Preparation method and application of hierarchical nanofiber membrane for nerve repair

Abstract

The application belongs to the field of medical biological materials, and relates to a preparation method of a hierarchical nanofiber composite film for nerve repair and application thereof. The method comprises the following steps: preparing multilayer nanofibers with different internal stresses through continuous multiple electrostatic spinning units, stacking the multilayer nanofibers with different internal stresses to form a composite nanofiber film with an internal stress gradient; chemically crosslinking the composite nanofiber film; performing adhesion molecule adhesion treatment on the upper surface of the composite nanofiber film; and preparing the hierarchical nanofiber composite film with an internal stress gradient and capable of self-deforming under the action of liquid to wrap nerves. The film provided by the application has excellent flexibility, biocompatibility and degradation performance, can efficiently and quickly adhere to the surface of damaged nerves, provides a good support microenvironment for nerve cell growth, promotes nerve regeneration and repair, and has a wide application prospect in the field of peripheral nerve injury repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical biological materials, and particularly relates to a preparation method and application of hierarchical nanofibers for nerve repair. BACKGROUND

[0002] Peripheral nerve injury is a difficult problem in clinical treatment, especially for patients with long nerve gap or absence (such as nerve defect or brachial plexus root avulsion injury). The traditional direct suture method often leads to repair failure due to technical limitations or material adaptation problems, and there are problems such as large suture tension, nerve epineurium stripping and neuroma formation, and the repair effect depends on the operation of high-skilled physicians. At present, peripheral nerve injury has become a major scientific problem in the field of nerve repair. Although autologous transplantation is the "gold standard" in clinical practice, it is difficult to be widely applied due to the limitations of damage to the donor site, limited source, size mismatch and the need for secondary surgery.

[0003] With the progress of science and technology, nerve epineurium sleeve and chitosan sleeve are developed to assist nerve repair. However, these sleeves need skilled physicians to select the appropriate size and perform nerve end-to-end docking and suture during use, which has high technical requirements and complex steps, and also causes secondary damage to the damaged sleeve and prolongs the recovery time. Therefore, it is of important clinical significance and application prospect to develop an artificial nerve conduit stent with adaptability, no suture and rapid closure as a substitute.

[0004] Nanofiber membranes, produced through precise control of various parameters such as thickness, cross-linking degree, diameter, density, and pore structure using electrospinning technology, can be mass-produced at a low cost. Nanofiber membranes possess high porosity, large specific surface area, and a nanoscale structure similar to the natural extracellular matrix (ECM). They also offer significant advantages in raw material sourcing, technological integration, and macroscopic preparation, making them an ideal and highly sought-after method for preparing fibrous membranes, as evidenced by patents CN 116099044 A, CN 117018290 B, CN 117504006 A, CN 114808276B, CN 115957379 A, ​​CN 117626654 A, and CN118718068 A. These nanofiber films utilize their high porosity and large specific surface area for drug loading or to mimic the extracellular matrix for therapeutic applications. Furthermore, patent CN 116099044 A utilizes electrospinning technology to obtain nanofibers, which are then further coiled into tubular shapes at high temperatures before being sutured by doctors. Similarly, patent CN 117504006 A uses electrospinning technology to first obtain a nanofiber film, then uses ultraviolet light to irradiate it to obtain a tubular nerve scaffold before suturing. Patent CN 114808276 B obtains three layers of nanofibers, which are then bonded together with a solution to form a cannula scaffold. Therefore, all the above preparation methods involve mechanically or manually coiling the nanofibers into tubular scaffolds. However, the thickness of nerves varies in different locations, and pre-constructing a cannula makes it difficult to match the nerve thickness. Therefore, when using nanofiber cannulas, medical personnel still need to manually insert the nerve into the cannula before suturing, which does not reduce the difficulty. Combining the advantages of nanofibers, designing and developing an adaptive nerve nanopatch that can quickly adhere to and wrap the nerve under physiological conditions and reduce surgical complexity is of significant value and has broad clinical demand. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention discloses a method for preparing and applying a hierarchical nanofiber membrane for nerve repair. By using multiple sets of electrospinning units, composite nanofiber films with different internal stress gradients are constructed. Then, through cross-linking and adhesion treatments, a hierarchical nanofiber composite membrane for nerve repair is prepared. Under the action of liquid and different driving forces, the hierarchical nanofiber composite membrane undergoes self-deformation to encapsulate the severed nerve ends, rapidly closing the nerve ends, eliminating the need for sutures, effectively reducing secondary damage, shortening surgical time, and improving repair efficiency and effectiveness.

[0006] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0007] A method for preparing a hierarchical nanofiber composite membrane for nerve repair, the method comprising:

[0008] (1) Multilayer nanofibers with different internal stresses are prepared by continuous multiple sets of electrospinning units, and multilayer nanofibers with different internal stresses are stacked layer by layer to form a composite nanofiber film with internal stress gradient; wherein, the preparation of continuous multiple sets of electrospinning units refers to the continuous setting of multiple sets of electrospinning units on the same production line so that the nanofibers generated by different sets of electrospinning units are stacked layer by layer, and the electrospinning unit is a conventional electrospinning equipment.

