Regenerative neural electrodes and methods of making and using the same

Abstract

The application provides a regenerative nerve electrode and a preparation method and use thereof. The regenerative nerve electrode comprises a base layer, a directional structure layer adhered to a surface of the base layer, and the directional structure layer is interwoven by fiber filaments; and a metal thin film electrode formed on a side of the directional structure layer away from the base layer by using a metal adhesive layer; preferably, the regenerative nerve electrode can be at least partially rolled to form a rolled structure. The regenerative nerve electrode has excellent degradation performance, and the impedance of the regenerative nerve electrode is low, and the electrode shows good stability. The cell signal recorded by the regenerative nerve electrode can significantly promote the dispersion of nerve tissue, and has a basis for improving the recording accuracy of nerve signals in the tissue structure. The regenerative nerve electrode has faster regeneration ability.

Description

Technical Field

[0001] This invention relates to a regenerative neural electrode, its preparation method, and its uses, belonging to the field of medical materials. Background Technology

[0002] Peripheral nerve injury or limb loss leading to loss of function in the nerve innervation area can severely affect daily life activities and may significantly reduce the patient's quality of life. After losing a limb due to amputation or congenital deficiency, cosmetic prostheses can help patients alleviate psychological distress over the missing part of their body and improve their self-image. However, such prostheses cannot help patients restore motor and sensory functions. Therefore, the ideal prosthesis is (1) the same in appearance as a cosmetic prosthesis; (2) controllable by the patient; (3) allowing multiple degrees of freedom of movement; and (4) able to interact with the patient, read the patient's nerve signals, and provide sensory feedback. In order to enable natural control of the prosthesis, an interface for information transmission is needed between the peripheral nervous system (PNS) and the prosthesis, namely peripheral nerve electrodes.

[0003] Electrodes placed proximal to the severed nerve can record commands transmitted from the central nervous system (CNS) and decode these pulse signals into signals that the prosthesis can recognize, thereby controlling the prosthesis's movement. Furthermore, for amputees, the formation of proximal neuromas in nearby tissues at the nerve transection site can lead to increased nerve excitability, resulting in unwanted complications such as hyperreflexia, hyperalgesia, and excessive pain. Therefore, connecting the nerve endings to electrodes to control the prosthesis can significantly reduce patient suffering.

[0004] Researchers have developed various types of electrodes for PNS interfaces to record electrophysiological activities from different biomedical applications. Most peripheral nerve electrodes are implanted around or inside the peripheral nerve to reduce tissue impedance and thus increase the signal-to-noise ratio of the recorded signal. Based on their invasiveness, nerve electrodes can be divided into three main categories: extrafascicular electrodes, intrafascicular electrodes, and regenerative electrodes.

[0005] As implants become more invasive, higher selectivity for individual nerves can be achieved, thus shortening the distance to individual nerves, which has a positive impact on high-quality signal recording. However, high interface selectivity comes at a cost; the more invasive the electrode, the greater the potential for nerve damage. Extrafascicular electrodes, after implantation, come into contact with many axons within the nerve bundle simultaneously, resulting in poorer selectivity but minimal nerve damage. Intrafascicular electrodes can connect to discrete nerve axons within the nerve bundle, achieving very high selectivity, but with a high risk of nerve damage.

[0006] Regenerative electrodes represent a completely different approach to peripheral nerve interface contact. These electrodes are typically implanted onto severed nerves, allowing the nerve to grow internally and make contact with the internal signal recording sites. Compared to the previously described types of electrodes, ideal regenerative nerve electrodes may offer the highest spatial resolution (nerve fiber selectivity) and long-term stability of signal recording. Because these electrodes need to be implanted onto severed nerves, they are well-suited for integration with neuroprosthetics and application to the severed nerve stumps in amputees.

[0007] Currently, there are two main types of regenerative neural electrodes: a sieve structure and a microchannel structure. The sieve electrode is composed of a planar surface with densely packed pores, some or all of which are surrounded by metal electrodes. The proximal and distal ends of the severed nerve rest against the sieve, with an external polymer tube providing mechanical support to allow axons to grow through the sieve pores and reconnect the two ends.

[0008] The main factor to consider when designing mesh electrodes is porosity, which is the ratio of the total area of ​​the pores to the total area of ​​the plane. Higher porosity allows for greater axon growth but reduces mechanical strength. Aperture size affects the selectivity of electrode signal recording and the speed at which the interface forms between the nerve and the electrode recording site. Larger pores result in faster nerve regeneration but lower selectivity for electrode signal recording.

