Electronic functional fibroin nerve repair conduit with electronic structure, preparation method and application thereof

Abstract

The invention discloses an electronic functional fibroin nerve repair conduit with an electronic structure, a preparation method and application thereof. An electric conductor is embedded in the neural restoration catheter and is used for applying electrical stimulation in situ or collecting neural electrophysiological signals; the preparation method comprises the following steps: manufacturing a conduit wall with electronic functionalization by utilizing a silk fibroin solution and a nerve conduit core layer material, performing directional freezing treatment on the conduit wall by utilizing a plastic pipe, a specific copper block and the nerve conduit core layer material, and then performing freeze drying on the whole conduit subjected to the directional freezing treatment. Therefore, an oriented porous material structure is formed in the conduit, and finally the electronic functional fibroin nerve repair conduit with an electronic structure is formed. The electronic functional catheter provides stable and lasting electrical stimulation conduction, promotes nerve fiber regeneration and functional recovery, promotes neuron repair, prevents secondary mechanical damage to nerves, and can also be used for collecting nerve activity conditions in a repair area and monitoring the nerve repair process.

Description

Technical Field

[0001] The present invention relates to the field of biomedical devices, and in particular to an electronically functionalized silk protein nerve repair conduit with an electronic structure, a preparation method and applications thereof. Background Art

[0002] Repairing nerve damage is a major challenge in clinical medicine and tissue engineering. Traditional autologous nerve transplantation has shown certain efficacy in bridging nerve defects, but its clinical application is significantly restricted due to the limited source of donor nerves and the possibility of functional damage in the donor area. Biomaterial-based nerve conduits are considered to be a potential alternative to autologous transplantation, which can provide structural support and the microenvironment required for regeneration at the site of damaged nerves, showing good prospects for filling nerve defects. However, most existing nerve conduits have a single cavity structure and lack effective physical and biological signal guidance, making it difficult to achieve precise migration of neurons and directional growth of axons. Their repair effect is still not comparable to that of autologous transplantation.

[0003] As a natural nerve conduit core material, silk fibroin has excellent mechanical properties, adjustable degradability and good biocompatibility. It can provide an ideal growth microenvironment for nerve cells and shows great application potential in the field of nerve repair. Studies have shown that silk conduits can optimize the delivery of cell migration nutrients and substances through their torsional structure and directional fiber arrangement, thereby significantly promoting the directional regeneration of nerve fibers (CN 112263714 B, Progress in Neurobiology 131 (2015) 87-104). In addition, the surface of silk materials can be optimized through neurochemical modification to enhance their interaction with nerve cells, thereby improving biological functions such as cell adhesion, proliferation and differentiation. For example, foreign scholars have combined silk with bioactive molecules (such as nerve growth factor) to further enhance the effect of nerve repair (ACs Appl. Mater. interfaces 2022, 14, 46188-46200).

[0004] As an important auxiliary means in nerve regulation and repair, electrical stimulation enhances changes in cell membrane potential, activates electrosensitive ion channels, and regulates key signal pathways (such as calcium ion flow and nerve growth factor expression) by applying electric fields or electrical signals in nerve conduits, thereby promoting the proliferation and differentiation of neurons, significantly promoting nerve fiber regeneration, accelerating axon extension, and enhancing nerve function recovery. In the silk fibroin-based nerve conduit technology system, there has been no design breakthrough in integrating functional electrodes directly into the conduit matrix. Existing technologies mostly focus on using the excellent biocompatibility and degradability of silk fibroin to construct a three-dimensional growth microenvironment. How to embed a stable electrode system in this biological matrix so that the conduit has both the dual functions of a nerve-directed regeneration scaffold and in situ electrical stimulation regulation remains a key scientific issue that needs to be urgently addressed in this field. Summary of the Invention

[0005] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide an electronic functionalized silk protein nerve repair conduit with an electronic structure and its preparation method and application, to provide stable and long-lasting electrical stimulation conduction, promote nerve fiber regeneration and functional recovery.

[0006] The technical solution adopted by the present invention is as follows, comprising the following steps:

[0007] 1. An electronically functionalized silk protein nerve repair conduit with electronic structure:

[0008] An electric conductor is embedded in the nerve repair conduit, which can be used to apply electric stimulation in situ or collect nerve electrophysiological signals. The nerve repair conduit is subjected to a directional freezing treatment to form a nerve conduit core layer with a directional arranged porous structure.

[0009] 2. A method for preparing an electronically functionalized silk protein nerve repair conduit having an electronic structure, the method comprising:

[0010] S1. Using silk fibroin solution to fabricate an electronically functionalized catheter wall for use as a conductive shell;

[0011] S2. The prefabricated catheter wall is subjected to a directionally frozen treatment using a plastic tube, a specific copper block and a nerve catheter core layer material having biological activity. The whole catheter subjected to the directionally frozen treatment is then placed in a freeze dryer for freeze drying, thereby forming a porous material structure with a directionally oriented arrangement inside the catheter, and finally forming an electronically functionalized silk protein nerve repair catheter with an electronic structure, wherein both the catheter wall and the nerve catheter core layer have a porous structure.

[0012] like Figure 1 and Figure 2 As shown, the step S1 is specifically as follows:

[0013] S1.1. Preparing a film material using a silk fibroin solution, and obtaining a silk fibroin film by natural drying or freeze drying;

[0014] S1.2. Prepare degradable electrical conductors and embed them into the silk film;

[0015] S1.3. A silk film containing electrical conductors is prepared by a film winding process to form and serve as an electronically functionalized catheter wall of a catheter.

