Multilayer composite capacitive film, method of making, and use in nerve repair scaffolds

Abstract

The application belongs to the technical field of nerve grafts, and discloses a multilayer composite capacitive film, a preparation method and application of the multilayer composite capacitive film in nerve repair scaffolds. The scaffold is composed of three layers, namely, an A layer, a B layer and an A layer. The A layer is a sandwiched layer on both sides, and comprises a mixture of chitosan (or silk fibroin or a decellularized matrix) and a conductive polymer PEDOT:PSS and a crosslinking agent polyethylene glycol diglycidyl ether (PEGDE). The B layer is a middle hydrogel sandwiched layer, and comprises methacrylated gelatin (Gel-MA). The A layers on both sides are bonded through the B layer to form a viscoelastic hydrogel after ultraviolet light irradiation, and the composite capacitive scaffold in a multilayer structure with a through middle cavity is obtained after curing and air drying. The formed scaffold has good electrical conductivity, mechanical properties and biological safety, and can effectively induce the proliferation, migration and differentiation of RSC96 Schwann cells and nerve system cells PC12, thereby providing a good physical stable support and a favorable microenvironment for peripheral nerve injury repair.

Description

Technical Field

[0001] This invention belongs to the field of neural graft technology, and relates to a multilayer composite capacitor film, its preparation method, and its application in neural repair scaffolds. Background Technology

[0002] Peripheral nerve injury (PNI) is a global clinical problem, primarily caused by traction injuries, lacerations, gunshot wounds, compression injuries, ischemia, and other factors, resulting in temporary or permanent neurological dysfunction. Injuries to the nervous system typically lead to nerve transection, disruption of communication between neurons and their supporting cells, and damage to the blood-nerve barrier. Unlike the central nervous system, peripheral nerves exhibit some regenerative capacity after injury. While this inherent regenerative capacity helps alleviate the surgical burden of peripheral nerve injuries, this spontaneous regenerative process is very limited and insufficient to support spontaneous regeneration after severe trauma, such as long interneural gaps following nerve transection, to achieve complete functional recovery. Given the large number of peripheral nerve injury cases, surgical repair of peripheral nerve injuries remains a top priority.

[0003] To date, autologous nerve transplantation remains the most effective form of nerve grafting and is still considered the "gold standard" for evaluating the efficacy of various nerve grafts. However, its limited availability, difficulty in matching tissue structure and size, long-term denervation of the donor site, and variability in transplant efficacy restrict its widespread application. Allogeneic nerve transplantation is also limited due to the need for long-term immunosuppressant use and its low success rate. These limitations necessitate research into the application of nerve substitutes.

[0004] To overcome the drawbacks of autologous nerve transplantation, artificial nerve conduits are increasingly being considered an alternative treatment. Nerve guide conduits (NGCs) are tissue-engineered tubular structures made of natural and / or synthetic biopolymers that possess the mechanical and biochemical properties required for nerve regeneration. NGCs can overcome the limitations of nerve transplantation and suturing methods. NGCs act as bridges between damaged nerve endings, providing structural and nutritional support to both ends, supporting the invasion of surrounding tissues and axonal regeneration along the conduit.

[0005] Electrical signals have been shown to provide crucial cues for bioactivity, promoting axonal extension and accelerating neurological function recovery. Existing research has demonstrated that alternating current (AC) or direct current (DC) electric fields can promote the growth and functional recovery of the sciatic nerve in rodents, while continuous electrical stimulation not only stimulates calcium activity and axonal growth in DRG neurons but also promotes Schwann cell proliferation and upregulation of neurotrophic factors. Among numerous tissue-engineered conduits, chitosan (or silk fibroin or decellularized matrix) conduits have been used for peripheral nerve injury repair due to their biocompatibility, non-toxicity, hydrophilicity, biodegradability, and high processability. However, due to the poor conductivity of natural biomaterials, they cannot perfectly mimic the oscillatory transmission rhythm of autologous nerve signals, resulting in poor signal response to damaged downstream nerve cells and an inability to promptly promote specific cellular event sequences that influence the regeneration process. Conductive polymers, such as PEDOT:PSS, as novel intelligent biomaterials, have shown great potential in recent years for biomedical applications such as tissue regeneration, drug delivery, and neural interfaces because they can directly deliver electrical stimulation and electrochemical signals to cells to regulate cell fate. Summary of the Invention

[0006] In view of this, the present invention aims to provide a multilayer composite capacitor film, a preparation method thereof, and its application in a nerve repair scaffold. In addition to providing sufficient mechanical structural support, the multilayer composite capacitor film also provides good nutritional permeability and conductivity, which can better promote the regeneration and repair of peripheral nerves.