[0009] The design of microchannel electrodes can be considered as the result of stretching a mesh electrode longitudinally, creating a tube with numerous tiny pores. Axons grow along these pores. Microchannel electrodes share advantages with Cuff electrodes, achieving external insulation around the nerve. However, unlike Cuff electrodes, microchannel electrodes have smaller recording sites within the tube, allowing contact with smaller nerve fiber bundles and significantly improved signal recording selectivity. Furthermore, multiple recording sites can be placed within a single channel, enhancing noise suppression and signal selectivity. However, microchannel electrodes also have drawbacks, such as a significant difference in morphology from nerve morphology, and their high structural rigidity leading to a mismatch with neuromechanical properties and potentially triggering inflammatory responses.

[0010] Currently, the primary recipients of neural prostheses are patients with limb disabilities, most of whom have undergone amputation surgery, leaving nerve stumps at the ends of their limbs. While existing regenerative neural electrodes can improve the degree of nerve discretization and record more precise nerve fiber signals within the nerve, they also present the problem of being overly invasive to nerve tissue, potentially triggering severe inflammatory and rejection reactions.

[0011] Therefore, researching a regenerative neural electrode for use at nerve stumps, which can not only significantly improve the degree of nerve discretization but also avoid causing secondary damage to the nerve, has become an urgent technical problem to be solved. Summary of the Invention

[0012] The problem the invention aims to solve

[0013] In view of the technical problems existing in the prior art, the present invention first provides a regenerative neural electrode. The regenerative neural electrode of the present invention has excellent degradation performance and low impedance, exhibiting good stability. The regenerative neural electrode of the present invention can record cellular signals, which can significantly promote the discretization of neural tissue, providing a basis for improving the accuracy of recording neural signals in terms of tissue structure. The regenerative neural electrode of the present invention has faster regeneration capability.

[0014] Furthermore, the present invention also provides a method for preparing a regenerative neural electrode, which is simple and easy to implement, uses readily available raw materials, and is suitable for mass production.

[0015] Solution for solving the problem

[0016] [1] A regenerative neural electrode, wherein the regenerative neural electrode comprises:

[0017] grassroots

[0018] An oriented structural layer, the oriented structural layer being adhered to the surface of the base layer, and the oriented structural layer being composed of interwoven fiber filaments; and

[0019] A metal thin film electrode, wherein the metal thin film electrode utilizes a metal adhesion layer formed on the side of the orientation structure layer opposite to the base layer;

[0020] Preferably, the regenerative neural electrode is at least partially curled to form a curled structure.

[0021] [2] According to the regenerative neural electrode described in [1] above, a portion of the metal thin film electrode extends in the direction of nerve growth and is parallel to the orientation of the orientation structure layer; another portion of the metal thin film electrode extends in the direction perpendicular to nerve growth and is perpendicular to the orientation of the orientation structure layer.

[0022] [3] The regenerative neural electrode according to [1] or [2] above, wherein the base layer is derived from a biodegradable polylactic acid copolymer; preferably, the material of the base layer includes polylactic acid-polytrimethylene carbonate copolymer.

[0023] [4] The regenerative neural electrode according to any one of [1]-[3] above, wherein the directional structural layer is derived from a biodegradable polymer material; preferably, the material of the directional structural layer includes polycaprolactone.

[0024] [5] The regenerative neural electrode according to any one of [1]-[4] above, wherein the metal thin film electrode is derived from a noble metal material, preferably one or a combination of two or more of gold, silver and platinum.

[0025] [6] The regenerative neural electrode according to any one of [1]-[5] above, wherein the metal adhesion layer is derived from an adhesive metal material; preferably, the material of the metal adhesion layer includes titanium and / or chromium.

[0026] [7] The regenerative neural electrode according to any one of [1]-[6] above, wherein the thickness of the substrate is 100 μm to 300 μm; and / or

[0027] The thickness of the oriented structural layer is 20–40 μm; and / or

[0028] The thickness of the metal adhesion layer is 1–20 nm; and / or

[0029] The thickness of the metal thin film electrode is 50–300 nm.

[0030] [8] A method for preparing a regenerative neural electrode according to any one of [1]-[7] above, comprising the step of compositing a base layer, a directional structural layer and a metal thin film electrode.

[0031] [9] The preparation method according to [8] above, wherein the preparation method includes the following steps:

[0032] The oriented structural layer is prepared on one side of the base layer using electrospinning technology;

[0033] A metal adhesion layer is formed on the side of the oriented structure layer opposite to the base layer using magnetron sputtering technology, and a metal thin film electrode is formed on the metal adhesion layer.

[0034] Preferably, the regenerative neural electrode is at least partially curled to form a curled structure.

[0035]

[10] Use of a regenerative neural electrode according to any one of [1]-[7] above in the preparation of a neural repair product.

[0036] The effects of the invention

[0037] The regenerative neural electrode of the present invention has excellent degradation performance, low impedance, and good stability.