[0016] The step S2 specifically comprises placing the catheter wall in a prefabricated plastic tube, and making the outer periphery of the catheter wall close to the inner wall of the plastic tube, with one end of the catheter wall flush with one end of the plastic tube and sealed at the port with a copper block, and placing or not placing a preformed rod in the middle of the catheter wall, injecting a nerve catheter core layer material solution from the other end of the catheter wall and the plastic tube, so that the nerve catheter core layer material solution fills the hollow space inside the catheter wall or the hollow space between the inner wall of the catheter wall and the preformed rod, and then immersing the end of the copper block that does not participate in the sealing in a low-temperature environment, so that the nerve catheter core layer material solution in the catheter is directionally frozen in the low-temperature environment by transferring the low temperature through the copper block, so that a nerve catheter core layer with a porous structure is formed in the catheter wall, and the catheter wall also has a porous structure.

[0017] In a specific implementation, the nerve conduit can be prepared into two forms, hollow and solid, without placing the preform rod. The hollow conduit needs to add the preform rod during preparation, while the solid conduit does not need the preform rod.

[0018] By not inserting a preform rod, a solid nerve repair conduit can be prepared to fill in the long-distance nerve loss. Instead, the nerve repair conduit with the preform rod inserted can be used as a cannula to connect the two stumps of the nerve or connect the stump of the nerve to the solid nerve conduit.

[0019] The copper block has a base portion, a raised portion and a peripheral portion, the raised portion and the peripheral portion are concentrically fixed on the same side surface of the base portion, and an annular gap is provided between the raised portion and the peripheral portion, the conduit wall and one end of the plastic tube are inserted into the annular gap, so that the raised portion is embedded in one end of the conduit wall, the peripheral portion surrounds the outer circumference of the same end of the plastic tube, and the surface of the base portion between the raised portion and the peripheral portion is in close contact with the end surface of the conduit wall and one end of the plastic tube.

[0020] The temperature of the low-temperature environment should be lower than -70°C, including liquid nitrogen, dry ice, ethanol cold bath or temperature-controllable low-temperature freezer.

[0021] The conduit wall is actually similar to silk fibroin with a porous structure, wherein the pore size of the pore structure is 1-10 μm.

[0022] The electric conductors are made of degradable conductive materials, including degradable metals (magnesium, zinc, iron and alloys thereof), conductive polymers (polypyrrole, polythiophene derivatives, polyaniline), and conductive carbon-based materials (carbon nanotubes, graphene and derivatives thereof).

[0023] The catheter wall is further loaded with anti-fibrotic drugs or factors, including dexamethasone, antibodies (such as TGF-β inhibitors) or anti-proliferative molecules (such as mitomycin C);

[0024] The catheter wall can also be loaded with anti-inflammatory molecules (such as IL-10 or IL-4) to reduce inflammatory cell infiltration.

[0025] The anti-fibrosis drugs or factors and anti-inflammatory molecules are loaded by immersing the whole catheter in a solution containing the anti-fibrosis drugs or factors / anti-inflammatory molecules after directional freezing, specifically loaded in the porous structure of the catheter wall and the nerve catheter core layer.

[0026] The material of the nerve guide core layer is mainly composed of a base material and an active material added into the base material.

[0027] The nerve conduit core layer material solution comprises a base material and an active material added to the base material as solutes, wherein the base material uses deionized water as a solvent, the mass concentration of the base material is 2.5% to 10%, and the active material uses PBS as a solvent, the mass concentration of the active material is 4 μg / ml;

[0028] The base material is one or more of collagen, gelatin, hyaluronic acid, chitosan, silk fibroin, polylactic acid-co-glycolic acid, polycaprolactone, and polyethylene glycol;

[0029] The active material is a mixture of seed cells with therapeutic effects and one or both of cytokines with therapeutic effects, wherein the seed cells include one or more of neural stem cells, bone marrow mesenchymal stem cells, fibroblasts, Schwann cells or olfactory ensheathing cells, and the cytokines include one or more of nerve growth factor, brain-derived growth factor or basic fibroblast growth factor.

[0030] In the step S1, the method of embedding the electrical conductors in the silk film material includes a casting and curing method and a stacking molding method;

[0031] The winding process adopts loose winding and utilizes the wetting effect after solution injection to make the layers of the silk protein film adhere to each other, so as to maintain the porous structure after freeze-drying.

[0032] When the method is implemented, the hollow structure can also be prepared by adding a preformed rod when the solution is frozen.

[0033] 3. The electronically functionalized silk protein nerve repair catheter is used to apply electrical stimulation to promote nerve function repair, or to collect nerve activity in the nerve function repair area and monitor the progress of nerve repair.

[0034] The electrical conductor in the electronically functionalized silk protein nerve repair conduit is a degradable electrical conductor, which is used to apply electrical stimulation and is tightly connected to biological tissue, thereby promoting nerve function repair.

[0035] 4. An application method of an electronically functionalized silk protein nerve repair conduit, the specific application method includes the following two aspects:

[0036] The electronically functionalized silk protein nerve repair conduit is implanted and arranged at the site of nerve injury:

[0037] Solid nerve repair conduit, used to fill in long-distance nerve gaps.