[0007] The technical solution provided by this invention is as follows:

[0008] A method for preparing a multilayer composite capacitor film includes the following steps:

[0009] S1. Add biomaterial, conductive polymer and crosslinking agent to deionized water, stir in a water bath at 40-50℃ for 2-4 hours, add acetic acid, continue stirring in a water bath at 40-50℃ for 2-4 hours; sonicate for 30-45 minutes, let stand for 2-3 hours, remove residual bubbles, and obtain a mixed solution; the biomaterial is one of chitosan, silk fibroin and decellularized matrix.

[0010] S2. The mixed solution is uniformly coated onto an adhesive glass slide, placed in an ultra-clean fume hood, and air-dried for 8-12 hours. The film layer is then removed and washed with NaOH and deionized water in sequence, and air-dried to obtain film A.

[0011] S3. Dissolve methacrylamide gelatin in a photoinitiated LAP solution to obtain layer B solution. After coating layer B solution onto film A, place another layer of film A and cure by ultraviolet irradiation to obtain a multilayer composite capacitor film.

[0012] Furthermore, the biomaterial is chitosan with a molecular weight of 200–300 kDa.

[0013] Furthermore, in step S1, the ratio of the amount of biomaterial, conductive polymer, crosslinking agent, deionized water and acetic acid is (0.6-1.0)g:(0.02-0.06)g:(0.1-0.2)g:(10-20)mL:(0.1-0.2)mL.

[0014] Furthermore, the degree of amino substitution of the methacrylamide gelatin is 30-90±5%, and the molecular weight is 100-200 kDa.

[0015] Furthermore, the concentration of the methacrylamide gelatin is 5–10 wt%.

[0016] Furthermore, the removed membrane layer is washed sequentially with NaOH and deionized water, specifically: soaked in 5-10% NaOH for 1-2 hours to remove residual acetic acid; after removal, soaked in deionized water, with the water changed every 30-60 minutes until the pH of the soaking solution is the same as that of the deionized water, to remove residual NaOH and acetic acid.

[0017] The present invention also provides a multilayer composite capacitor film prepared by the above-described preparation method.

[0018] The present invention also provides an application of the above-mentioned multilayer composite capacitor film in the preparation of a multilayer composite capacitor scaffold for nerve injury repair.

[0019] The present invention also provides a multilayer composite capacitor support, which is prepared from the above-mentioned multilayer composite capacitor film. The preparation method is as follows: the multilayer composite capacitor film is wound around a stainless steel round rod, and methacrylamide gelatin is coated at the side opening. The film is then cured by ultraviolet irradiation to form a multilayer composite capacitor support with a through cavity.

[0020] Compared with existing technologies, this invention uses natural polymer chitosan (or silk fibroin or decellularized matrix) and conductive polymer PEDOT:PSS through chemical crosslinking with polyethylene glycol diglycidyl ether (PEGDE), and then combines them with a photocurable gelatin (Gel-MA) hydrogel layer to form a capacitor scaffold. The natural polymer chitosan (or silk fibroin or decellularized matrix) serves as the main supporting structure of the scaffold, providing excellent mechanical support and a porous structure for material exchange throughout the entire conduit. PEDOT:PSS particles continuously dispersed in the chitosan (or silk fibroin or decellularized matrix) layer act as the conductive medium. The methacrylamide gelatin (Gel-MA) hydrogel layer serves as the elastic buffer layer and energy storage layer of the entire composite capacitor scaffold. This invention combines the crosslinking agent polyethylene glycol diglycidyl ether (PEGDE) with PEDOT:PSS, further improving the conductivity and biocompatibility of PEDOT:PSS. Chemically cross-linked PEDOT:PSS exhibits improved stability, enabling more uniform dispersion and fixation within the chitosan (or silk fibroin or decellularized matrix) layer, thus enhancing scaffold conductivity. This ensures signal continuity during neuronal electrical signal transmission and promotes signal exchange between the proximal and distal ends of damaged nerves. The introduction of a Gel-MA hydrogel layer as an intermediate insulating layer in the capacitor structure provides high elastic support and enriches surrounding tissue fluid with beneficial nutrients and ionic liquids. Based on this, a sandwich structure consisting of chitosan (or silk fibroin or decellularized matrix) with poor conductivity but good mechanical properties and biocompatibility, and PEDOT:PSS with excellent conductivity, combined with an elastic buffer layer of methacrylamide gelatin (Gel-MA), forms a conductive and non-conductive layer, allowing for the implantation of multilayer composite capacitors. The non-conductive hydrogel layer acts as a power station for the bio-microcapacitor, storing excess charge transmitted from the upstream nerve by the conductive layer when the neuron fires. When cells grow, migrate, and proliferate, the stored charge released by the cellular mechanical stimulation material serves as an activation signal, stimulating the initiation of regeneration-related cellular functions. This plays a crucial role in recruiting the nutritional factors required for nerve regeneration and stimulating target organs. Furthermore, the substrates selected in this invention are all biodegradable and biocompatible green and harmless materials. The fabrication process is simplified by neutralizing the acid and alkali in a neutral environment, greatly avoiding the adverse effects of residual toxic organic solvents in in vitro cell culture and scaffold implantation. Examples demonstrate that the multilayer composite capacitive scaffold described in this invention has a porous structure with better mechanical support and material exchange, which is more conducive to axonal electrical signal transmission, providing a better microenvironment for cell adhesion, proliferation, and differentiation. This results in a better promoting effect on peripheral nerve regeneration and repair, and it can be more widely applied to the repair of different types of peripheral nerve injuries. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the fabrication process of the multilayer composite capacitor support described in this invention;