[0038] The regenerative neural electrode of the present invention can record cellular signals that can significantly promote the discretization of neural tissue, providing a basis for improving the accuracy of recording neural signals in terms of tissue structure.

[0039] The regenerative neural electrode of the present invention has a faster regenerative capacity.

[0040] Furthermore, the preparation method of the regenerative neural electrode of the present invention is simple and easy to implement, the raw materials are readily available, and it is suitable for mass production. Attached Figure Description

[0041] Figure 1 A schematic diagram of the regenerative neural electrode and the neural growth direction of the present invention is shown;

[0042] Figure 2 SEM images of the surface of the regenerative neural electrode are shown; the left image is a characterization of the metal thin film electrode on the directional structure layer, and the right image is a micrograph of the metal thin film electrode within the red dashed box.

[0043] Figure 3 A schematic diagram of the fabrication process for regenerative neural electrodes is shown.

[0044] Figure 4 A schematic diagram showing the impedance results of the regenerative neural electrode is presented.

[0045] Figure 5 This diagram illustrates how neurons are guided to regenerative neural electrodes to grow in a directed manner.

[0046] Figure 6 This invention illustrates a quantitative characterization of the orientation of DRG neuronal cells on the directional structure layer in the regenerative neural electrode of the present invention;

[0047] Figure 7 A schematic diagram illustrating the degradation process of a simulated regenerative neural electrode in vivo is shown.

[0048] Figure 8 A schematic diagram illustrating the impedance stability characterization of a simulated regenerative neural electrode is shown.

[0049] Figure 9 A schematic diagram of the two-dimensional planar electrode structure of the regenerative neural electrode of the present invention is shown;

[0050] Figure 10 A schematic diagram of the DRG neuron cell signal recording device of the present invention is shown;

[0051] Figure 11The diagram shows the spontaneous signals generated by DRG neurons without external stimulation when the neurons are cultured on two-dimensional planar electrodes; the top image shows the original signal, and the bottom image shows the signal after filtering and noise reduction.

[0052] Figure 12 The classification of signal characteristics of DRG neurons after they were cultured on two-dimensional planar electrodes is shown.

[0053] Figure 13 The statistical analysis of the signal firing frequency of DRG neurons after they were cultured on two-dimensional planar electrodes is shown.

[0054] Figure 14 A schematic diagram of the two-dimensional planar electrode of the regenerative neural electrode of the present invention being rolled into a three-dimensional tubular electrode is shown.

[0055] Figure 15 The diagram shows the DRG neuron cell signal measured by the regenerative neural electrode of the present invention after DRG neuron cells were cultured at the opening of a three-dimensional tubular electrode.

[0056] Figure 16 The statistical analysis of the signal emission frequency of DRG neurons after they were cultured at the opening of a three-dimensional tubular electrode is shown.

[0057] Figure 17 A surgical photograph of the regenerative neural electrode of the present invention sutured at the sciatic nerve resection site is shown;

[0058] Figure 18 The electromyographic signals of rats at 7 weeks of age are shown in this invention.

[0059] Figure 19 A slice of nerve tissue 6 weeks post-surgery is shown. Detailed Implementation

[0060] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0061] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0062] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0063] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0064] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0065] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0066] <First Aspect>

[0067] A first aspect of the present invention provides a regenerative neural electrode, wherein the regenerative neural electrode comprises:

[0068] grassroots

[0069] An oriented structural layer, the oriented structural layer being adhered to the surface of the base layer, and the oriented structural layer being composed of interwoven fiber filaments; and

[0070] A metal thin film electrode, wherein the metal thin film electrode utilizes a metal adhesion layer formed on the side of the orientation structure layer opposite to the base layer;

[0071] Preferably, the regenerative neural electrode is at least partially curled to form a curled structure.

[0072] The regenerative neural electrode of this invention exhibits excellent degradation performance, low impedance, and good stability. The cellular signals recorded by this regenerative neural electrode can significantly promote neural tissue discretization, providing a basis for improving the accuracy of neural signal recording in terms of tissue structure. Furthermore, the regenerative neural electrode of this invention has faster regeneration capabilities.

[0073] The regenerative neural electrode of the present invention can at least partially curl to form a curled structure. Therefore, the regenerative neural electrode can maximize neural discretization and increase the number of signal recording channels per unit cross-sectional area.

[0074] grassroots

[0075] The substrate of this invention possesses excellent toughness, flexibility, and tensile properties. Furthermore, the substrate exhibits good biocompatibility, mechanical properties similar to those of nerve tissue, and biodegradability. In this invention, the substrate primarily provides mechanical support for the entire device, while its good toughness also facilitates surgical suturing.