[0038] The hollow nerve repair catheter acts as a cannula, connecting the two stumps of the damaged nerve or connecting the stump of the damaged nerve to the solid nerve catheter. One end of the electrical wire is connected to an external neuromodulator, while the other end contacts the inner core of the electronically functionalized silk protein nerve repair catheter. The wire receives electrical signals from the external neuromodulator, applying electrical stimulation to the damaged area to promote nerve function repair.

[0039] The main innovation of the present invention is that conductive material is bonded to the catheter wall to increase the conductivity of the nerve repair catheter; and a directional structure that is beneficial to nerve repair is formed by directional freezing of the inner core material.

[0040] The beneficial effects of the present invention are:

[0041] (1) The present invention utilizes the principle of silk fibroin self-assembly and directional freeze-drying to produce oriented silk fibroin nanofibers with a size between 10 and 200 μm. These fibers can provide the physical guidance signals required for constructing cells and nerve axons within the conduit, promoting the directional extension of axons. They can also be loaded with therapeutic seed cells or cytokines to promote neuronal repair. The structural strength of silk fibroin also provides appropriate support to prevent secondary mechanical damage to nerves.

[0042] (2) The porous structure of the nerve conduit wall of the present invention can promote the exchange of nutrients and oxygen, while preventing fluid accumulation, providing a suitable microenvironment for nerve regeneration; the conduit wall can be loaded with anti-fibrosis drugs or factors and anti-inflammatory molecules to reduce adverse reactions such as fibrosis and inflammatory cell infiltration.

[0043] (3) The introduction of conductive materials enables the silk nerve conduit to achieve stable and long-lasting electrical stimulation conduction, which can significantly promote the regeneration of nerve fibers, accelerate axonal extension, and enhance the recovery of nerve function. It can also be connected to external electrophysiological data acquisition equipment to collect information about nerve activity in the repair area and monitor the progress of nerve repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a schematic diagram of a typical preparation process of the electronically functionalized silk protein nerve repair conduit of the present invention;

[0045] Figure 2 This is a second schematic diagram of a typical preparation process of the electronically functionalized silk protein nerve repair conduit of the present invention;

[0046] Figure 3 This is a schematic structural diagram of the electronically functionalized silk protein nerve repair conduit of the present invention;

[0047] Figure 4 This is a schematic diagram of the preparation process of the electronically functionalized silk protein nerve repair conduit of the present invention;

[0048] Figure 5 This is a schematic structural diagram of the copper block in the electronically functionalized silk protein nerve repair conduit of the present invention;

[0049] Figure 6 This is a schematic diagram of the application state of the electronically functionalized silk protein nerve repair conduit of the present invention for nerve connection;

[0050] Figure 7 is a scanning electron micrograph of Example 1;

[0051] Figure 8 is the digital photograph of Example 2;

[0052] Figure 9 is a digital photograph of the catheter wall of Example 5;

[0053] Figure 10 is a scanning electron micrograph of Comparative Example 1.

[0054] In the figure: electronic functionalized silk protein nerve repair catheter 0, catheter wall 1, plastic tube 2, copper block 3, nerve catheter core layer material solution 4, low temperature environment 5, preform rod 6, damaged nerve 71, hollow nerve catheter 72, solid nerve catheter 73; base part 31, raised part 32, outer part 33.

[0055] PET substrate 11 , porous silk film 12 , electric wire 13 , hot roller 14 , PEDOTPSS electrode 15 , electronic porous silk film 16 . DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solutions and advantages of the present application clearer, the present application is further described in detail below in conjunction with the accompanying drawings and Examples. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present application and are not intended to limit the present application. In general, the terms and phrases used herein have generally recognized meanings in their respective fields and can be found by reference to standard texts, periodicals or texts known in the art. The following definitions are provided to clarify their specific applications in the present invention.

[0057] In the specific implementation of the present invention, a special device for directional freezing is provided, including a copper block, a preformed rod, a plastic tube, etc.

[0058] like Figure 4 As shown, the catheter wall 1 is first placed in a prefabricated plastic tube 2, which is open at both ends to form a through-hole pipe structure. The axial lengths of the catheter wall 1 and the plastic tube 2 are the same. The outer periphery of the catheter wall 1 is closely attached to the inner wall of the plastic tube 2, and one end of the catheter wall 1 is flush with one end of the plastic tube 2 and sealed with a copper block 3. A preform rod 6 is placed in the middle of the catheter wall 1. A nerve conduit core layer material solution 4 is injected from the other end of the catheter wall 1 and the plastic tube 2, so that the nerve conduit core layer material solution 4 fills the hollow space between the inner wall of the catheter wall 1 and the preform rod 6. Then, the end of the catheter wall 1 and the plastic tube 2 sealed by the copper block 3 is vertically immersed in a low-temperature environment, while the other end is not in the low-temperature environment. The nerve conduit core layer material solution in the catheter is directionally frozen in the low-temperature environment 5, so that a nerve conduit core layer with a porous structure is formed in the catheter wall, and the catheter wall also has a porous structure.

[0059] like Figure 3 As shown, the directional and oriented porous material structure means that the catheter wall of the catheter and the inner part of the nerve catheter core layer both have multiple pore structures, each pore structure is arranged along the axial direction of the catheter, and the multiple pore structures are arranged at intervals along the circumference of the catheter wall.