[0009] (2) The composite nanofiber film is chemically crosslinked; in step (2), the chemical crosslinking method includes: ultraviolet light polymerization or crosslinking with glutaraldehyde and genipin. Chemical crosslinking is used to enhance structural stability and deformability, forming a nanofiber film with stable mechanical properties, thereby effectively avoiding delamination or interfacial peeling caused by the mismatch of interlayer mechanical properties;

[0010] (3) Adhesive molecules are used to adhere the upper surface of the composite nanofiber membrane after step (3) to prepare a hierarchical nanofiber composite membrane with internal stress gradient and self-deformation under the action of liquid to wrap nerves; specifically, the liquid includes physiological saline, human tissue fluid, blood, etc.

[0011] Furthermore, step (1) specifically includes the following steps:

[0012] (1.1) Use one or more polymer materials as raw materials to prepare the spinning solution;

[0013] (1.2) The spinning solution is spun using multiple sets of electrospinning units to obtain multilayer nanofibers stacked on top of each other; by controlling the conditions of electrospinning, the diameter and / or porosity of the nanofibers in the different nanofiber layers are different, thereby obtaining a composite nanofiber film with internal stress gradient.

[0014] Further, in step (1.1), the polymeric material includes polyethylene glycol diacrylate, methacrylic anhydride gelatin, methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide hyaluronic acid, methacrylamide cellulose polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, chitosan, cellulose, silk fibroin, gelatin, polyacrylic acid, or polyamide; the concentration of the polymeric material in the spinning solution ranges from 2% to 10%.

[0015] In step (1.2), the electrospinning conditions are as follows: the injection speed of the spinning solution is 2-10 mL / min, the voltage is 18-25 kV, the air pressure is 0.2-2 MPa, the receiving distance is 15-45 cm, the temperature is 20-50℃, and the humidity is 40%-80%.

[0016] The prepared composite nanofiber film has 2 to 3 layers of nanofibers, with the diameter gradient difference between adjacent layers of nanofibers maintained at 5% to 35%; the porosity gradient difference between adjacent layers is maintained at 5% to 35%.

[0017] Furthermore, step (1) specifically includes the following steps:

[0018] (1.1) Prepare at least two different spinning solutions, each containing one or more polymeric materials; the different spinning solutions contain different polymeric materials;

[0019] (1.2) Different spinning solutions are spun using different sets of electrospinning units to obtain multilayer nanofibers stacked on top of each other; by controlling the type of polymer material in different spinning solutions, different nanofiber layers have different internal stresses, thereby obtaining composite nanofiber films with internal stress gradients.

[0020] Further, in step (1.1), the polymeric material includes polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polyamide, polyethylene glycol diacrylate, methacrylic anhydride gelatin, methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide hyaluronic acid, methacrylamide cellulose polyvinyl alcohol, chitosan, cellulose, silk fibroin, gelatin, polyacrylic acid, or polyamide;

[0021] The concentration range of the polymer material in the spinning solution is 2% to 10%;

[0022] The number of polymer materials in the same spinning solution is 1-3.

[0023] Furthermore, step (1.2) also includes: by controlling the conditions of electrospinning, the diameter and / or porosity of the nanofibers in the different nanofiber layers prepared are different, thereby obtaining a composite nanofiber film with an internal stress gradient.

[0024] The electrospinning conditions are as follows: the spinning solution injection speed is 2-10 mL / min, the voltage is 18-25 kV, the air pressure is 0.2-2 MPa, the receiving distance is 15-45 cm, the temperature is 20-50℃, and the humidity is 40%-80%.

[0025] The prepared composite nanofiber film has 2 to 3 layers of nanofibers, with the diameter gradient difference between adjacent layers of nanofibers maintained at 5% to 35%; the porosity gradient difference between adjacent layers is maintained at 5% to 35%.

[0026] Further, in step (3), the adhesion treatment specifically includes: spraying an adhesion molecule solution onto the upper surface of the cross-linked composite nanofiber membrane; preparing a hierarchical nanofiber composite membrane with an internal stress gradient that self-deforms under the action of liquid to encapsulate nerves; specifically, the mass fraction of the adhesion molecule solution is 0.1-0.5 wt%; after this adhesion treatment, the adhesion force of the hierarchical nanofiber composite nerve repair membrane is 0.01 N / cm. 2 ~0.1N / cm 2 ;

[0027] Further, in step (3), the adhesion treatment specifically includes:

[0028] (3.1) The upper surface of the cross-linked composite nanofiber membrane is modified by introducing nitrogen gas through plasma surface treatment technology to activate the upper surface of the composite nanofiber membrane and introduce amino groups into the upper surface of the composite nanofiber membrane. In this step, the upper surface of the cross-linked composite nanofiber membrane is modified by plasma surface treatment technology to activate the nanofibers on the upper surface of the composite nanofiber membrane and introduce functional active groups amino groups during the activation process.

[0029] (3.2) The adhesion molecules are dissolved in an EDC / NHS system to form an adhesion molecule activation solution. The EDC / NHS system is used to activate the carboxyl groups in the adhesion molecules. The composite nanofiber membrane treated in step (3.1) is immersed in the adhesion molecule activation solution. The amino groups on the surface of the composite nanofiber membrane and the activated carboxyl groups in the adhesion molecules undergo an amidation reaction. After the reaction, the membrane is washed with deionized water to remove unreacted adhesion molecules, so that the adhesion molecules are stably bonded to the nanofibers on the surface of the composite nanofiber membrane. Then, it is freeze-dried, cut, sterilized, and packaged to obtain a graded nanofiber composite membrane. The adhesion force of the graded nanofiber composite nerve repair membrane after this adhesion treatment is 0.08 N / cm. 2 ~0.2N / cm 2 ;

[0030] Furthermore, the adhesion molecules include any one of levodopa, tannic acid, gallic acid, anthocyanins, and secretions from giant salamanders.