[0076] In some specific embodiments, the base layer is made of polylactic acid copolymers. Considering that the regenerative neural electrode of the present invention needs to be biodegradable, the base layer can be prepared using a biodegradable polylactic acid copolymer. The present invention does not particularly limit the specific material used for the base layer; it can be any feasible biodegradable polylactic acid copolymer commonly used in the art.

[0077] Preferably, the base layer material is polylactic acid-polytrimethylene carbonate copolymer (PLLA-PTMC). Specifically, for the polylactic acid-polytrimethylene carbonate copolymer, its copolymerization ratio can be 50-70:30-50, for example: 55:45, 58:42, 60:40, 62:48, 65:35, 68:32, etc.

[0078] Furthermore, the base layer of the present invention can be prepared by pre-obtaining a base layer solution and then by drop coating.

[0079] Furthermore, in this invention, in order to effectively utilize the function of the base layer, the thickness of the base layer is 100μm to 300μm, for example: 120μm, 150μm, 180μm, 200μm, 220μm, 250μm, 280μm, etc.

[0080] Oriented structural layer

[0081] The directional structural layer of this invention has good orientation and is oriented. Furthermore, the oriented structural layer can guide the directional growth of cells. Using this directional structural layer as the innermost layer of a regenerative neural electrode aims to guide the proximal end of the nerve stump to grow distally, promoting nerve tissue regeneration. Simultaneously, during the growth process, it comes into contact with the metal thin-film electrode, thereby accelerating the formation of the neural-electrode interface.

[0082] In some specific implementations, the oriented structural layer is prepared using electrospinning. The principle of electrospinning is that a high voltage is applied to the polymer liquid during the electrospinning process, introducing charge into the liquid. When the charge in the liquid accumulates to a certain amount, the liquid forms a Taylor cone at the nozzle. Under the action of an applied electric field, it overcomes surface tension to form a liquid jet. Then, under the combined action of electrostatic repulsion, Coulomb force, and surface tension, the polymer jet moves along an irregular spiral trajectory. The jet is stretched and pulled in a very short time, and as the solvent evaporates or heat dissipates, the polymer jet solidifies to form micro / nanofibers. During the electrospinning process, many parameters affect the final electrospun fibers. By controlling the process parameters, micro / nanofibers of different sizes, morphologies, and structures can be prepared.

[0083] In the electrospinning process of this invention, the process parameters affect the oriented structural layer obtained by electrospinning. By controlling the process parameters, oriented structural layers of different sizes, shapes, and structures can be prepared. This invention does not have special requirements for the electrospinning method; it can be any electrospinning method commonly used in the art. Specifically, this invention dissolves a biodegradable polymer material in a suitable solvent to prepare a biodegradable polymer spinning solution; then, electrospinning is used to spin the spinning solution into an oriented structural layer composed of interwoven fibers. This oriented structural layer also has a porous structure suitable for cell growth.

[0084] Furthermore, the regenerative neural electrode of the present invention needs to be biodegradable. Therefore, the directional structural layer can be prepared using a biodegradable polymer material. The material of the directional structural layer can be the same as or different from the material of the base layer. The present invention does not particularly limit the specific material used for the directional structural layer; it can be any feasible biodegradable polymer material commonly used in the art. Preferably, the material of the directional structural layer includes polycaprolactone.

[0085] Furthermore, in this invention, in order to effectively utilize the function of the oriented structure layer, the thickness of the oriented structure layer is 20-40 μm, for example: 22 μm, 25 μm, 28 μm, 30 μm, 32 μm, 35 μm, 38 μm, etc.

[0086] Furthermore, in this invention, the average diameter of the fibers in the oriented structural layer can be 500nm-1500nm, which is more conducive to cell growth.

[0087] Metal thin film electrode

[0088] The metal thin film electrode of the present invention utilizes a metal adhesion layer formed on the side of the orientation structure layer opposite to the base layer. Figure 3SEM images of the surface of the regenerative neural electrode of the present invention are shown. Figure 3 As can be seen, the metal thin film electrode is completely attached to the oriented structure, thus the regenerative neural electrode of the present invention has a porous characteristic.

[0089] Furthermore, the metal thin-film electrodes of the present invention can exist in the form of a conductive array. In some specific embodiments, such as Figure 1 As shown, a portion of the metal thin-film electrode extends in the direction of nerve growth and is parallel to the orientation of the directional structure layer; another portion of the metal thin-film electrode extends in a direction perpendicular to nerve growth and is perpendicular to the orientation of the directional structure layer.

[0090] By setting it in the form of a conductive array, and with the metal thin film electrode in an L-shaped structure, the rear end of the electrode remains planar after it is rolled into a tube, which facilitates connection to a flexible circuit board (FPC) to achieve signal transmission.