[0060] The nerve conduit core layer has directional parallel channels along the length direction, and the pore size is 10-200 μm;

[0061] The wall of the conduit has a porous structure and also has oriented parallel pores along the length direction, and the pore diameter is 10-200 μm.

[0062] like Figure 5As shown, more specifically, the copper block 3 has a base portion 31, a raised portion 32 and a peripheral portion 33. The raised portion 32 and the peripheral portion 33 are concentrically fixed on the same side surface of the base portion 31, and an annular gap is provided between the raised portion 32 and the peripheral portion 33. One end of the conduit wall 1 and the plastic tube 2 that is flush is inserted into the annular gap, so that the raised portion 32 is embedded in one end of the conduit wall 1, and the peripheral portion 33 surrounds the outer periphery of the same end of the plastic tube 2, and the surface of the base portion 31 between the raised portion 32 and the peripheral portion 33 is in close contact with the end surface of the conduit wall 1 and the plastic tube 2 that are flush.

[0063] Specifically, the electronic functionalized silk protein nerve repair conduit is implanted and arranged at the site of nerve damage, such as Figure 6 As shown: For example, a hollow nerve conduit 72 is used as a sleeve, connected between the two broken ends of the damaged nerve 71, or connected between one broken end of the damaged nerve 71 and the solid nerve conduit 73. One end of the electrical conductor is connected to an external nerve regulation device, and the other end contacts the inner core of the electronic functionalized silk protein nerve repair conduit; the electrical conductor receives the electrical signal from the external nerve regulation device and applies electrical stimulation to the damaged area to promote nerve function repair. The electronic functionalized silk protein nerve repair conduit is implanted and placed at the site of nerve injury, with one end of the electrical conductor connected to an external electrophysiological acquisition device and the other end contacting the inner core of the functionalized silk protein nerve repair conduit; the electrical conductor receives the electrical signal from the external electrophysiological acquisition device and then collects the nerve activity in the nerve repair area for monitoring the nerve repair process.

[0064] The embodiments of the present invention are as follows:

[0065] Example 1

[0066] (1) 0.5 g of calcium chloride was added to 8.2 ml of formic acid for dissolution. After the dissolution was completed, a calcium chloride-formic acid solution with a concentration of 6.1 wt% was obtained. 1.5 g of degummed silk was dissolved in the above 8.2 ml of calcium chloride-formic acid solution and placed in an ultrasonic cleaning machine for oscillation for 1 hour. After oscillation, a regenerated silk fibroin solution was obtained. A clean PET (polyethylene terephthalate) substrate was taken and the regenerated silk fibroin solution was scraped and coated on the PET substrate using a four-sided preparation device with a thickness of 200 μm. After curing at room temperature, the regenerated silk fibroin solution formed a silk fibroin gel film, and the silk fibroin gel film was naturally dried.

[0067] (2) The silk fibroin gel film and the silver wire are bonded and compressed in sequence by hot roller pressing, that is, a silk fibroin film containing an electric wire is prepared on a PET substrate, and then the silk fibroin film containing an electric wire is peeled off from the PET substrate by mechanical stripping.

[0068] (3) The silk film containing the electrical conductors is subjected to a film winding process to prepare a catheter wall with electronic functionality.

[0069] (4) 80.77 g of lithium bromide was added to 50 ml of deionized water and stirred to dissolve. After full dissolution, the volume was adjusted to 100 ml. After the volume was adjusted to 9.3 M lithium bromide solution, 20 g of degummed silk was dissolved in the above 100 ml of lithium bromide solution and placed in a 60°C oven to dissolve for 1 h. After full dissolution, the solution was dialyzed using a 14 kD dialysis membrane for three days to obtain a regenerated silk fibroin solution. The concentration was measured and adjusted to 2.5 wt% as the nerve conduit core material solution.

[0070] (5) The prefabricated catheter wall is tightly placed in a prefabricated plastic tube, one end of which is sealed with a copper block. A 2.5 wt% regenerated silk fibroin solution is injected into the hollow structure in the catheter wall that is not sealed with a copper block. A prefabricated rod is inserted into the regenerated silk fibroin solution to prepare an inner core hollow structure.

[0071] (6) Immerse one end of the copper block base in liquid nitrogen to directionally freeze the regenerated silk solution in the catheter.

[0072] (7) The frozen catheter is placed in a freeze dryer for freeze drying to form an oriented porous material structure inside the catheter, thereby obtaining a hollow electronically functionalized silk protein nerve repair catheter with an electronic structure. Figure 3 The structure diagram of this embodiment is shown in FIG. The nerve repair catheter of this embodiment was tested using a scanning electron microscope. The cross section of the catheter is shown in FIG. Figure 7 As shown in a, the cross-sectional structure of the inner core is as follows Figure 7 As shown in b, the longitudinal section structure of the inner core is as follows Figure 7 As shown in c, the inner structure of the catheter wall is as follows Figure 7 As shown in d.

[0073] Example 2

[0074] (1) 0.5 g of calcium chloride was added to 8.2 ml of formic acid for dissolution. After the dissolution was completed, a calcium chloride-formic acid solution with a concentration of 6.1 wt% was obtained. 1.5 g of degummed silk was dissolved in the above 8.2 ml of calcium chloride-formic acid solution and placed in an ultrasonic cleaning machine for oscillation for 1 hour. After oscillation, a regenerated silk fibroin solution was obtained. A clean PET (polyethylene terephthalate) substrate was taken and the regenerated silk fibroin solution was scraped and coated on the PET substrate using a four-sided preparation device with a thickness of 200 μm. After curing at room temperature, the regenerated silk fibroin solution formed a silk fibroin gel film, and the silk fibroin gel film was naturally dried.