[0031] A hierarchical nanofiber for nerve repair, wherein the hierarchical nanofiber composite nerve repair membrane comprises at least two nanofiber layers with different internal stresses, and the internal stress of the nanofiber layers is distributed from low to high from the upper surface to the lower surface of the composite nanofiber membrane; the upper surface of the composite nanofiber membrane includes adhesive molecules; preferably, the hierarchical nanofiber composite nerve repair membrane comprises 2 to 3 nanofiber layers; the thickness of the hierarchical nanofiber composite nerve repair membrane is 50 μm-2000 μm, and the adhesion force of the hierarchical nanofiber composite nerve repair membrane is 0.01 N / cm. 2 ~0.2N / cm 2 The deformation time is 10s to 100s.

[0032] An application of a graded nanofiber composite nerve repair membrane, wherein the self-driven curling nanofiber adhesive patch is used in products for repairing sciatic nerve injuries or traumatic peripheral nerve injuries.

[0033] The beneficial effects of this invention are:

[0034] Design of internal stress gradient: For the first time, a controllable internal stress gradient is introduced into the nanofiber membrane, enabling the prepared hierarchical nanofiber composite membrane to spontaneously deform under the action of liquid. This is an innovative extension of the function of traditional nanofiber membranes.

[0035] Construction of adhesive nanofibers: By performing an adhesive treatment on the inner surface of the composite nanofiber membrane, the hierarchical nanofiber composite membrane is endowed with the ability to rapidly adhere to nerve tissue, thereby improving the efficiency of nerve repair.

[0036] The graded nanofiber composite membrane provided by this invention serves as a novel alternative to artificial nerve conduit scaffolds. It offers advantages such as ease of operation, reduced surgical time, and minimized secondary damage, demonstrating significant research value and broad clinical application prospects. Attached Figure Description

[0037] Figure 1 These are two layers of nanofibers with different porosity structures. A is the upper layer (small diameter, low porosity), and B is the lower layer (large diameter, low porosity).

[0038] Figure 2 This is a scanning electron microscope cross-sectional image of two layers of nanofibers;

[0039] Figure 3 Images showing the process of rolling up nanofiber membranes;

[0040] Figure 4 The change in the curling angle of the nanofiber film over time;

[0041] Figure 5 The stress-strain curve of the hierarchical nanofiber composite membrane;

[0042] Figure 6 The adhesion force of the hierarchical nanofiber composite membrane to the tissue is shown in Figure A, which is an actual adhesion image, and Figure B is an adhesion curve.

[0043] Figure 7 The following images demonstrate the sciatic nerve repair effect in the treatment group rats: A shows NF200 staining of the rat sciatic nerve, indicating good axonal growth; B shows S100 staining of the rat sciatic nerve, indicating good axonal growth; C shows toluidine blue staining of the rat sciatic nerve, indicating good nerve recovery; D shows a scanning electron microscope image of the rat sciatic nerve repair.

[0044] Figure 8 The study demonstrates the functional effects of sciatic nerve repair in rats. A shows Masson staining of the muscles; B shows good distal gastrocnemius and soleus muscles after nerve innervation; C shows good signal transduction function in the results of neurophysiological tests; and D shows good recovery in SD rats after nerve regeneration in Carwalk paw print images.

[0045] Figure 9 To characterize the diameter, porosity, and layer-by-layer stacking structure of nanofibers using scanning electron microscopy; A shows the structure of the nanofiber layer (upper layer) prepared with a 2% (w / w) chitosan spinning solution; B shows the structure of the nanofiber layer (middle layer) prepared with a 5% (w / w) chitosan spinning solution; C shows the structure of the nanofiber layer (lower layer) prepared with an 8% (w / w) chitosan spinning solution. Detailed Implementation

[0046] The technical solution of the present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0047] Example 1: A method for preparing a hierarchical nanofiber composite membrane for nerve repair, the method comprising:

[0048] (1) Using methacrylic anhydride gelatin as raw material, two spinning solutions with a mass fraction of 5% and 10% were prepared respectively.

[0049] (2) Electrospinning of a 5% spinning solution was carried out in the first electrospinning unit under the conditions of injection speed of 5 mL / min, voltage of 24 kV, receiving distance of 30 cm, temperature of 37 °C and humidity of 50%.

[0050] Electrospinning was performed on a 10% (w / w) spinning solution in the second electrospinning unit under the following conditions: injection speed of 8 mL / min, voltage of 18 kV, receiving distance of 15 cm, temperature of 37 °C, and humidity of 70%.

[0051] Composite nanofiber films with internal stress gradients were obtained by continuously spinning two sets of electrospinning units.

[0052] The diameter, porosity, and layer-by-layer stacking structure of the nanofibers in the composite nanofiber film were characterized by scanning electron microscopy. Figure 1 In Figure A, the structure (upper layer) of the nanofiber layer prepared using a 5% by mass spinning solution is shown. Figure 1 In Figure B, the structure (lower layer) of the nanofiber layer prepared with a spinning solution of 10% by mass is shown.

[0053] The diameter and porosity of nanofibers in different layers were statistically analyzed using ImageJ. Furthermore, point-by-point testing was conducted on different layers of the composite membrane using nanoindentation technology to measure the Young's modulus distribution, reflecting changes in internal stress, as shown in Table 1.