[0091] The metal thin film electrode of the present invention almost completely covers each fiber of the highly oriented structural layer, so that the metal thin film electrode does not affect the original three-dimensional morphology of the surface of the oriented structural layer. Furthermore, due to the porous structure of the surface of the oriented structural layer, the metal thin film electrode also has a high specific surface area, which also gives the regenerative neural electrode a low impedance.

[0092] Furthermore, the metal thin-film electrode is derived from a noble metal material, preferably one or a combination of two or more of gold, silver, and platinum. The metal adhesion layer is derived from a metal material with adhesive properties; preferably, the material of the metal adhesion layer includes titanium and / or chromium.

[0093] Furthermore, in order to enable the metal thin film electrode of the present invention to function most effectively, the thickness of the metal adhesion layer is 1-20 nm, for example: 2 μm, 5 μm, 8 μm, 10 μm, 12 μm, 15 μm, 18 μm, etc.; and / or, the thickness of the metal thin film electrode is 50-300 nm, for example: 80 μm, 100 μm, 120 μm, 150 μm, 180 μm, 200 μm, 220 μm, 250 μm, 280 μm, etc.

[0094] In some specific embodiments, the present invention can use magnetron sputtering technology to prepare the metal thin film electrode.

[0095] The working principle of magnetron sputtering is that, under the influence of an electric field E, electrons collide with argon atoms as they fly towards the substrate, ionizing them to produce Ar ions and new electrons. The new electrons fly towards the substrate, while the Ar ions are accelerated by the electric field and fly towards the cathode target, bombarding the target surface with high energy, thus sputtering the target material. In the sputtered particles, neutral target atoms or molecules are deposited on the substrate to form a thin film.

[0096] In the magnetron sputtering process of this invention, the process parameters affect the metal thin film electrodes obtained by magnetron sputtering. By controlling the process parameters, metal thin film electrodes of different sizes, shapes, and structures can be prepared. This invention does not have special requirements for the magnetron sputtering method; any magnetron sputtering method commonly used in the art can be used.

[0097] <Second aspect>

[0098] A second aspect of the present invention provides a method for preparing a regenerative neural electrode according to the first aspect of the present invention, comprising the step of composite molding a base layer, a directional structural layer and a metal thin film electrode.

[0099] Specifically, the preparation method includes the following steps:

[0100] The oriented structural layer is prepared on one side of the base layer using electrospinning technology;

[0101] A metal adhesion layer is formed on the side of the oriented structure layer opposite to the base layer using magnetron sputtering technology, and a metal thin film electrode is formed on the metal adhesion layer.

[0102] Preferably, the regenerative neural electrode is at least partially curled to form a curled structure.

[0103] Preparation of base layer

[0104] For the base layer, a base layer solution can be prepared first, and then the base layer can be prepared by drop coating.

[0105] For the base layer solution, a biodegradable polylactic acid copolymer is dissolved in a solvent to prepare a base layer solution containing the biodegradable polylactic acid copolymer. The base layer solution is then drop-coated onto the substrate, and the solvent is allowed to evaporate and solidify into a film. The mass-to-volume ratio of the biodegradable polylactic acid copolymer to the solvent is 1:5-20, for example: 1:8, 1:10, 1:12, 1:15, 1:18, etc.

[0106] The solvent can be a commonly used solvent in this field, such as chloroform or trichloromethane. Additionally, to accelerate dissolution, mechanical stirring can be used to assist dissolution for 1-2 hours. Furthermore, to prevent contamination, stirring should be carried out in a sealed environment.

[0107] Furthermore, to achieve a tighter bond between the base layer and the oriented structural layer, adhesive chemicals can be used as raw materials for the base layer. For example, some biodegradable polylactic acid copolymers are inherently adhesive. Using self-adhesive polylactic acid copolymers can enhance the adhesion between the base layer and the oriented structural layer, making them more tightly bonded. The polylactic acid copolymer can be, for example, a polylactic acid-polytrimethylene carbonate copolymer.

[0108] The present invention does not particularly limit the substrate, which can be a material commonly used in the art with a smooth surface and almost no defects, such as a glass plate or a petri dish.

[0109] After the base solution is applied to the substrate, it can be cured at a temperature below 10°C for 5-24 hours. After the solvent evaporates, the base layer is obtained.

[0110] Preparation of oriented structural layers

[0111] On one side of the base layer, the oriented structural layer is prepared using electrospinning technology. In the electrospinning step, a biodegradable polymer material is dissolved in a suitable solvent to prepare a spinning solution of the biodegradable polymer material; wherein, the biodegradable polymer material can be the biodegradable polymer material suitable for the oriented structural layer in the first aspect.