[0075] (2) The silk fibroin gel film and the silver wire are bonded and compressed in sequence by hot roller pressing, that is, a silk fibroin film containing silver wires is prepared on a PET substrate, and then the silk fibroin film containing silver wires is peeled off from the PET substrate by mechanical stripping.

[0076] (3) The silk film containing the electrical conductors is subjected to a film winding process to prepare a catheter wall with electronic functionality.

[0077] (4) Take 10 g of gelatin and add it to 100 ml of PBS solution and stir to dissolve. Take 8 ml of methacrylic anhydride and add it to the above 100 ml of gelatin solution. Place it in a heating mantle at 50°C and stir to react for 3 hours. After sufficient reaction, dialyze it with a 14 kD dialysis membrane for one week to obtain GELMA solution. After measuring the concentration, adjust it to 2.5 wt%.

[0078] (5) The prefabricated catheter wall is tightly placed in a prefabricated plastic tube, one end of which is sealed with a copper block. A 2.5 wt% GELMA solution is injected into the hollow structure in the catheter wall that is not sealed with a copper block. A prefabricated rod is inserted into the regenerated silk fibroin solution to prepare an inner core hollow structure.

[0079] (6) Immerse one end of the copper block in liquid nitrogen to allow the GELMA solution in the catheter to undergo directionally freezing.

[0080] (7) The frozen catheter is placed in a freeze dryer for freeze drying to form an oriented porous material structure inside the catheter, thereby obtaining a hollow electronically functionalized silk protein nerve repair catheter with an electronic structure.

[0081] Figure 8 A digital photo of this embodiment is shown, in which electrical wires are bonded to the outer wall of the nerve repair catheter, and the inner wall has a clear hollow structure.

[0082] Example 3

[0083] (1) 0.5 g of calcium chloride was added to 8.2 ml of formic acid for dissolution. After the dissolution was completed, a calcium chloride-formic acid solution with a concentration of 6.1 wt% was obtained. 1.5 g of degummed silk was dissolved in the above 8.2 ml of calcium chloride-formic acid solution and placed in an ultrasonic cleaning machine for oscillation for 1 hour. After oscillation, a regenerated silk fibroin solution was obtained. A clean PET (polyethylene terephthalate) substrate was taken and the regenerated silk fibroin solution was coated on the PET substrate using a four-sided preparation device with a thickness of 200 μm. After curing at room temperature, the regenerated silk fibroin solution formed a silk fibroin gel film.

[0084] (2) The silk fibroin gel film is frozen at a vacuum degree of 0.05 MPa and a cold trap temperature of -50°C, so that the surface of the silk fibroin gel film spontaneously nucleates and freezes, and then in-situ freeze-dried to obtain a porous silk fibroin film after freeze-drying.

[0085] (3) The silk fibroin gel film and the silver wire are bonded and compressed in turn by hot roller pressing, that is, a silk fibroin film containing electric wires is prepared on a PET substrate, and then the silk fibroin film containing electric wires is peeled off from the PET substrate by mechanical stripping.

[0086] (4) The silk film containing the electrical conductors is subjected to a film winding process to prepare a catheter wall with electronic functionality.

[0087] (5) According to the method disclosed in patent CN106421902A, a fast-gelling silk fibroin solution was prepared.

[0088] (6) The prefabricated catheter wall is tightly placed in a prefabricated plastic tube, one end of which is sealed with a copper block. The fast-gelling silk fibroin solution is injected into the hollow structure in the catheter wall that is not sealed with a copper block, and placed at 37°C for 12 hours to induce gelation.

[0089] (7) Immerse one end of the copper block in liquid nitrogen to directionally freeze the regenerated silk solution in the catheter.

[0090] (8) The frozen catheter is placed in a freeze dryer for freeze drying to form an oriented porous material structure inside the catheter, thereby obtaining a solid electronically functionalized silk protein nerve repair catheter with an electronic structure.

[0091] (8) A 4 μg / mL neurotrophic factor solution was prepared using PBS buffer in a sterile environment. The solid electronically functionalized silk protein nerve repair conduit with electronic structure was immersed in the 4 μg / mL neurotrophic factor solution at 37°C for 3 h to obtain an electronically functionalized silk protein nerve repair conduit loaded with neurotrophic factors.

[0092] Example 4

[0093] (1) 0.5 g of calcium chloride was added to 8.2 ml of formic acid for dissolution. After the dissolution was completed, a calcium chloride-formic acid solution with a concentration of 6.1 wt% was obtained. 1.5 g of degummed silk was dissolved in the above 8.2 ml of calcium chloride-formic acid solution and placed in an ultrasonic cleaning machine for oscillation for 1 hour. After oscillation, a regenerated silk fibroin solution was obtained. A clean PET (polyethylene terephthalate) substrate was taken and the regenerated silk fibroin solution was coated on the PET substrate using a four-sided preparation device with a thickness of 200 μm. After curing at room temperature, the regenerated silk fibroin solution formed a silk fibroin gel film.

[0094] (2) The silk fibroin gel film is frozen at a vacuum degree of 0.05 MPa and a cold trap temperature of -50°C, so that the surface of the silk fibroin gel film spontaneously nucleates and freezes, and then in-situ freeze-dried to obtain a porous silk fibroin film after freeze-drying.