[0022] Figure 2 These are SEM feature images of the stents in Embodiments 1, 2, 3 and Comparative Example 1 of the present invention;

[0023] Figure 3 This is a statistical chart of the Young's modulus of the stents in Embodiments 1, 2, 3 and Comparative Example 1 of the present invention;

[0024] Figure 4 These are statistical graphs of the stent conductivity of Embodiments 1, 2, 3 and Comparative Example 1 of the present invention;

[0025] Figure 5 This is a statistical chart of the cytotoxicity verification of various types of scaffolds using the MTT assay in Test Example 1 of this invention;

[0026] Figure 6 These are images showing the state of RSC96 cell growth and differentiation promoted by various types of scaffold membranes in Test Example 2 of this invention after 48 hours.

[0027] Figure 7 These are images showing the state of PC12 cell growth and differentiation promoted by various types of scaffold membranes in Test Example 2 of this invention after 48 hours. Detailed Implementation

[0028] To more clearly demonstrate the research objectives, experimental technical solutions, and advantages of this invention, the technical solutions of this invention will be described in more detail below. The described examples are only some embodiments of this invention, not all embodiments. Therefore, the technical routes involved are not limited to the mentioned embodiments, and all related combinations are protected by this invention.

[0029] This invention provides a method for preparing a multilayer composite capacitor film and a multilayer composite capacitor support, comprising the following steps:

[0030] (1) Add biomaterials, conductive polymers and crosslinking agents to deionized water, stir in a water bath at 40-50°C for 2-4 hours, add acetic acid, and continue stirring in a water bath at 40-50°C for 2-4 hours; sonicate for 30-45 minutes, let stand for 2-3 hours, remove residual bubbles, and obtain a mixed solution; the preferred biomaterials are chitosan, silk fibroin and decellularized matrix.

[0031] (2) The mixed solution obtained in step (1) is uniformly coated on an adhesive glass slide, placed in an ultra-clean fume hood, and air-dried for 8-12 hours. The film layer is removed and washed with NaOH and deionized water in sequence, and then air-dried to obtain film A.

[0032] (3) Dissolve methacrylamide gelatin in a photoinitiated LAP solution to obtain layer B solution;

[0033] (4) After coating the B layer solution onto the A film, place another layer of the A film and cure it by ultraviolet irradiation to obtain a multilayer composite capacitor film.

[0034] (5) The multilayer composite capacitor film obtained in step (4) is wound onto a stainless steel rod, and methacrylamide gelatin is coated at the side opening. It is then cured by ultraviolet irradiation to form a multilayer composite capacitor support with a through-cavity. A schematic diagram of the fabrication process is shown below. Figure 1 As shown.

[0035] Example 1

[0036] A multilayer composite capacitor film and a multilayer composite capacitor support, and a method for preparing the same, comprising the following steps:

[0037] (1) Add 0.8g of chitosan, 0.02g of conductive polymer PEDOT:PSS and 0.1g of crosslinking agent PEGDE to 20ml of deionized water and stir in a water bath at 40℃ for 2h to prepare a mixed solution of 4% chitosan + 0.1% PEDOT:PSS + 0.5% PEGDE.

[0038] (2) Add 0.2 ml of acetic acid to the completely dissolved mixed solution obtained in step (1) while stirring slowly dropwise, and then continue stirring in a 50°C water bath for 2 hours.

[0039] (3) Place the solution from step (2) in a water bath ultrasonic instrument and sonicate for 30 minutes, then let it stand for 2 hours to remove residual air bubbles from the solution.

[0040] (4) Apply 1.5 ml of the solution from step (3) evenly to a 2 cm × 4 cm adhesive glass slide. The glass slide has been hydrophilically treated and rinsed and dried in a muffle furnace at 500 °C. Place the glass slide coated with the solution on a horizontal platform and let it stand for 30 minutes to completely remove air bubbles. Then, keep it horizontal and place it in an ultra-clean fume hood. Turn on the lowest setting (1) and let it air dry for 8 hours.

