mg content from the inner to the outer layers. Figures 2B to 2D The existence of a multi-gradient structure was verified by integrating cross-sectional SEM images and EDS analysis.

[0066] (2) The degradation performance of MgO / MgCO3 / PCL electrospun membrane was evaluated by in vitro degradation test incubation at 37℃.

[0067] The in vitro degradation assay at 37°C was performed according to the research paper "Biosynthesized bandages carrying magnesium oxide nanoparticles induce cortical bone formation by modulating endogenous periosteal cells". ACS nano, 2022, 16(11): 18071-18089.

[0068] The concentration of Mg in the solution was determined using an inductively coupled plasma atomic emission spectrometer (ICP-9820, Schimdzu, Japan). 2+ The concentration of Mg was determined, and a plot of Mg was drawn. 2+ The sustained-release curve.

[0069] Mg 2+ The sustained release curve is as follows Figure 3B As shown: 10% MgO / MgCO3 / PCL group releases Mg 2+ The most effective and stable method, with a release time of 1-6 weeks, reaching a plateau after 7 weeks. The 20% MgO / MgCO3 / PCL group and the 30% MgO / MgCO3 / PCL group showed slower release rates within 8 weeks compared to the 10% MgO / MgCO3 / PCL group, and did not reach the target MgO / MgCO3 / PCL level within the detection period (within 8 weeks). 2+ Release peak.

[0070] The above results demonstrate that MgO / MgCO3 / PCL multigradient nanofiber membranes can achieve Mg 2+Adjustable and sustained release. By adding different proportions of rapidly degrading MgO and slowly degrading MgCO3 to the inner, middle, and outer layers of a MgO / MgCO3 / PCL multi-gradient nanofiber membrane, the release of Mg can be effectively regulated. 2+ The release of Mg affects the sustained-release performance of multi-gradient nanofiber membranes. High concentrations of mixed Mg monomers or a large proportion of MgCO3 can slow down the release of Mg. 2+ The release of MgO was weaker in the 20% and 30% MgO / MgCO3 / PCL groups during the first 8 weeks compared to the 10% MgO / MgCO3 / PCL membrane. In fiber membranes with higher magnesium monomer concentrations, Mg... 2+ The slow release could be due to several factors. An increase in the total amount of MgCO3 may lead to a decrease in Mg... 2+ Release delay. In addition, a higher total Mg monomer concentration leads to enhanced crosslinking between PCL and Mg, which may also cause slight changes in material thickness, ultimately resulting in a nonlinear relationship between the concentration of magnesium monomer and the sustained release rate of the material.

[0071] (3) Structural fiber parameters

[0072] The control group, the 10% MgO / MgCO3 / PCL group, the 20% MgO / MgCO3 / PCL group, and the 30% MgO / MgCO3 / PCL group were treated with 2.5% glutaraldehyde for 6 h, then rinsed three times with deionized water for 30 min each time; followed by dehydration in gradient concentrations of ethanol solutions (30%, 50%, 75%, and 100% ethanol) for 30 min each time. To enhance conductivity and contrast, a thin layer of platinum mixture was coated onto the surface, and then the fibers were accelerated using a scanning electron microscope (SEM; S-8010, Hitachi, Japan) at an accelerating voltage of 5 keV. The structural fiber parameters were evaluated using image analysis software (ImageJ; Cybernetics, Bethesda, MD, USA).

[0073] The network structure of each group of fiber membranes was analyzed by scanning electron microscopy, such as... Figure 3C As shown: the multi-gradient nanofiber membranes (10% MgO / MgCO3 / PCL group, 20% MgO / MgCO3 / PCL group, 30% MgO / MgCO3 / PCL group) are networks of interconnected nanofibers distributed with MgO / MgCO3 nanoparticles. For example... Figure 3D As shown: the average diameter of nanofibers in the control group (PCL) was 126.8±39.6 nm, in the 10% MgO / MgCO3 / PCL group it was 229.4±102.3 nm, in the 20% MgO / MgCO3 / PCL group it was 217.9±65.8 nm, and in the 30% MgO / MgCO3 / PCL group it was 253.6±92.3 nm.

[0074] (4) Mechanical properties

[0075] The contact angles of the control group, the 10% MgO / MgCO3 / PCL group, the 20% MgO / MgCO3 / PCL group, and the 30% MgO / MgCO3 / PCL group were measured to further characterize the surface properties (wetting properties) of the MgO / MgCO3 / PCL multi-gradient nanofiber membrane.