[0054] Table 1. Structural parameters of the upper and lower layers in the composite nanofiber film of Example 1.

[0055] Level Fiber diameter (nm) Porosity (%) Internal stress (MPa) Upper layer 750 nm 50 0.15 Lower layer 900 nm 70 0.27

[0056] (3) Crosslink the composite nanofiber film obtained in step (2) under ultraviolet light for 5 min.

[0057] (4) The cross-linked composite nanofiber membrane is subjected to internal surface adhesion treatment:

[0058] First, the cross-linked composite nanofiber membrane was placed in a plasma chamber, then evacuated to 0.1 Pa and maintained at that pressure for 30 min. Nitrogen gas was then introduced at a flow rate of 2 mL / min for 10 min to fill the chamber. Next, a low-voltage radio frequency (RF) glow discharge was activated, and plasma was formed by exciting the gas through an electromagnetic field, thus modifying the surface of the nanofibers on the upper surface of the composite nanofiber membrane with amino groups. This step is primarily referenced in the literature: Amino modification of pure titanium surface by radio frequency plasma, Journal of Vacuum Science and Technology, 2014.

[0059] The adhesive molecules are dissolved in the EDC / NHS system to form an adhesive molecule activation solution. The EDC / NHS system is used to activate the carboxyl groups in the adhesive molecules. The composite nanofiber membrane treated in step (3) is immersed in the adhesive molecule activation solution. The amino groups on the surface of the composite nanofiber membrane and the activated carboxyl groups in the adhesive molecules undergo an amidation reaction. After the reaction is completed, the membrane is washed with deionized water to remove unreacted adhesive molecules, so that the adhesive molecules are stably combined with the nanofibers on the surface of the composite nanofiber membrane. Then, the membrane is freeze-dried, cut, sterilized, and packaged to obtain a graded nanofiber composite membrane.

[0060] Specifically, the EDC / NHS system is prepared using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), and water; the EDC concentration in the EDC / NHS system is (0.01-0.02) g / mL, and the mass fraction ratio of EDC to NHS is 1:(1.2-1.5); the mass fraction of the adhesion molecule in the adhesion molecule activation solution is 0.1-0.5 wt%, the activation time is 15-30 min, and the reaction time is 2-6 h; in this example, the adhesion molecule used is levodopa, and the mass percentage concentration of the adhesion molecule in the adhesion molecule activation solution is 0.2%.

[0061] In this invention, the graded nanofiber composite membrane is cut to a suitable size, and its self-deformation, mechanical properties, adhesion properties, biocompatibility, and therapeutic effects on severed nerve ends are further verified to ensure the safety, functionality, and usability of the nanofiber patch under physiological conditions.

[0062] The self-deformability of the hierarchical nanofiber composite membrane was verified by a wet immersion method: the hierarchical nanofiber composite membrane was first immersed in an aqueous solution, and it was found that it could self-deform and curl into a cylindrical sleeve. Figure 3 ), and recorded the curling angle at different times, such as Figure 4 As shown, the hierarchical nanofiber composite membrane curls into a tubular shape in 25s-30s;

[0063] According to the international standard ASTM D3039 (Standard Test Method for Tensile Properties of Polymer-Based Composite Materials), the mechanical properties of the prepared graded nanofiber composite membrane were tested using a universal tensile testing machine, and the stress-strain curve was calculated using the following formula.

[0064] Stress = F (force) / A (area of ​​application of the force)

[0065] Strain = ΔL (deformation distance) / L (initial length)

[0066] Stress-strain curves as follows Figure 5 As shown in the figure, the maximum tensile strength of the hierarchical nanofiber composite membrane is 3.2 MPa, which fully meets the mechanical performance requirements for peripheral nerve repair.

[0067] According to the literature "Dry double-sided tape for adhesion of wet tissues and devices" (Nature 2019, 575(7781):169-174), the adhesion force of the hierarchical nanofiber composite membrane to tissues was determined by peeling using a universal tensile testing machine.Figure 6 In the figure, A is the actual adhesion image, and B is the adhesion curve; as shown in the figure, 0.15 N / cm is required during the peeling process. 2 The adhesive force allows the hierarchical nanofiber composite membrane to adhere well to the nerve, preventing it from falling off and failing without damaging the nerve, thus effectively treating nerve damage.

[0068] The graded nanofiber composite membrane described in this embodiment was used for the repair and treatment of the sciatic nerve in rats; for example... Figure 7 The treatment group rats showed improved sciatic nerve repair. Figure 7 Image A shows NF200 staining of the rat sciatic nerve, indicating good axonal growth; Image B shows S100 staining of the rat sciatic nerve, indicating good axonal growth; Image C shows toluidine blue staining of the rat sciatic nerve, indicating good nerve recovery; Image D shows a scanning electron microscope image of the rat sciatic nerve repair.

[0069] like Figure 8 The functional effects of treatment on the sciatic nerve in rats after repair were demonstrated: Figure 8 Image A shows Masson staining of the muscle; Image B shows good distal gastrocnemius and soleus muscles after nerve innervation; Image C shows good signal transduction function as indicated by neurophysiological testing results; Image D shows good recovery of SD rats after nerve regeneration as shown by Carwalk paw print image. Experiments demonstrate that the graded nanofiber composite membrane described in this embodiment has a good therapeutic effect on nerve injury.