[0112] There are no specific limitations on the type and concentration of the solvent used to form the solution, as long as it meets the requirements of the subsequent electrospinning process. For example, suitable solvents can be one or a combination of two or more of trifluoroethanol, hexafluoroisopropanol, trifluoroacetic acid, cyclohexanone, acetone, butanone, and tetrahydrofuran. Specifically, in the spinning solution, the mass-to-volume ratio of the biodegradable polymer to the solvent is 0.05-0.5:1, for example: 0.1:1, 0.15:1, 0.2:1, 0.25:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1, etc.

[0113] The desired oriented structural layer can be prepared by adjusting the spinning parameters during electrospinning. These parameters include, for example, voltage, syringe advance speed, receiver rotation speed, and spinning environment. Preferably, the electrospinning process parameters described in this invention are: a metal needle voltage of 10–20 kV, a syringe advance speed of 0.005–2 mm / min, a receiver rotation speed of 700–1000 rpm, a negative voltage of -10–-1 kV applied to the receiver, and an ambient temperature generally of 10–40°C, such as room temperature.

[0114] Fabrication of metal thin film electrodes

[0115] This invention utilizes magnetron sputtering technology to fabricate metal thin-film electrodes. For all metal thin-film electrodes, magnetron sputtering can be performed on the oriented structure layer using a mask.

[0116] The present invention does not impose specific limitations on the photomask; generally, it is a photomask with an electrode pattern. The photomask can be tightly attached to the orientation structure layer, and metal thin film electrodes can be deposited by magnetron sputtering.

[0117] To ensure a tight bond between the metal thin film electrode and the orientation structure layer, a metal adhesion layer, such as a titanium layer and / or a chromium layer, can be deposited on the orientation structure layer using magnetron sputtering. Then, the metal thin film electrode can be deposited on the metal adhesion layer using magnetron sputtering.

[0118] Specifically, this invention does not impose particular limitations on the conditions for depositing the metal adhesion layer using magnetron sputtering technology, as long as a metal adhesion layer can be obtained. Preferably, to obtain a more suitable metal adhesion layer, the vacuum degree of the magnetron sputtering can be 0.5 × 10⁻⁶ e⁻¹. -3 -5×10e -3 Torr, for example: 1×10e -3 Torr, 1.5×10e -3 Torr, 2×10e -3 Torr, 2.5×10e -3 Torr, 3×10e -3 Torr, 3.5×10e -3 Torr, 4×10e -3 Torr, 4.5×10e -3 Torr, etc.; power can be 10-100W, such as 20W, 40W, 60W, 80W, etc.; speed is 0.1-5A / s, such as 0.5A / s, 1A / s, 1.5A / s, 2A / s, 2.5A / s, 3A / s, 3.5A / s, 4A / s, 4.5A / s, etc.

[0119] Furthermore, the present invention does not impose particular limitations on the conditions for depositing metal thin film electrodes using magnetron sputtering technology, as long as a more suitable metal thin film electrode can be obtained. Specifically, in order to obtain the desired metal thin film electrode, the vacuum degree of the magnetron sputtering can be 0.5 × 10⁻⁶ e⁻¹. -3 -5×10e -3 Torr, for example: 1×10e -3 Torr, 1.5×10e -3 Torr, 2×10e -3 Torr, 2.5×10e -3 Torr, 3×10e-3 Torr, 3.5×10e -3 Torr, 4×10e -3 Torr, 4.5×10e -3 Torr, etc.; power can be 10-80W, such as: 20W, 40W, 60W, 80W, etc.; speed is 0.1-5A / s, such as: 0.5A / s, 1A / s, 1.5A / s, 2A / s, 2.5A / s, 3A / s, 3.5A / s, 4A / s, 4.5A / s, etc.

[0120] In magnetron sputtering, an inert gas is generally used as the ionization atmosphere. Suitable inert gases include nitrogen and argon, with argon being the preferred choice. After introducing the inert gas, the chamber pressure is typically 0.05-1 Torr, for example: 0.1 Torr, 0.2 Torr, 0.3 Torr, 0.4 Torr, 0.5 Torr, 0.6 Torr, 0.7 Torr, 0.8 Torr, etc.

[0121] Next, the membrane with the metal thin-film electrode is removed from the glass slide, and the metal thin-film electrode and flexible cable are hot-pressed together. After encapsulation with an encapsulant, a two-dimensional planar electrode is obtained. During hot pressing, silver paste zebra paper can be used, and the hot pressing temperature can be 250-500℃, for example, 300℃ or 350℃, and the hot pressing time can be 0.5-5 minutes.

[0122] Finally, as a preferred embodiment, the prepared two-dimensional planar electrode containing a base layer, a directional structural layer, and a metal thin film electrode can be rolled into a three-dimensional tubular structure to obtain the regenerative neural electrode of the present invention.