[0095] (3) The silk fibroin gel film and the silver wire are bonded and compressed in turn by hot roller pressing, that is, a silk fibroin film containing electric wires is prepared on a PET substrate, and then the silk fibroin film containing electric wires is peeled off from the PET substrate by mechanical stripping.

[0096] (4) The silk film containing the electrical conductors is subjected to a film winding process to prepare a catheter wall with electronic functionality.

[0097] (5) Take 80.77g of lithium bromide and add it to 50ml of deionized water, stir and dissolve it. After it is fully dissolved, the volume is adjusted to 100ml. After the volume is adjusted, a lithium bromide solution with a concentration of 9.3M is obtained. Take 20g of degummed silk and dissolve it in the above 100ml lithium bromide solution. Place it in a 60℃ oven to dissolve for 1h. After it is fully dissolved, dialyze it with a 14kd dialysis membrane for three days to obtain a regenerated silk fibroin solution. After measuring the concentration, adjust it to 2.5wt%.

[0098] (6) The prefabricated catheter wall is tightly placed in a prefabricated plastic tube, one end of which is sealed with a copper block, and a 2.5 wt% regenerated silk fibroin solution is injected into the hollow structure in the catheter wall that is not sealed with a copper block.

[0099] (7) Immerse one end of the copper block in liquid nitrogen to directionally freeze the regenerated silk solution in the catheter.

[0100] (8) The frozen catheter is placed in a freeze dryer for freeze drying to form an oriented porous material structure inside the catheter, thereby obtaining a solid electronically functionalized silk protein nerve repair catheter with an electronic structure.

[0101] Example 5

[0102] (1) 0.5 g of calcium chloride was added to 8.2 ml of formic acid for dissolution. After the dissolution was completed, a calcium chloride-formic acid solution with a concentration of 6.1 wt% was obtained. 1.5 g of degummed silk was dissolved in the above 8.2 ml of calcium chloride-formic acid solution and placed in an ultrasonic cleaning machine for oscillation for 1 hour. After oscillation, a regenerated silk fibroin solution was obtained. A clean PET (polyethylene terephthalate) substrate was taken and the regenerated silk fibroin solution was scraped and coated on the PET substrate using a four-sided preparation device with a thickness of 200 μm. After curing at room temperature, the regenerated silk fibroin solution formed a silk fibroin gel film, and the silk fibroin gel film was naturally dried.

[0103] (2) Mix 5 g of PDMS (polydimethylsiloxane) and 0.5 g of PDMS crosslinker, stir thoroughly, and use a vacuum pump to remove bubbles to obtain a PDMS mixed solution;

[0104] (3) Take a clean PET substrate and use a four-sided preparation device with a thickness of 200 μm to scrape and coat the PDMS mixed solution on the PET substrate, and heat and cure it on a hot plate at 60°C to form a PDMS film.

[0105] (4) PEDOT:PSS (poly(2,3-dihydrothieno-1,4-dioxane)-poly(styrene sulfonate)) solution was scraped onto the surface of the PDMS film using a four-sided preparation device with a thickness of 400 μm and allowed to dry naturally to form a PEDOT:PSS film;

[0106] (5) Using a laser engraving machine to engrave a patterned electrode on the PEDOT:PSS film, placing the patterned PEDOT electrode opposite to the freeze-dried film, performing hot roller bonding and compression in sequence, and transferring the patterned electrode to the surface of the silk film by mechanical peeling;

[0107] (6) The silk fibroin film and the silk fibroin film with patterned electrodes printed on the surface are sequentially hot-rolled and compressed to obtain a silk fibroin film containing patterned electrodes on a PET substrate. The silk fibroin film containing patterned electrodes is then peeled off from the PET substrate by mechanical stripping.

[0108] (7) The silk film containing patterned electrodes is subjected to a film winding process to prepare a catheter wall with electronic functionalization.

[0109] (8) 80.77 g of lithium bromide was added to 50 ml of deionized water and stirred to dissolve. After complete dissolution, the volume was adjusted to 100 ml. After the volume was adjusted to 9.3 M lithium bromide solution, 20 g of degummed silk was dissolved in the above 100 ml lithium bromide solution and placed in a 60°C oven to dissolve for 1 h. After complete dissolution, the solution was dialyzed for three days using a 14 kD dialysis membrane to obtain a regenerated silk fibroin solution. The concentration was measured and adjusted to 2.5 wt%.

[0110] (9) The prefabricated catheter wall is tightly placed in a prefabricated plastic tube, one end of which is sealed with a copper block, and a 2.5 wt% regenerated silk fibroin solution is injected into the hollow structure in the catheter wall that is not sealed with a copper block.

[0111] (10) Immerse one end of the copper block in liquid nitrogen to directionally freeze the regenerated silk solution in the catheter.

[0112] (11) The frozen catheter is placed in a freeze dryer for freeze drying, forming an oriented porous material structure inside the catheter, and obtaining a hollow electronically functionalized silk protein nerve repair catheter with an electronic structure.