[0070] Example 2: A method for preparing a hierarchical nanofiber composite membrane for nerve repair, the method comprising:

[0071] (1) Using chitosan as raw material, spinning solutions with mass fractions of 2%, 5%, and 8% were prepared respectively.

[0072] (2) The spinning solution with a chitosan mass fraction of 2% was electrospun in the first electrospinning unit under the conditions of injection speed of 5 mL / min, voltage of 24 kV, receiving distance of 30 cm, temperature of 37 °C and humidity of 50%.

[0073] Electrospinning of a 5% chitosan spinning solution was carried out in the second electrospinning unit under the following conditions: injection speed of 8 mL / min, voltage of 18 kV, receiving distance of 15 cm, temperature of 37 °C, and humidity of 70%.

[0074] Electrospinning was performed in the third electrospinning unit under the following conditions: an injection speed of 8 mL / min, a voltage of 18 kV, a receiving distance of 15 cm, a temperature of 37 °C, and a humidity of 70%.

[0075] Composite nanofiber films with internal stress gradients were obtained by continuously spinning three sets of electrospinning units.

[0076] The diameter, porosity, and layer-by-layer stacking structure of the nanofibers were characterized by scanning electron microscopy. Figure 9 In the diagram, A shows the structure of the nanofiber layer (upper layer) prepared with a chitosan spinning solution of 2% by mass; B shows the structure of the nanofiber layer (middle layer) prepared with a chitosan spinning solution of 5% by mass; and C shows the structure of the nanofiber layer (lower layer) prepared with a chitosan spinning solution of 8% by mass.

[0077] The diameter and porosity of nanofibers in different layers were statistically analyzed using ImageJ. Furthermore, point-by-point testing was conducted on different layers of the composite membrane using nanoindentation technology to measure the Young's modulus distribution, reflecting changes in internal stress, as shown in Table 2.

[0078] Table 2 shows the structural parameters of the upper and lower layers of the composite nanofiber film in Example 2.

[0079] Level Fiber diameter (nm) Porosity (%) Internal stress (MPa) Upper layer 300 35 0.06 Middle layer 600 45 0.17 Bottom layer 800 65 0.32

[0080] (3) Crosslink the composite nanofiber film obtained in step (2) with glutaraldehyde for 15 min.

[0081] (4) The cross-linked composite nanofiber membrane is subjected to internal surface adhesion treatment: This step is the same as step (4) in Example 1, except that the adhesion molecules in this example are the secretions of the giant salamander; the mass fraction of the adhesion molecules in the adhesion molecule activation solution is 0.3 wt%.

[0082] Furthermore, it is cut to a suitable size for use; the adhesion force of the hierarchical nanofiber composite nerve repair membrane prepared in this embodiment is 0.12 N / cm. 2 The deformation time is 20-28 seconds.

[0083] Comparative Example 1

[0084] (1) Using methacrylic anhydride gelatin as raw material, two spinning solutions with a mass fraction of 5% and 10% were prepared respectively.

[0085] (2) Electrospinning of a 5% spinning solution was carried out in the first electrospinning unit under the conditions of injection speed of 5 mL / min, voltage of 30 kV, receiving distance of 24 cm, temperature of 30 °C and humidity of 50%.

[0086] Electrospinning was performed on a 10% (w / w) spinning solution in the second electrospinning unit under the following conditions: injection speed of 8 mL / min, voltage of 30 kV, receiving distance of 15 cm, temperature of 37 °C, and humidity of 70%.

[0087] Table 3 shows the structural parameters of the upper and lower layers in the composite nanofiber film of Comparative Example 1.

[0088] Level Fiber diameter (nm) Porosity (%) Internal stress (MPa) Middle layer 700 30 0.10 Bottom layer 1100 70 0.40

[0089] (3) Crosslink the composite nanofiber film obtained in step (2) with glutaraldehyde for 15 min.

[0090] However, the 40% porosity difference between the upper and lower layers leads to inconsistent deformation and uneven stress distribution. This prevents the layers from maintaining their integrity during deformation, resulting in slippage or fracture between them. Furthermore, experiments have shown that when the porosity difference falls below 5%, deformation also fails due to the small difference between the two layers.

[0091] Example 3: A method for preparing a hierarchical nanofiber composite membrane for nerve repair, the method comprising:

[0092] (1) Prepare a chitosan spinning solution with a mass fraction of 5% using chitosan as raw material; and prepare a silk fibroin spinning solution with a mass fraction of 10% using silk fibroin as raw material.

[0093] (2) The chitosan spinning solution was electrospun in the first electrospinning unit under the conditions of injection speed of 5 mL / min, voltage of 24 kV, receiving distance of 30 cm, temperature of 37 ℃ and humidity of 50%.

[0094] The silk fibroin spinning solution was electrospun in the second electrospinning unit under the conditions of an injection speed of 10 mL / min, a voltage of 20 kV, a receiving distance of 15 cm, a temperature of 37 °C, and a humidity of 70%.

[0095] Composite nanofiber films with internal stress gradients were obtained by continuously spinning two-component electrospinning units.

[0096] Table 4. Structural parameters of the upper and lower layers in the composite nanofiber film of Example 3.

[0097] Level Fiber diameter (nm) Porosity (%) Internal stress (MPa) Upper layer 800 nm 40 0.16 Lower layer 600 nm 70 0.28

[0098] (3) The composite nanofiber film obtained in step (2) is cross-linked with glutaraldehyde for 30 min to enhance mechanical strength;

[0099] (4) The cross-linked composite nanofiber membrane is subjected to internal surface adhesion treatment: a 0.5 wt% giant salamander solution is sprayed onto the inner surface of the composite nanofiber membrane using an electrostatic sprayer to obtain a graded nanofiber composite membrane with adhesion function, and then cut into suitable sizes for use.