[0041] (5) Immerse the completely air-dried composite membrane from step (4) in a 10% NaOH solution for 1 hour to remove residual acetic acid.

[0042] (6) Soak the composite membrane from step (5) in deionized water. Replace the deionized water every 30 minutes for the first 4 times and every 60 minutes for the next 4 times to remove residual NaOH and acetic acid, so that the pH of the soaking solution is the same as that of the deionized water.

[0043] (7) Place the composite film from step (6) into a cleanroom fume hood, turn on the lowest setting (1), and air dry for 8 hours. Then, place the composite film together with the adhesive glass slide into a sealed plastic bag to dry and store for later use.

[0044] (8) Under light-protected conditions, 0.05 g of photoinitiator LAP was added to 20 ml of 1x PBS buffer and stirred in a 50°C water bath for 20 min to prepare a 0.25% LAP solution. 1 g of methacrylated gelatin (Gel-MA(30)) solid was added to 20 ml of the 0.25% LAP solution and stirred in a 60°C water bath for 30 min to prepare a 5% methacrylated gelatin (Gel-MA(30)). The prepared solution was used immediately, and the remainder was stored at 4°C in the dark. When using, it was reheated to 40°C to dissolve.

[0045] (9) Take the composite film in step (7) as layer A. Under light-proof environment, apply 100 μL of the 5% Gel-MA (30) solution in step (8) as layer B on layer A placed on an adhesive glass slide. Allow it to stretch freely for 10 seconds. Then place the same layer A on layer B, cover it with a glass slide to fix the shape, and place it on a portable curing light source. Turn on the UV lamp to irradiate both sides for 30 seconds each, so that Gel-MA (30) can be completely cured into glue to form a multilayer composite capacitor film.

[0046] (10) Wrap the multilayer composite capacitor film from step (9) around a stainless steel rod with a diameter of 2 mm, coat the side interface with 30 μL Gel-MA (30), and cure it by UV light for 30 seconds to form a multilayer composite capacitor support with a through cavity.

[0047] (11) Take a microscopic SEM image of the multilayer composite capacitor support from step (10) using a transmission electron microscope (SEM) (e.g., ...). Figure 2 As shown), the Young's modulus, measured by the MTS universal testing machine, is 16.53 ± 1.166 MPa. Figure 3 As shown in the figure, the conductivity, measured by a four-probe conductivity meter, is 0.1308 ± 0.5520 × 10⁻⁶. -2 S / m (e.g.) Figure 4 (As shown).

[0048] Example 2

[0049] (1) Add 0.8g of chitosan, 0.04g of conductive polymer PEDOT:PSS and 0.1g of crosslinking agent PEGDE to 20ml of deionized water and stir in a water bath at 40℃ for 2h to prepare a mixed solution of 4% chitosan + 0.2% PEDOT:PSS + 0.5% PEGDE.

[0050] (2) Add 0.2 ml of acetic acid to the completely dissolved solution in step (1) while stirring slowly, and then continue stirring in a water bath at 50°C for 2 hours.

[0051] (3) Place the solution from step (2) in a water bath ultrasonic instrument and sonicate for 30 minutes, then let it stand for 2 hours to remove residual air bubbles from the solution.

[0052] (4) Apply 1.5 ml of the solution from step (3) evenly to a 2 cm × 4 cm adhesive glass slide. The glass slide has been hydrophilically treated and rinsed and dried in a muffle furnace at 500 °C. Place the glass slide coated with the solution on a horizontal platform and let it stand for 30 minutes to completely remove air bubbles. Then, keep it horizontal and place it in an ultra-clean fume hood. Turn on the lowest setting (1) and let it air dry for 8 hours.

[0053] (5) Immerse the completely air-dried composite membrane from step (4) in a 10% NaOH solution for 1 hour to remove residual acetic acid.

[0054] (6) Soak the composite membrane from step (5) in deionized water. Replace the deionized water every 30 minutes for the first 4 times and every 60 minutes for the next 4 times to remove residual NaOH and acetic acid, so that the pH of the soaking solution is the same as that of the deionized water.

[0055] (7) Place the composite film from step (6) into a cleanroom fume hood, turn on the lowest setting (1), and air dry for 8 hours. Then, place the composite film together with the adhesive glass slide into a sealed plastic bag to dry and store for later use.