[0076] The contact angle is measured using the ring pull-out method: During the measurement, the surface of the sample to be analyzed is suspended from a force sensor instead of a metal sheet and gradually immersed in the liquid phase. Changes in force are recorded to calculate the advancing contact angle between the liquid and solid surfaces. Subsequently, the solid sample is slowly withdrawn from the liquid phase, while changes in force are recorded to determine the retreating contact angle.

[0077] Measurement results as follows Figure 3E As shown: the average contact angle of the control group (PCL) membrane was 124.9°±0.52°, the 10% MgO / MgCO3 / PCL group was 122.1°±1.61°, the 20% MgO / MgCO3 / PCL group was 120.2°±2.13°, and the 30% MgO / MgCO3 / PCL group was 120.9°±2.53°.

[0078] The results above show that there was no statistically significant difference in the contact angles among the different groups of membrane materials, indicating that the interaction levels between the membrane surface and the test liquid were comparable. This further confirms that the bioactive material mainly works by releasing Mg... 2+ It promotes peripheral nerve regeneration, rather than its physical properties.

[0079] Experimental Example 2: Animal Experiment with Sciatic Nerve Defect

[0080] The autologous nerve transplantation group was set as the blank control group; the PCL electrospun membrane was set as the PCL nerve conduit control group; the 10% MgO / MgCO3 / PCL multigradient nanofiber membrane of Example 1 was set as the 10% MgO / MgCO3 / PCL group, the 20% MgO / MgCO3 / PCL multigradient nanofiber membrane of Example 2 was set as the 20% MgO / MgCO3 / PCL group, and the 30% MgO / MgCO3 / PCL multigradient nanofiber membrane of Example 3 was set as the 30% MgO / MgCO3 / PCL group; the 10% MgO / MgCO3 / PCL group, the 20% MgO / MgCO3 / PCL group, and the 30% MgO / MgCO3 / PCL group were cut into 0.5mm×12mm multigradient fibers, which were respectively set as the 10% MgO / MgCO3 / PCL nerve conduit group, the 20% MgO / MgCO3 / PCL nerve conduit group, and the 30% MgO / MgCO3 / PCL nerve conduit group.

[0081] 1. 10mm sciatic nerve defect model

[0082] Female SD rats (2 months old, weighing 200g-250g) were housed under standard laboratory conditions with a 12-hour light / dark cycle, an ambient temperature of 25℃, and a humidity of 50%. The rats were randomly divided into 5 groups: autologous nerve transplantation group, PCL nerve conduit group, 10% MgO / MgCO3 / PCL nerve conduit group, 20% MgO / MgCO3 / PCL nerve conduit group, and 30% MgO / MgCO3 / PCL nerve conduit group.

[0083] Rats were anesthetized with sodium pentobarbital (50 mg / kg body weight) combined with isoflurane, and then microsurgically operated on by the same experienced surgeon. Specifically:

[0084] 1) Fix and disinfect the surgical site (lateral aspect of the right thigh), then make an incision of about 4 cm parallel to the lower edge of the femur; carefully expose the sciatic nerve through this incision using a surgical microscope;

[0085] 2) In the autologous transplantation group, a 10mm sciatic nerve was removed, everted, and reinserted. The autologous nerve was fixed to the epidermis of the proximal and distal ends of the nerve using 10-0 monofilament nylon sutures.

[0086] The PCL, 10% MgO / MgCO3 / PCL, 20% MgO / MgCO3 / PCL, and 30% MgO / MgCO3 / PCL nerve conduit groups were first cut at the distal end of the exposed sciatic nerve. Then, the nerve stump was moved 1 mm proximally to the 12 mm nerve conduit, creating a 10 mm gap. These were then fixed in place with 10-0 nylon sutures. Multi-gradient fibrous membranes containing 10%, 20%, and 30% MgO / MgCO3 / PCL, cut into 0.5 mm × 12 mm pieces, were inserted into the nerve conduits (4 membranes per conduit, with the inner layer of the multi-gradient fibrous membrane adhering to the inner wall of the nerve conduit). The other end was connected to the distal nerve stump. The conduit diameter (approximately 2 mm) was anastomosed with the sciatic nerve.

[0087] 3) Use 4-0 silk sutures to suture the skin wound.

[0088] 2. Gait analysis and neurophysiological monitoring to assess nerve regeneration and motor function recovery.

[0089] Footprints were captured at 2, 4, 6, and 12 weeks post-surgery to assess the functional recovery of peripheral nerves. Figure 4A(B) The Sciatic Function Index (SFI) is a well-established and sophisticated method for assessing sciatic nerve function in rats. Calculating the SFI provides a non-invasive way to evaluate the degree of recovery of motor function in target muscles after injury to the sciatic nerve or its branches. SFI values ​​range from 0 (representing normal) to -100 (representing complete nerve rupture). Lower absolute SFI values ​​indicate better nerve function recovery. Rats in all five experimental groups showed improvement in overall motor function over time.