[0100] The adhesion force of the hierarchical nanofiber composite nerve repair membrane prepared in this embodiment is 0.15 N / cm. 2 The deformation time is 30-40 seconds.

[0101] Example 4: A method for preparing a hierarchical nanofiber composite membrane for nerve repair, the method comprising:

[0102] (1) A gelatin spinning solution with a mass fraction of 10% was prepared using gelatin as the raw material; and a cellulose spinning solution with a mass fraction of 5% was prepared using cellulose as the raw material.

[0103] (2) The gelatin spinning solution was electrospun in the first electrospinning unit under the conditions of injection speed of 8 mL / min, voltage of 24 kV, receiving distance of 25 cm, temperature of 37 ℃ and humidity of 40%.

[0104] The cellulose spinning solution was electrospun in the second electrospinning unit under the conditions of an injection speed of 10 mL / min, a voltage of 20 kV, a receiving distance of 18 cm, a temperature of 37 °C, and a humidity of 60%.

[0105] Composite nanofiber films with internal stress gradients were obtained by continuously spinning two-component electrospinning units.

[0106] Table 5. Structural parameters of the upper and lower layers in the composite nanofiber film of Example 4.

[0107]

[0108]

[0109] (3) The composite nanofiber film prepared in step (2) is cross-linked with genipin for 10 min to enhance mechanical strength;

[0110] (4) The cross-linked composite nanofiber membrane is subjected to internal surface adhesion treatment: In this step, a spraying method is used to spray the inner surface of the above nanofiber membrane with a dopamine nanoparticle solution of 0.2 wt% by mass through an electrostatic sprayer to obtain a hierarchical nanofiber composite membrane with adhesion effect, and then cut it into a suitable size for use.

[0111] The adhesion force of the hierarchical nanofiber composite nerve repair membrane prepared in this embodiment is 0.08 N / cm. 2 The deformation time is 20-30 seconds.

[0112] Example 5: A method for preparing a hierarchical nanofiber composite membrane for nerve repair, the method comprising:

[0113] (1) A gelatin spinning solution with a mass fraction of 2% was prepared using gelatin as the raw material; and a chitosan spinning solution with a mass fraction of 5% was prepared using chitosan as the raw material.

[0114] (2) Electrospinning of gelatin spinning solution and cellulose spinning solution were carried out in the first electrospinning unit and the second electrospinning unit respectively under the conditions of injection speed of 5 mL / min, voltage of 22 kV, receiving distance of 18 cm, temperature of 37 ℃ and humidity of 60%.

[0115] Composite nanofiber films with internal stress gradients were obtained by continuously spinning two-component electrospinning units.

[0116] Table 6. Structural parameters of the upper and lower layers in the composite nanofiber film of Example 5.

[0117]

[0118]

[0119] (3) The composite nanofiber film obtained in step (2) is cross-linked with glutaraldehyde for 10 min to enhance mechanical strength;

[0120] (4) The cross-linked composite nanofiber membrane is subjected to internal surface adhesion treatment: This step is the same as step (4) in Example 1, except that anthocyanins are used as the adhesion molecules in this example; the mass percentage concentration of anthocyanins in the adhesion molecule activation solution is 0.2%; a hierarchical nanofiber composite membrane with adhesion effect is obtained, and it is cut into a suitable size for use. The adhesion force of the hierarchical nanofiber composite nerve repair membrane prepared in this example is 0.15 N / cm. 2 The deformation time is 30-40 seconds.

[0121] Example 6: A method for preparing a hierarchical nanofiber composite membrane for nerve repair, the method comprising:

[0122] (1) A first mixed spinning solution with a mass fraction of 5% was prepared using polyvinylpyrrolidone and chitosan in a ratio of 9:1; and a second mixed spinning solution with a mass fraction of 5% was prepared using polyvinyl alcohol and chitosan in a ratio of 9:1.

[0123] (2) The first mixed spinning solution was electrospun in the first electrospinning unit under the conditions of injection speed of 8 mL / min, voltage of 24 kV, receiving distance of 25 cm, temperature of 37 °C and humidity of 40%.

[0124] The second mixed spinning solution was electrospun in the second electrospinning unit under the conditions of an injection speed of 10 mL / min, a voltage of 20 kV, a receiving distance of 18 cm, a temperature of 37 °C, and a humidity of 60%.

[0125] Composite nanofiber films with internal stress gradients were obtained by continuously spinning two-component electrospinning units.

[0126] Table 7 Structural parameters of the upper and lower layers in the composite nanofiber film of Example 6 Table 7 Structural parameters of the upper and lower layers in Example 6

[0127] Level Fiber diameter (nm) Porosity (%) Internal stress (MPa) Upper layer 880 nm 44 0.13 Lower layer 580 nm 75 0.25

[0128] (3) The composite nanofiber film obtained in step (2) is cross-linked with glutaraldehyde for 10 min to enhance mechanical strength;

[0129] (4) The cross-linked composite nanofiber membrane is subjected to internal surface adhesion treatment: This step is the same as step (4) in Example 1, except that gallic acid is used as the adhesion molecule in this example; the mass fraction of gallic acid in the adhesion molecule activation solution is 0.5 wt%; a hierarchical nanofiber composite membrane with adhesion effect is obtained, and it is cut into a suitable size for use. The adhesion force of the hierarchical nanofiber composite nerve repair membrane prepared in this example is 0.14 N / cm. 2 The deformation time is 30-40 seconds.