[0056] (8) Under light-protected conditions, 0.05 g of photoinitiator LAP was added to 20 ml of 1x PBS buffer and stirred in a 50°C water bath for 20 min to prepare a 0.25% LAP solution. 1 g of methacrylated gelatin (Gel-MA(30)) solid was added to 20 ml of the 0.25% LAP solution and stirred in a 60°C water bath for 30 min to prepare a 5% methacrylated gelatin (Gel-MA(30)). The prepared solution was used immediately, and the remainder was stored at 4°C in the dark. When using, it was reheated to 40°C to dissolve.

[0057] (9) Take the composite film in step (7) as layer A. Under light-proof environment, apply 100 μL of the 5% Gel-MA (30) solution in step (8) as layer B on layer A placed on an adhesive glass slide. Allow it to stretch freely for 10 seconds. Then place the same layer A on layer B, cover it with a glass slide to fix the shape, and place it on a portable curing light source. Turn on the UV lamp to irradiate both sides for 30 seconds each, so that Gel-MA (30) can be completely cured into glue to form a multilayer composite capacitor film.

[0058] (10) Wrap the multilayer composite capacitor film from step (9) around a stainless steel rod with a diameter of 2 mm, coat the side interface with 30 μL Gel-MA (30), and cure it by UV light for 30 seconds to form a multilayer composite capacitor support with a through cavity.

[0059] (11) Take a microscopic SEM image of the multilayer composite capacitor support from step (10) using a transmission electron microscope (SEM) (e.g., ...). Figure 2 As shown), the Young's modulus, measured by the MTS universal testing machine, is 18.59 ± 0.987 MPa. Figure 3 As shown in the figure, the conductivity was measured using a four-probe conductivity meter and was 0.1551 ± 0.5081 × 10⁻⁶. -2 S / m (e.g.) Figure 4 (As shown).

[0060] Example 3

[0061] (1) Add 0.8g of chitosan, 0.06g of conductive polymer PEDOT:PSS and 0.1g of crosslinking agent PEGDE to 20ml of deionized water and stir in a water bath at 40℃ for 2h to prepare a mixed solution of 4% chitosan + 0.3% PEDOT:PSS + 0.5% PEGDE.

[0062] (2) Add 0.2 ml of acetic acid to the completely dissolved solution in step (1) while stirring slowly, and then continue stirring in a water bath at 50°C for 2 hours.

[0063] (3) Place the solution from step (2) in a water bath ultrasonic instrument and sonicate for 30 minutes, then let it stand for 2 hours to remove residual air bubbles from the solution.

[0064] (4) Apply 1.5 ml of the solution from step (3) evenly to a 2 cm × 4 cm adhesive glass slide. The glass slide has been hydrophilically treated and rinsed and dried in a muffle furnace at 500 °C. Place the glass slide coated with the solution on a horizontal platform and let it stand for 30 minutes to completely remove air bubbles. Then, keep it horizontal and place it in an ultra-clean fume hood. Turn on the lowest setting (1) and let it air dry for 8 hours.

[0065] (5) Immerse the completely air-dried composite membrane from step (4) in a 10% NaOH solution for 1 hour to remove residual acetic acid.

[0066] (6) Soak the composite membrane from step (5) in deionized water. Replace the deionized water every 30 minutes for the first 4 times and every 60 minutes for the next 4 times to remove residual NaOH and acetic acid, so that the pH of the soaking solution is the same as that of the deionized water.

[0067] (7) Place the composite film from step (6) into a cleanroom fume hood, turn on the lowest setting (1), and air dry for 8 hours. Then, place the composite film together with the adhesive glass slide into a sealed plastic bag to dry and store for later use.

[0068] (8) Under light-protected conditions, 0.05 g of photoinitiator LAP was added to 20 ml of 1x PBS buffer and stirred in a 50°C water bath for 20 min to prepare a 0.25% LAP solution. 1 g of methacrylated gelatin (Gel-MA(30)) solid was added to 20 ml of the 0.25% LAP solution and stirred in a 60°C water bath for 30 min to prepare a 5% methacrylated gelatin (Gel-MA(30)). The prepared solution was used immediately, and the remainder was stored at 4°C in the dark. When using, it was reheated to 40°C to dissolve.

[0069] (9) Take the composite film in step (7) as layer A. Under light-proof environment, apply 100 μL of the 5% Gel-MA (30) solution in step (8) as layer B on layer A placed on an adhesive glass slide. Allow it to stretch freely for 10 seconds. Then place the same layer A on layer B, cover it with a glass slide to fix the shape, and place it on a portable curing light source. Turn on the UV lamp to irradiate both sides for 30 seconds each, so that Gel-MA (30) can be completely cured into glue to form a multilayer composite capacitor film.

[0070] (10) Wrap the multilayer composite capacitor film from step (9) around a stainless steel rod with a diameter of 2 mm, coat the side interface with 30 μL Gel-MA (30), and cure it by UV light for 30 seconds to form a multilayer composite capacitor support with a through cavity.