[0090] The results showed that the autologous transplantation group had the lowest absolute SFI value among the five groups, followed by the 10% MgO / MgCO3 / PCL group. No statistically significant differences were observed among the other three groups. Electrophysiological assessments were performed 12 weeks post-surgery. CMAP (compound muscle action potential) was recorded by placing signal receiving electrodes on the gastrocnemius muscle of the five groups of rats. The autologous transplantation group showed the highest CMAP amplitude, followed by the 10% MgO / MgCO3 / PCL group. No statistically significant differences were observed among the other three groups. Figure 4C (D).

[0091] Following nerve injury, muscle atrophy was observed in all five affected gastrocnemius (triceps surae) muscles, and nerve regeneration improved over time. Gastrocnemius muscle samples were collected 12 weeks post-surgery for Masson's trichrome staining to assess muscle structure. Figure 4E Of the five experimental groups, the autologous transplantation group showed the best results, characterized by an increase in muscle fiber cross-sectional area, a decrease in collagen fiber area, and a reduction in the percentage of collagen fiber area. Among the other four groups, the 10% MgO / MgCO3 / PCL group had the largest muscle fiber cross-sectional area, the greatest reduction in collagen fiber area, and the lowest percentage of collagen fiber area. However, no significant differences were observed among the 20% MgO / MgCO3 / PCL, 30% MgO / MgCO3 / PCL, and PCL nerve conduit groups. The mean percentages of collagen fiber area in the five groups were: 12.2% ± 2.3% in the autologous transplantation group, 26.1% ± 5.5% in the PCL group, 16.5% ± 4.7% in the 10% MgO / MgCO3 / PCL group, 20.1% ± 3.3% in the 20% MgO / MgCO3 / PCL group, and 21.1% ± 4.0% in the 30% MgO / MgCO3 / PCL group. Figure 4F -H).

[0092] 3. At 6 and 12 weeks post-surgery, regenerated nerves were obtained by perfusing the rat circulatory system with 4% paraformaldehyde solution.

[0093] like Figure 5BAs shown, nerve continuity was observed to be restored 6 weeks postoperatively, and regenerated nerve tissue was visible within the nerve conduit in the experimental group. The nerve graft was divided into three equal parts: proximal, middle, and distal. A 2mm mid-segment nerve specimen was harvested at 12 weeks postoperatively for histological evaluation of the regenerated nerve tissue.

[0094] 1) Using a Leica CM3050S microtome, cross sections of the central region were cut to a thickness of 7 μm. The sections were stained with hematoxylin and eosin. Then, five random field images of each section were analyzed.

[0095] Hematoxylin-eosin staining was used to evaluate the newly formed nerve fibers in each group, and the diameter of the newly formed nerves was measured in cross-section. Figure 5C and 5D As shown, the autologous nerve transplantation group exhibited the most significant size increase, which was statistically significant, followed by the 10% MgO / MgCO3 / PCL, 20% MgO / MgCO3 / PCL, 30% MgO / MgCO3 / PCL, and PCL nerve conduit. The results indicate that the nerve repair effect of the MgO / MgCO3 / PCL multi-gradient nanofiber conduits implanted in all three groups was significantly better than that in the negative control group (PCL nerve conduit). Among the three experimental groups, MgO / MgCO3 / PCL... 2+ The lowest concentration (10%) of MgO / MgCO3 / PCL showed the best repair effect. During the first 6 weeks of nerve regeneration, the 10% MgO / MgCO3 / PCL group exhibited higher Mg levels. 2+ Effective release rate.

[0096] 2) The distal tissue structure of the nerve graft was observed using toluidine blue staining and transmission electron microscopy. A 2mm portion of the regenerated nerve was treated with 2.5% glutaraldehyde for 2 hours, followed by fixation with 1% osmium tetroxide for 1.5 hours. Tissue samples were dehydrated in ethanol, embedded in Epon 812 resin (Ted Pella, Redding, CA, USA), and then cut into 1μm semi-thin sections and 50nm ultrathin sections. The semi-thin sections were stained with toluidine blue solution, and the ultrathin sections were examined using TEM (JEOL, Tokyo, Japan). Five regions were randomly selected from each area for image acquisition. The axonal diameter and myelin sheath thickness of the myelinated nerve were measured using Photoshop CS6 software (Adobe Systems). The G ratio was the ratio of axonal diameter to fiber diameter. Five random-field images of each ultrathin nerve section were analyzed, and statistical analysis was performed on the five samples in each group.