[0130] Example 7: A method for preparing a hierarchical nanofiber composite membrane for nerve repair, the method comprising:

[0131] (1) A first mixed spinning solution with a mass fraction of 2% was prepared using polyvinylpyrrolidone, methacrylamide hyaluronic acid, and methacrylamide chitosan (in a ratio of 8:1:1) as raw materials; and a second mixed spinning solution with a mass fraction of 5% was prepared using polyvinylpyrrolidone, methacrylamide hyaluronic acid, and methacrylamide chitosan (in a ratio of 8:1:1) as raw materials.

[0132] (2) The first mixed spinning solution and the second mixed spinning solution were electrospun in the first group of electrospinning units and the second group of electrospinning units respectively under the conditions of injection speed of 8 mL / min, voltage of 24 kV, receiving distance of 25 cm, temperature of 37 ℃ and humidity of 40%.

[0133] Composite nanofiber films with internal stress gradients were obtained by continuous two-component electrospinning units and stacked layer by layer.

[0134] Table 8. Structural parameters of the upper and lower layers in the composite nanofiber film of Example 7.

[0135] Level Fiber diameter (nm) Porosity (%) Internal stress (MPa) Upper layer 820 nm 38 0.12 Lower layer 580 nm 75 0.26

[0136] (3) The composite nanofiber film obtained in step (2) is copolymerized and crosslinked under ultraviolet light for 10 min;

[0137] (4) The cross-linked composite nanofiber membrane is subjected to internal surface adhesion treatment: This step is the same as step (4) in Example 1, except that gallic acid is used as the adhesion molecule in this example; the mass fraction of gallic acid in the adhesion molecule activation solution is 0.5 wt%; a hierarchical nanofiber composite membrane with adhesion effect is obtained, and it is cut into a suitable size for use. The adhesion force of the hierarchical nanofiber composite nerve repair membrane prepared in this example is 0.15 N / cm. 2 The deformation time is 30-40 seconds.

[0138] The present invention provides a method for preparing a hierarchical nanofiber composite nerve repair membrane using electrospinning technology to construct nanofiber membranes with different internal stress gradients. Under different driving forces, this nanofiber membrane can deform spontaneously, encapsulating the severed nerve ends, rapidly closing the nerve ends, effectively reducing secondary damage, shortening surgical time, and improving repair efficiency and effectiveness. Therefore, as an alternative to artificial nerve conduit scaffolds, this nanofiber membrane has significant research value and broad clinical application prospects.

[0139] The method for preparing hierarchical nanofiber composite membranes provided by this invention utilizes the structural characteristics of controllable nanofiber diameter and porosity to obtain nanofiber layers with different diameters and porosities. Multilayer nanofibers with different internal stresses are stacked to form a composite nanofiber film with an internal stress gradient. Each layer has a continuous porosity gradient. In the presence of liquid, the hierarchical nanofiber composite membrane can rapidly change shape to wrap around severed nerve ends for treatment.

[0140] The graded nanofiber composite membrane provided by this invention does not require consideration of the nerve diameter during use, nor does it require pre-processing into a tube of a specific diameter through complex technology. During use, it can be in close contact with the nerve without the complicated surgical procedure of connecting the nerve to the cannula, making it easy to operate and reducing surgical time.

[0141] The graded nanofiber composite membrane provided by this invention has the advantage that, when in use, the adhesion layer on the inner surface can make close contact with the nerve, has good fit, and can provide the mechanical strength required for the nerve repair process, without the need for sutures, and can reduce secondary damage.

[0142] The graded nanofiber composite membrane provided by this invention has a thin film thickness (50um-2000um), only at the micrometer level, requires less material, is lightweight, and is easily deformable to encapsulate nerves. It can also match nerve repair degradation, thereby reducing the medical safety risks during the nerve repair process.

[0143] The hierarchical nanofiber composite membrane provided by this invention uses multiple sets of continuous spinning units to prepare nanofibers with different porosities, which are stacked layer by layer. The process is a one-time multi-layer composite molding process that can be completed on a single production line, making the process simple.

[0144] The hierarchical nanofiber composite membrane provided by this invention has a relatively smooth inner wall after it transforms into a tube. The nerve side can be in close contact with the cannula wall, but the nerve section does not contact the cannula. This does not hinder the radial growth of the nerve, which is conducive to the radial growth and repair of the nerve and improves the repair efficiency.

[0145] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, modifications or variations can still be made to the technical solutions described above, and these modifications and variations all fall within the protection scope of the present invention.

Claims

1. A method for preparing a hierarchical nanofiber composite membrane for nerve repair, characterized in that, The method includes: (1) Multilayer nanofibers with different internal stresses are prepared by continuous electrospinning units. The multilayer nanofibers with different internal stresses are stacked layer by layer to form a composite nanofiber film with internal stress gradient. The prepared composite nanofiber film has 2 to 3 layers of nanofibers, and the diameter gradient difference between adjacent nanofibers is maintained at 5% to 35%. The porosity gradient difference between adjacent layers is maintained at 5% to 35%. (2) The composite nanofiber film is chemically cross-linked; (3) Adhesive molecules are used to adhere the upper surface of the composite nanofiber membrane after step (2) to prepare a hierarchical nanofiber composite membrane with internal stress gradient and self-deformation under the action of liquid to wrap nerves.