[0123] <Third aspect>

[0124] A third aspect of the present invention provides the use of the regenerative neural electrode according to the first aspect of the present invention in the preparation of neural repair articles.

[0125] Example

[0126] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0127] Example

[0128] Polylactic acid-polytrimethylene carbonate copolymer (PLLA-PTMC) films were prepared using a drop-coating method, with a copolymerization ratio of 60:40. First, a PLLA-PTMC solution was prepared by dissolving PLLA-PTMC particles (Jinan Daigang Biomaterials Co., Ltd.) in chloroform at a mass / volume ratio of 1:10 (g / mL). Then, 4 mL of the PLLA-PTMC solution was drop-coated onto a glass slide and cured at 4°C for 12 hours. After solvent evaporation, a PLLA-PTMC film (200 μm thick, 1.5 cm × 3 cm) was obtained.

[0129] Polycaprolactone (PCL) oriented fibers were prepared by electrospinning. First, PCL particles were dissolved in hexafluoroisopropanol at a mass / volume ratio of 0.9:10 (g / mL). The solution was then drawn up using a 10 mL syringe with a metal needle attached. The syringe was placed on the electrospinning feeder and advanced at a rate of 0.05 mL / min. A positive voltage of 15 kV was applied to the metal needle of the syringe, and a negative voltage of -4 kV was applied to the rotating receiver (850 r / min) with the PLLA-PTMC film. After 30 min, an oriented PCL fiber film (30 μm thick) was obtained on the PLLA-PTMC film.

[0130] A mask with an electrode pattern (300 μm linewidth) was tightly attached to a oriented PCL thin film. A 10 nm thick Ti (metal adhesion layer) and a 100 nm thick Au thin film electrode were then continuously deposited by magnetron sputtering. The magnetron sputtering conditions for the metal adhesion layer were: a vacuum level of 1 × 10⁻⁶ e⁻¹. -3 Torr, power 76W, speed 2.8A / s; conditions for magnetron sputtering of metal thin film electrodes: vacuum degree 1×10e -3 The Torr has a power of 33.6W and a speed of 1.9A / s. Ar gas is used as the ionization atmosphere in all cases, and the pressure inside the chamber after filling with Ar gas is 0.15 Torr.

[0131] The diaphragm with the metal thin film electrode was removed from the glass slide and connected to the metal thin film electrode and the flexible polyimide cable (manufacturer: Shenzhen Xingchen Feifan Technology Co., Ltd.) by hot pressing with silver paste zebra paper (350℃, 2min). The interface was then sealed with encapsulant AB glue (mass ratio of A glue to B glue is 1:10) to obtain a two-dimensional planar electrode.

[0132] Finally, the flat two-dimensional planar electrode can be rolled into a three-dimensional spiral tubular structure with three layers, and fixed with the aforementioned encapsulant to obtain a three-dimensional tubular electrode.

[0133] Example 2

[0134] Polylactic acid-polytrimethylene carbonate copolymer (PLLA-PTMC) films were prepared using a drop-coating method, with a copolymerization ratio of 60:40. First, a PLLA-PTMC solution was prepared by dissolving PLLA-PTMC particles (Jinan Daigang Biomaterials Co., Ltd.) in chloroform at a mass / volume ratio of 1:10 (g / mL). Then, 4 mL of the PLLA-PTMC solution was drop-coated onto a glass slide and cured at 4°C for 12 hours. After solvent evaporation, a PLLA-PTMC film (200 μm thick, 1.5 cm × 3 cm) was obtained.

[0135] Polycaprolactone (PCL) oriented fibers were prepared by electrospinning. First, PCL particles were dissolved in hexafluoroisopropanol at a mass / volume ratio of 0.9:10 (g / mL). The solution was then drawn up using a 10 mL syringe with a metal needle attached. The syringe was placed on the electrospinning feeder and advanced at a rate of 0.05 mL / min. A positive voltage of 15 kV was applied to the metal needle of the syringe, and a negative voltage of -4 kV was applied to the rotating receiver (850 r / min) with the PLLA-PTMC film. After 30 min, an oriented PCL fiber film (30 μm thick) was obtained on the PLLA-PTMC film.

[0136] A mask with an electrode pattern (300 μm linewidth) was tightly attached to a oriented PCL thin film. A 10 nm thick Cr (metal adhesion layer) and a 100 nm thick Au were then continuously deposited by magnetron sputtering to fabricate a metal thin-film electrode. The magnetron sputtering conditions for the metal adhesion layer were: a vacuum level of 1 × 10⁻⁶ e⁻¹. -3 Torr, power 32W, speed 0.5A / s; conditions for magnetron sputtering of metal thin film electrodes: vacuum degree 1×10e -3 The Torr has a power of 33.6W and a speed of 1.9A / s. Ar gas is used as the ionization atmosphere in all cases, and the pressure inside the chamber after filling with Ar gas is 0.15 Torr.