[0071] (11) Take a microscopic SEM image of the multilayer composite capacitor support from step (10) using a transmission electron microscope (SEM) (e.g., ...). Figure 2 As shown), the Young's modulus was 20.63 ± 0.819 MPa, as determined by the MTS universal testing machine. Figure 3 As shown in the figure, the conductivity, measured by a four-probe conductivity meter, is 0.1086 ± 0.5283 × 10⁻⁶. -2 S / m (e.g.) Figure 4 (As shown).

[0072] Comparative Example 1

[0073] The preparation of a pure chitosan membrane conduit, the method comprising the following steps:

[0074] (1) Add 0.4g of chitosan to 10ml of deionized water and stir in a water bath at 40℃ for 2h to prepare a 4% chitosan solution.

[0075] (2) Add 0.1 ml of acetic acid to a 4% chitosan solution, and slowly add it dropwise while stirring. Then continue stirring in a 50°C water bath for 2 hours.

[0076] (3) Place the solution from step (2) in a water bath ultrasonic instrument and sonicate for 30 minutes, then let it stand for 2 hours to remove residual air bubbles from the solution.

[0077] (4) The solution from step (3) is injected into a stainless steel tube with an inner diameter of 2 mm and an outer diameter of 4 mm using a syringe. 1.5 ml is injected into each tube. After sealing both ends with caps, the tube is placed vertically into a beaker and then placed in a -20℃ freezer for 2 hours to solidify.

[0078] (5) Take out the sleeve that has been frozen and solidified in step (4), quickly remove the sealing cap and outer sleeve, and put the inner core of the sleeve back into the -20℃ freezer for 4 hours to freeze and solidify.

[0079] (6) The inner core of the sleeve that has been frozen and solidified in step (5) is transferred into a freeze dryer at -60°C and freeze-dried for 12 hours.

[0080] (7) Remove the completely dried inner core from the freeze dryer in step (6), slowly remove the pure chitosan membrane conduit from the inner core, and soak it in 10% NaOH solution for 2 hours to remove residual acetic acid.

[0081] (8) Remove the pure chitosan membrane tubing from step (7) from the 10% NaOH solution and place it in deionized water. Change the deionized water every 30 minutes for the first four times and every 60 minutes for the next four times to remove residual NaOH and acetic acid. The pH test paper showed that the pH of the soaking solution was the same as that of the deionized water.

[0082] (9) Place the pure chitosan membrane tube from step (8) back into a freeze dryer at -60°C and freeze dry for 12 hours.

[0083] (10) Remove the pure chitosan membrane conduit from the freeze dryer and take a microscopic SEM image using a transmission electron microscope (SEM) (e.g., ...). Figure 2 As shown), the Young's modulus, measured by the MTS universal testing machine, is 13.43 ± 1.323 MPa. Figure 3 As shown in the figure, the conductivity, measured by a four-probe conductivity meter, is 0.0908 ± 0.6325 × 10⁻⁶. -2 S / m (e.g.) Figure 4 (As shown).

[0084] Test Example 1: MTT assay for the cytotoxicity of composite capacitor scaffolds

[0085] (1) The multilayer composite capacitor films of Examples 1, 2, and 3 and the pure chitosan film of Comparative Example 1 were cut into pieces with an area of ​​3 cm². 2 After sterilizing the strips of various sizes with UV light for 24 hours, rinse them three times with sterile water and once with PBS, and then place them in 24-well plates for later use.

[0086] (2) In the well plate from step (1), add 1 ml of L-DMEM or RPMI-1640 basic culture medium to each well, and place the well plate containing the soaking material in a 5% CO2, 37°C constant temperature cell culture incubator for static extraction for 24 h. At the same time, seed 5000 rat Schwann cell line RSC96 cells and rat adrenal pheochromocytoma cell line PC12 cells into blank 96-well plates.

[0087] (3) Transfer the extract from step (2) into a centrifuge tube, centrifuge at 1000 rpm for 5 min, and add horse serum, FBS and PS to prepare complete culture medium for the extract. The culture medium used for RSC96 cells is L-DMEM complete culture medium containing 10% FBS + 1% PS, and the culture medium used for PC12 cells is RPMI-1640 complete culture medium containing 10% horse serum + 5% FBS + 1% PS. The blank control group cells are in the above two complete culture media without extract.

[0088] (4) Remove the old culture medium from the 96-well plate in which RSC96 cells and PC12 cells were inoculated in step (3), rinse with PBS, add 100 μL of complete culture medium of extraction solution to each well, and place in a 5% CO2, 37°C constant temperature cell culture incubator for 48 h.