[0113] Figure 9a shows a digital photo of this embodiment before winding. In the picture, it can be seen that the patterned PEDOT electrode is transferred to the surface of the silk film. By controlling the size of the silk film in step (6), the exposed electrode position can be selected, and the remaining positions that need to be insulated are protected by bonding with the silk film. Figure 9 b shows a digital photo of this embodiment after winding. After winding, the signal collection point is located at the position on the inner wall of the nerve repair catheter closest to the inner core, which is used to transmit electrical signals to the neural stem cells at the inner core position or to collect electrical signals during the nerve repair process. The electrode at the outermost end of the nerve repair catheter is exposed for external power supply, application of electrical stimulation, or external signal collector to process the collected electrical signals.

[0114] Comparative Example 1: No directional freezing treatment such as copper blocks

[0115] (1) 0.5 g of calcium chloride was added to 8.2 ml of formic acid for dissolution. After the dissolution was completed, a calcium chloride-formic acid solution with a concentration of 6.1 wt% was obtained. 1.5 g of degummed silk was dissolved in the above 8.2 ml of calcium chloride-formic acid solution and placed in an ultrasonic cleaning machine for oscillation for 1 hour. After oscillation, a regenerated silk fibroin solution was obtained. A clean PET (polyethylene terephthalate) substrate was taken and the regenerated silk fibroin solution was scraped and coated on the PET substrate using a four-sided preparation device with a thickness of 200 μm. After curing at room temperature, the regenerated silk fibroin solution formed a silk fibroin gel film, and the silk fibroin gel film was naturally dried.

[0116] (2) The silk fibroin gel film and the silver wire are bonded and compressed in sequence by hot roller pressing, that is, a silk fibroin film containing an electric wire is prepared on a PET substrate, and then the silk fibroin film containing an electric wire is peeled off from the PET substrate by mechanical stripping.

[0117] (3) The silk film containing the electrical conductors is subjected to a film winding process to prepare a catheter wall with electronic functionality.

[0118] (4) 80.77 g of lithium bromide was added to 50 ml of deionized water and stirred to dissolve. After complete dissolution, the volume was adjusted to 100 ml. After the volume was adjusted to 9.3 M lithium bromide solution, 20 g of degummed silk was dissolved in the above 100 ml lithium bromide solution and placed in a 60°C oven to dissolve for 1 h. After complete dissolution, the solution was dialyzed for three days using a 14 kD dialysis membrane to obtain a regenerated silk fibroin solution. The concentration was measured and adjusted to 2.5 wt%.

[0119] (5) The prefabricated catheter wall was placed in a prefabricated plastic tube, one end of which was sealed with PDMS, and a 2.5 wt% regenerated silk fibroin solution was injected into the hollow structure in the catheter wall.

[0120] (6) Place the mold in a -80°C refrigerator to directly freeze the regenerated silk fibroin solution in the catheter.

[0121] (7) The frozen catheter is placed in a freeze dryer for freeze drying to obtain a silk protein nerve repair catheter comparison example.

[0122] The comparative example was tested using a scanning electron microscope, and the longitudinal section structure of the inner core was as follows: Figure 10 As shown, the sheet structure formed by silk fibroin is intertwined with each other and lacks directional parallel channels along the length direction.

[0123] Comparative Example 2: No electrical conductors

[0124] (1) 0.5 g of calcium chloride was added to 8.2 ml of formic acid for dissolution. After the dissolution was completed, a calcium chloride-formic acid solution with a concentration of 6.1 wt% was obtained. 1.5 g of degummed silk was dissolved in the above 8.2 ml of calcium chloride-formic acid solution and placed in an ultrasonic cleaning machine for oscillation for 1 hour. After oscillation, a regenerated silk fibroin solution was obtained. A clean PET (polyethylene terephthalate) substrate was taken and the regenerated silk fibroin solution was scraped and coated on the PET substrate using a four-sided preparation device with a thickness of 200 μm. After curing at room temperature, the regenerated silk fibroin solution formed a silk fibroin gel film, and the silk fibroin gel film was naturally dried.

[0125] (2) The naturally dried silk fibroin film is peeled off from the PET substrate by mechanical peeling without bonding or setting electrical conductors.

[0126] (3) The silk film without the electric wire is subjected to a film winding process to prepare a silk film conduit wall.

[0127] (4) Take 10 g of gelatin and add it to 100 ml of PBS solution and stir to dissolve. Take 8 ml of methacrylic anhydride and add it to the above 100 ml of gelatin solution. Place it in a 50°C heating mantle and stir to react for 3 hours. After sufficient reaction, dialyze it with a 14 kd dialysis membrane for one week to obtain GELMA solution. After measuring the concentration, adjust it to 2.5 wt%.

[0128] (5) The prefabricated catheter wall is placed in a prefabricated plastic tube, one end of which is sealed with a copper block, 2.5 wt% GELMA solution is injected into the hollow structure in the catheter wall, and a prefabricated rod is inserted to prepare an inner core hollow structure.

[0129] (6) Immerse the copper block in liquid nitrogen to directionally freeze the regenerated silk solution in the catheter.

[0130] (7) The frozen catheter is placed in a freeze dryer for freeze drying to form an oriented porous material structure inside the catheter, thereby obtaining a hollow silk protein nerve repair catheter without electronic structure.

[0131] The digital photos of the above embodiments show that the present invention can embed electrical conductors or conductive electrodes in the catheter wall and can control the local insulation protection of the electrical conductors and conductive electrodes; the scanning electron microscopy results reflect the difference between the inner core structures obtained by the directional freezing and non-directional freezing preparation methods.