[0097] like Figure 5E As shown, myelin regeneration of the regenerated nerve was assessed by toluidine blue staining and TEM analysis. Nerve graft specimens were collected 12 weeks postoperatively, and a transverse section of the regenerated nerve 2 mm distal to the graft was examined.

[0098] Assessment of the diameter and number of myelinated axons in TEM images ( Figure 5F G), and the thickness of the new myelin sheath ( Figure 5H In addition, the G ratio was calculated to measure the maturity of myelinated axons. Figure 5I In each group, the autologous nerve transplantation group showed significant myelin regeneration, with marked thickening of the myelin sheath around the regenerated axons. Compared with the control group, the 10% MgO / MgCO3 / PCL nerve conduit group showed a significant increase in the number and size of myelinated nerve axons and the thickness of the myelin sheath (p<0.05). Compared with the 30% MgO / MgCO3 / PCL nerve conduit group and the PCL nerve conduit group, the 20% MgO / MgCO3 / PCL nerve conduit group showed a significant improvement in the thickness of the newly formed myelin sheath (p<0.05). There were no statistically significant differences in nerve diameter, number of myelinated axons, and G-ratio among the 20% MgO / MgCO3 / PCL nerve conduit group, the 30% MgO / MgCO3 / PCL nerve conduit group, and the PCL nerve conduit group (p>0.05). These results indicate that MgO / MgCO3 / PCL nerve conduit is a suitable neurotransmitter for myelin regeneration. 2+ Schwann cells play a crucial role in promoting axonal myelination. They are primarily involved in nerve regeneration, and the continuous release of MgO from MgO / MgCO3 / PCL multigradient fibers... 2+ It is possible that during the mid-to-late stages of neurogenesis, the Shh / Ptch1 / Gli1 axis can influence the phenotypic transformation of Schwann cells, promoting the repair of peripheral nerve defects. Maintaining optimal Mg 2+ Balance is essential for supporting Schwann cell function and promoting efficient neural regeneration.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A multi-gradient nanofiber membrane, characterized in that, It comprises an inner layer, a middle layer, and an outer layer; the inner layer comprises a magnesium-containing compound and polycaprolactone in a mass ratio of (0.12-0.36):1; the middle layer comprises a magnesium-containing compound and polycaprolactone in a mass ratio of (0.1-0.3):1; and the outer layer comprises a magnesium-containing compound and polycaprolactone in a mass ratio of (0.08-0.24):

1. The magnesium-containing compounds include MgO and MgCO. 3； The mass ratio of MgO to MgCO3 in the inner layer is (0.3-0.7):1; the mass ratio of MgO to MgCO3 in the middle layer is (0.8-1.4):1; and the mass ratio of MgO to MgCO3 in the outer layer is (1.5-2.5):

1.

2. The multi-gradient nanofiber membrane according to claim 1, characterized in that, The inner layer contains 12.57%-24.54% magnesium by mass; the middle layer contains 11.42%-20.46% magnesium by mass; and the outer layer contains 9.43%-19.63% magnesium by mass.

3. The method for preparing the multi-gradient nanofiber membrane according to claim 1 or 2, characterized in that, Includes the following steps: (1) Add the polycaprolactone to the solvent and stir to obtain a mixed solution; (2) Add the magnesium-containing compounds to the inner, middle and outer layers respectively, stir evenly, and obtain the electrospinning solutions of the inner, middle and outer layers respectively; (3) Electrospinning is performed using the obtained inner, middle and outer electrospinning solutions. The electrospinning is stacked layer by layer to form a three-layer composite electrospinning membrane, which is a multi-gradient nanofiber membrane.

4. The preparation method according to claim 3, characterized in that, In step (1), the solvent is trifluoroethanol; the mass-to-volume ratio of polycaprolactone to the solvent is 5%-15% g / mL.

5. The preparation method according to claim 3, characterized in that, In step (3), the electrospinning uses a high-voltage electric field of 10kV-20kV.

6. A nerve conduit, characterized in that, It incorporates the multi-gradient nanofiber membrane as described in claim 1 or 2.

7. The nerve conduit according to claim 6, characterized in that, The inner layer of the multi-gradient nanofiber membrane faces the inner wall of the nerve conduit.

8. Use of the multi-gradient nanofiber membrane of claim 1 or 2, or the nerve conduit of claim 6 or 7, in any of the following fields, characterized in that, include: i. Preparation of agents that promote nerve regeneration; ii. Preparation of nerve conduits that promote nerve regeneration; iii. Preparation of formulations for the repair of peripheral nerve defects; iv. Prepare nerve conduits for peripheral nerve defect repair.

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