[0089] (5) Discard the extract and complete culture medium from the cells cultured to the corresponding time point in step (4), add 100ul of MTT (5mg / ml) working solution to each well, and incubate in a constant temperature cell culture incubator at 37℃ for 4h in the dark.

[0090] (6) Add 100 μL of formazan solution to each well in step (5), place the plate in the dark on a constant temperature shaker at 37°C, and dissolve for 4 hours at 50 rpm / min.

[0091] (7) Gently shake the well plate in step (6) to ensure there are no air bubbles in the wells, and measure the absorbance value at a wavelength of 570 nm. After comparing it with the blank control group (cells cultured alone in well plates without added material) and Comparative Example 1, the results show that the multilayer composite capacitor membrane provided in this embodiment of the invention has no significant cytotoxicity to rat Schwann cell line RSC96 cells and rat adrenal pheochromocytoma cell line PC12 cells (e.g., Figure 5 (As shown).

[0092] Test Example 2: Determining the effect of multilayer composite capacitor membranes on cell proliferation and differentiation

[0093] (1) Cut the multilayer composite capacitor membrane of Example 2 and the pure chitosan membrane of Comparative Example 1 into round pieces with a diameter of 1.5 cm. After sterilizing with ultraviolet light for 24 h, rinse them three times with sterile water and once with PBS, and place them in a 24-well plate for later use.

[0094] (2) The rat Schwann cell line RSC96 and the rat adrenal pheochromocytoma cell line PC12, which had been pre-cultured to the exponential growth phase, were digested with 0.25% trypsin for 45 seconds. After digestion, the cells were centrifuged at 1000 rpm for 5 minutes. After centrifugation, the cells were resuspended in complete culture medium and counted. The cells were then seeded onto the membrane material in step (1), with 3000 cells seeded onto each membrane. 200 μL of complete culture medium was added to each well. After the cells adhered to the membrane in a 5% CO2, 37°C incubator for 4 hours, 300 μL of complete culture medium was added, and the cells were incubated for another 48 hours. The culture medium used for RSC96 cells was L-DMEM complete culture medium containing 10% FBS and 1% PS, and the culture medium used for PC12 cells was RPMI-1640 complete culture medium containing 10% horse serum, 5% FBS, and 1% PS.

[0095] (3) After discarding the culture medium from the cells in step (2), add 1 ml of PBS to each well and wash 3 times for 5 min each time. Add 1 ml of 4% paraformaldehyde fixative to each well and fix for 1 h at room temperature. Discard the fixative and wash 3 times for 5 min each time with PBS. Add 1 ml of blocking buffer to each well and block for 2 h at room temperature. Discard the waste liquid. Add 200 μl of phalloidin (1:1000) to each well and incubate in the dark at room temperature for 1 h. Discard the waste liquid and wash 3 times for 5 min each time with PBS. Add 200 μl of DAPI (1:1000) to each well and incubate in the dark at room temperature for 10 min. Discard the waste liquid and wash 3 times for 5 min each time with PBS.

[0096] (4) Imaging the prepared cell immunofluorescence slides from step (3) under a microscope (e.g.) Figure 6 , Figure 7 As shown in the figure, after comparing the cell growth on the pure chitosan membrane in Comparative Example 1, it was found that the composite capacitor scaffold membrane was more able to promote the attachment, proliferation and differentiation of rat Schwann cell line RSC96 cells and rat adrenal pheochromocytoma cell line PC12 cells.

[0097] Based on the above embodiments, comparative examples, and test cases, it can be seen that:

[0098] 1. The Young's modulus of the multilayer composite capacitor scaffold provided in this embodiment of the invention is (20.63±0.819) MPa, which is 211 to 258% higher than that of the pure chitosan scaffold (8.91±1.24) MPa. In addition to providing more stable mechanical structural support and nutrient permeability, it still maintains good biodegradability.

[0099] 2. The conductivity of the multilayer composite capacitor support provided in this embodiment of the invention is (15.51±0.51)×10 -2S / m and conductivity of pure chitosan scaffold (9.08±0.63)×10 -2 Compared to S / m, it is 165-178% higher, which is more conducive to the AC transmission of axon electrical signals between upstream and downstream.

[0100] 3. The RSC96 and PC12 cells cultured on the multilayer composite capacitor scaffold described in this invention exhibited 80-90% of the cell viability of the blank control group, with no significant cytotoxicity. Their proliferation rate and axonal growth status reached 80-90% of the blank control group, providing a favorable microenvironment for cell growth, adhesion, proliferation, and differentiation in the neural regeneration and repair process.

[0101] 4. The multilayer composite capacitor stent described in this invention can be easily prepared into different sizes, making it more widely applicable to the repair of peripheral nerve damage of different distances and degrees.