[0132] From this implementation, it can be seen that the electronically functionalized catheter of the present invention provides stable and long-lasting electrical stimulation conduction, promotes nerve fiber regeneration and functional recovery, promotes neuronal repair, prevents secondary mechanical damage to nerves, and can also be used to collect nerve activity in the repair area and monitor the progress of nerve repair.

[0133] The above specific embodiments are used to illustrate the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

[0134] The above description is only a preferred embodiment of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics and principles described in the scope of the patent application of the present invention are included in the scope of the patent application of the present invention.

Claims

1. An electronically functionalized silk protein nerve repair conduit with an electronic structure, characterized by: An electric conductor is embedded in the nerve repair conduit, and is used for applying electric stimulation in situ or collecting nerve electrophysiological signals. The nerve repair conduit is subjected to a directional freezing treatment to form a nerve conduit core layer with a directional arranged porous structure.

2. A method for preparing the electronically functionalized silk protein nerve repair catheter having an electronic structure as claimed in claim 1, characterized in that: The method comprises: S1. Fabricating an electronically functionalized catheter wall for a catheter using a silk fibroin solution; S2. The catheter wall is subjected to a directionally frozen treatment using a plastic tube, a specific copper block and a biologically active nerve catheter core material, and then the entire catheter subjected to the directionally frozen treatment is freeze-dried, thereby forming a porous material structure with a directionally oriented arrangement inside the catheter, and finally forming an electronically functionalized silk protein nerve repair catheter with an electronic structure.

3. The preparation method according to claim 2, wherein: The step S1 is specifically as follows: S1.

1. Preparing a film material using a silk fibroin solution, and obtaining a silk fibroin film by natural drying or freeze drying; S1.

2. Prepare degradable electrical conductors and embed them into the silk film; S1.

3. A silk film containing electrical conductors is prepared through a film winding process to form a catheter wall with electronic functionality.

4. The preparation method according to claim 2, wherein: The step S2 specifically comprises placing the catheter wall (1) in a prefabricated plastic tube (2) so that the outer periphery of the catheter wall (1) is in close contact with the inner wall of the plastic tube (2), one end of the catheter wall (1) is flush with one end of the plastic tube (2) and sealed at the port with a copper block (3), and a preform rod (6) is placed or not placed in the middle of the catheter wall (1), injecting a nerve catheter core layer material solution (4) from the other end of the catheter wall (1) and the plastic tube (2) so that the nerve catheter core layer material solution (4) fills the hollow space inside the catheter wall (1) or the hollow space between the inner wall of the catheter wall (1) and the preform rod (6), and then immersing the end of the copper block (3) not involved in the sealing into a low-temperature environment, so that the nerve catheter core layer material solution in the catheter is directionally frozen in the low-temperature environment (5) by transferring the low temperature through the copper block, so that a nerve catheter core layer with a porous structure is formed in the catheter wall, and the catheter wall also has a porous structure.

5. The preparation method according to claim 4, characterized in that: The copper block (3) comprises a base portion (31), a raised portion (32) and a peripheral portion (33); the raised portion (32) and the peripheral portion (33) are fixed concentrically on the same side surface of the base portion (31), and an annular gap is provided between the raised portion (32) and the peripheral portion (33); one end of the conduit wall (1) and the plastic tube (2) are inserted into the annular gap, so that the raised portion (32) is embedded in one end of the conduit wall (1), the peripheral portion (33) surrounds the outer periphery of the same end of the plastic tube (2), and the surface of the base portion (31) between the raised portion (32) and the peripheral portion (33) is in close contact with the end surface of one end of the conduit wall (1) and the plastic tube (2).

6. The preparation method according to claim 2, wherein: The catheter wall is further loaded with anti-fibrosis drugs or factors, including dexamethasone, antibodies or anti-proliferation molecules; the catheter wall is also loaded with anti-inflammatory molecules to reduce inflammatory cell infiltration.

7. The preparation method according to claim 2, wherein: The nerve conduit core layer material solution comprises a base material and an active material added to the base material as solutes, wherein the base material uses deionized water as a solvent, the mass concentration of the base material is 2.5% to 10%, and the active material uses PBS as a solvent, the mass concentration of the active material is 4 μg / ml; The base material is one or more of collagen, gelatin, hyaluronic acid, chitosan, silk fibroin, polylactic acid-co-glycolic acid, polycaprolactone, and polyethylene glycol; The active material is a mixture of seed cells with therapeutic effects and one or both of cytokines with therapeutic effects, wherein the seed cells include one or more of neural stem cells, bone marrow mesenchymal stem cells, fibroblasts, Schwann cells or olfactory ensheathing cells, and the cytokines include one or more of nerve growth factor, brain-derived growth factor or basic fibroblast growth factor.

8. The preparation method according to claim 2, wherein: In the step S1, the method of embedding the electrical conductors in the silk film material includes a casting and curing method and a stacking molding method; The winding process adopts loose winding and utilizes the wetting effect after solution injection to make the layers of the silk protein film adhere to each other, so as to maintain the porous structure after freeze-drying.

9. An electronically functionalized silk protein nerve repair catheter, characterized in that: The invention is prepared by the preparation method according to any one of claims 2 to 8.

10. An application of the electronically functionalized silk protein nerve repair catheter according to claim 9, characterized in that: Used to apply electrical stimulation to promote nerve function repair, or to collect nerve activity in the nerve function repair area and monitor the progress of nerve repair.

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