2. The method for preparing a hierarchical nanofiber composite membrane for nerve repair according to claim 1, characterized in that, Step (1) specifically includes the following steps: (1.1) Use one or more polymer materials as raw materials to prepare the spinning solution; (1.2) The spinning solution is spun using multiple sets of electrospinning units to obtain multilayer nanofibers stacked on top of each other; by controlling the conditions of electrospinning, the diameter and / or porosity of the nanofibers in the different nanofiber layers are different, thereby obtaining a composite nanofiber film with internal stress gradient.

3. The method for preparing a hierarchical nanofiber composite membrane for nerve repair according to claim 2, characterized in that, In step (1.1), the polymeric material includes polyethylene glycol diacrylate, methacrylic anhydride gelatin, methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide hyaluronic acid, methacrylamide cellulose polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, chitosan, cellulose, silk fibroin, gelatin, polyacrylic acid, or polyamide; the concentration of the polymeric material in the spinning solution ranges from 2% to 10%. In step (1.2), the electrospinning conditions are as follows: the injection speed of the spinning solution is 2-10 mL / min, the voltage is 18-25kV, the air pressure is 0.2-2MPa, the receiving distance is 15-45cm, the temperature is 20-50℃, and the humidity is 40%-80%.

4. The method for preparing a hierarchical nanofiber composite membrane for nerve repair according to claim 1, characterized in that, Step (1) specifically includes the following steps: (1.1) Prepare at least two different spinning solutions, each containing one or more polymeric materials; the different spinning solutions contain different polymeric materials; (1.2) Different spinning solutions are spun using different sets of electrospinning units to obtain multilayer nanofibers stacked on top of each other; by controlling the type of polymer material in different spinning solutions, different nanofiber layers have different internal stresses, thereby obtaining composite nanofiber films with internal stress gradients.

5. The method for preparing a hierarchical nanofiber composite membrane for nerve repair according to claim 4, characterized in that, In step (1.1), the polymeric material includes polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polyamide, polyethylene glycol diacrylate, methacrylic anhydride gelatin, methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide hyaluronic acid, methacrylamide cellulose polyvinyl alcohol, chitosan, cellulose, silk fibroin, gelatin, polyacrylic acid, or polyamide; The concentration of the polymer material in the spinning solution ranges from 2% to 10%. The number of polymer materials in the same spinning solution is 1-3.

6. The method for preparing a hierarchical nanofiber composite membrane for nerve repair according to claim 1, characterized in that, In step (3), the adhesion treatment specifically includes: spraying an adhesion molecule solution onto the upper surface of the cross-linked composite nanofiber membrane; and preparing a hierarchical nanofiber composite membrane with an internal stress gradient that self-deforms under the action of liquid to wrap nerves after cutting, sterilization, and packaging.

7. The method for preparing a hierarchical nanofiber composite membrane for nerve repair according to claim 1, characterized in that, In step (3), the adhesion treatment specifically includes: (3.1) The upper surface of the cross-linked composite nanofiber membrane is modified by filling the cavity with nitrogen gas through plasma surface treatment technology to activate the upper surface of the composite nanofiber membrane and introduce amino groups into the upper surface of the composite nanofiber membrane. (3.2) The adhesive molecules are dissolved in the EDC / NHS system to form an adhesive molecule activation solution. The EDC / NHS system is used to activate the carboxyl groups in the adhesive molecules. The composite nanofiber membrane treated in step (3.1) is immersed in the adhesive molecule activation solution. The amino groups on the surface of the composite nanofiber membrane and the activated carboxyl groups in the adhesive molecules undergo an amidation reaction. After the reaction is completed, the membrane is washed with deionized water to remove unreacted adhesive molecules, so that the adhesive molecules are stably combined with the nanofibers on the surface of the composite nanofiber membrane. Then, the membrane is freeze-dried, cut, sterilized, and packaged to obtain a graded nanofiber composite membrane.

8. A method for preparing a hierarchical nanofiber composite membrane for nerve repair according to claim 6 or 7, characterized in that, The adhesion molecules include any one of levodopa, tannic acid, gallic acid, anthocyanins, and secretions from giant salamanders.

9. A hierarchical nanofiber composite nerve repair membrane for nerve repair, characterized in that, The hierarchical nanofiber composite nerve repair membrane comprises at least two nanofiber layers with different internal stresses, and the internal stress of the nanofiber layers is distributed from small to large from the upper surface to the lower surface of the hierarchical nanofiber composite nerve repair membrane; the upper surface of the hierarchical nanofiber composite nerve repair membrane includes adhesive molecules; in the hierarchical nanofiber composite nerve repair membrane, the composite nanofiber film has 2 to 3 nanofiber layers, the diameter gradient difference between adjacent nanofiber layers is maintained at 5% to 35%, and the porosity gradient difference between adjacent layers is maintained at 5% to 35%; The thickness of the hierarchical nanofiber composite nerve repair membrane is 50µm-2000µm, and the adhesion force of the hierarchical nanofiber composite nerve repair membrane is 0.01N / cm. 2 ~0.2 N / cm 2 The deformation time is 10s~100s.

10. An application of the hierarchical nanofiber composite nerve repair membrane according to claim 9, characterized in that, The graded nanofiber composite nerve repair membrane is used in the preparation of products for repairing traumatic peripheral nerve injuries.

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