[0102] In summary, compared with the pure chitosan scaffold, the multilayer composite capacitor scaffold described in the embodiments of this invention exhibits a 211-258% increase in Young's modulus and a 165-178% increase in conductivity, and shows no significant cytotoxicity in cultured RSC96 and PC12 cells. Compared with scaffolds prepared solely from biomaterials, the chemically cross-linked composite capacitor scaffold containing the conductive polymer PEDOT:PSS demonstrates improved stability. The conductive polymer is more uniformly dispersed and fixed within the biomaterial layer, acting as a conductive medium for nanoparticles, thereby increasing the scaffold's conductivity and promoting electrical signal exchange between the proximal and distal ends of damaged nerves. The introduction of a Gel-MA hydrogel layer as an intermediate insulating layer in the capacitor structure provides highly elastic support and enriches beneficial nutrients and ionic liquids from the surrounding tissue fluid, playing a crucial role in recruiting nutrients required for nerve regeneration and stimulating target organs. Furthermore, all selected substrates are biodegradable and biocompatible green and harmless materials. Therefore, the multilayer composite capacitor scaffold described in this invention has a porous structure with better mechanical support and material exchange, which is more conducive to the transmission of axonal electrical signals, provides a better microenvironment for cell adhesion, proliferation and differentiation, and is more widely applicable to the repair of different types of peripheral nerve injuries.

Claims

1. A method for preparing a multilayer composite capacitor film, characterized in that, Includes the following steps: S1. Add the biomaterial, conductive polymer, and crosslinking agent to deionized water, stir in a water bath at 40-50°C for 2-4 hours, add acetic acid, and continue stirring in a water bath at 40-50°C for 2-4 hours; sonicate for 30-45 minutes, let stand for 2-3 hours to remove residual bubbles, and obtain a mixed solution; the biomaterial is one of chitosan, silk fibroin, and decellularized matrix; the conductive polymer is PEDOT:PSS; the crosslinking agent is polyethylene glycol diglycidyl ether; S2. The mixed solution is uniformly coated onto an adhesive glass slide, placed in an ultra-clean fume hood, and air-dried for 8-12 hours. The film layer is then removed and washed with NaOH and deionized water in sequence, and air-dried to obtain film A. S3. Dissolve methacrylamide gelatin in a photoinitiated LAP solution to obtain layer B solution. After coating layer B solution onto film A, place another layer of film A and cure by ultraviolet irradiation to obtain a multilayer composite capacitor film.

2. The preparation method according to claim 1, characterized in that, The biomaterial is chitosan with a molecular weight of 200-300 kDa.

3. The preparation method according to claim 2, characterized in that, In step S1, the ratio of biomaterial, conductive polymer, crosslinking agent, deionized water and acetic acid is (0.6~1.0)g:(0.02~0.06)g:(0.1~0.2)g:(10~20)mL:(0.1~0.2)mL.

4. The preparation method according to claim 1, characterized in that, The methacrylamide gelatin has an amino substitution degree of 30~90±5% and a molecular weight of 100~200 Kda.

5. The preparation method according to claim 4, characterized in that, The concentration of the methacrylamide gelatin is 5-10%.

6. The preparation method according to claim 1, characterized in that, The removed membrane layer is washed sequentially with NaOH and deionized water. Specifically, it is soaked in 5-10% NaOH for 1-2 hours to remove residual acetic acid; after removal, it is soaked in deionized water, with the water changed every 30-60 minutes, until the pH of the soaking solution is the same as that of the deionized water to remove residual NaOH and acetic acid.

7. A multilayer composite capacitor film, characterized in that, The multilayer composite capacitor film is prepared by the preparation method of any one of claims 1-6, and the multilayer composite capacitor film is composed of an outer layer of composite conductive biomaterial and a hydrogel interlayer.

8. The multilayer composite capacitor film according to claim 7, characterized in that, The outer layer of the composite conductive biomaterial is formed by chemical cross-linking of chitosan and conductive polymer PEDOT:PSS with polyethylene glycol diglycidyl ether (PEGDE), and the hydrogel interlayer is methacrylamide gelatin.

9. The application of the multilayer composite capacitor film prepared by the preparation method according to any one of claims 1-6 or the multilayer composite capacitor film according to claim 7 or 8 in the preparation of a multilayer composite capacitor scaffold for nerve injury repair.

10. A multilayer composite capacitor support, characterized in that, The multilayer composite capacitor support is prepared by the multilayer composite capacitor film prepared by the preparation method of any one of claims 1-6 or the multilayer composite capacitor film of claim 7 or 8. The preparation method is as follows: the multilayer composite capacitor film is wound around a stainless steel round rod, and methacrylamide gelatin is coated at the side opening. The film is cured by ultraviolet light to form a multilayer composite capacitor support with a through cavity.

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