[0137] The diaphragm with the metal thin film electrode was removed from the glass slide and connected to the metal thin film electrode and the flexible polyimide cable (manufacturer: Shenzhen Xingchen Feifan Technology Co., Ltd.) by hot pressing with silver paste zebra paper (350℃, 2min). The interface was then sealed with encapsulant AB glue (mass ratio of A glue to B glue is 1:10) to obtain a two-dimensional planar electrode.

[0138] Finally, the flat two-dimensional planar electrode can be rolled into a three-dimensional spiral tubular structure with three layers, and fixed with the aforementioned encapsulant to obtain a three-dimensional tubular electrode.

[0139] Performance testing

[0140] 1. Impedance test

[0141] The impedance of the regenerative neural electrode of this invention was measured using a Gamry potentiostat. A standard three-electrode system was established in PBS solution, wherein the two-dimensional planar electrode of Example 1 was used as the working electrode, Ag / AgCl was used as the reference electrode, and a Pt electrode was used as the counter electrode. The impedance of each channel of the regenerative neural electrode was measured at frequencies ranging from 10 Hz to 100 kHz with an amplitude of 5 mV and an OCV bias of 0 V. The results are as follows: Figure 4 As shown.

[0142] Depend on Figure 4 It can be seen that the regenerative neural electrode of the present invention has low impedance.

[0143] 2. DRG neuron cell-guided test

[0144] Figure 5 The process of guiding DRG neurons onto the two-dimensional planar electrode of Example 1 is illustrated. Specifically, SD rats were euthanized and disinfected by immersion in 75% alcohol for 15 minutes. DRG neurons were then removed from the intervertebral foramen of the rat vertebrae under a stereomicroscope and isolated using trypsin digestion. The isolated DRG neurons were seeded with 2.5 mL of DRG culture medium (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 [DMEM / F-12], 2 mM GlutalMAX-I, 1% penicillin-streptomycin, and 1% B-27 factor). Figure 5 Within the red dashed box, and cultured for 7 days at 37°C and 5% CO2, DRG neurons underwent directional growth, with the results as follows: Figure 5 As shown in the figure. Then, the Fourier transform in ImageJ was used to calculate the quantitative characterization of the DRG neuron cell orientation on the orientation structure layer, and the results are as follows. Figure 6 As shown.

[0145] Depend on Figure 5 and Figure 6 It can be seen that the directional structure layer can guide the directional growth of neurons and provide fiber type support for nerves, thereby guiding DRG neurons to the metal electrode and realizing the ideal neuro-electrode interface for nerve growth along the electrode.

[0146] 3. Degradation experiment

[0147] The two-dimensional planar electrode of Example 1 is attached to a flexible curved substrate. Figure 7 In step a), by slowly adding 4 mL of hexafluoroisopropanol solution to the two-dimensional planar electrode, the PLLA-PTMC film and the PCL film slowly dissolved. After continuous solvent addition, the substrate dissolved after about 30 seconds, and finally only the metal film electrode remained attached to the flexible curved substrate. Figure 7 (b)

[0148] Therefore, the regenerative neural electrode of the present invention will slowly degrade after implantation, eventually leaving only the metal thin film electrode attached to the nerve tissue.

[0149] 4. Impedance stability test

[0150] The impedance stability of the two-dimensional planar electrode of Example 1 was measured using a Gamry potentiostat. A standard three-electrode system was established in PBS solution, in which the two-dimensional planar electrode was used as the working electrode, Ag / AgCl as the reference electrode, and Pt as the counter electrode. The impedance of each channel of the two-dimensional planar electrode was measured at frequencies ranging from 10 Hz to 100 kHz with an amplitude of 5 mV and an OCV bias of 0 V. The results are as follows. Figure 8 As shown.

[0151] Depend on Figure 8 It can be seen that the regenerative neural electrode exhibits good stability in impedance after culturing DRG neurons in the initial state and in impedance simulation after degradation (mid-frequency 1kHz).

[0152] 5. In vitro cell experiments

[0153] 5.1 Two-dimensional planar structure experiment

[0154] DRG neurons were cultured in a medium such as Figure 9 On the two-dimensional planar structure shown in Example 1, cell signal recording began after 7 days of culture. A DRG neuron cell signal recording device was used for recording the cell signals, specifically as follows... Figure 10 As shown. To induce stronger neuronal cell signals, a microfluidic device was incorporated into the recording apparatus to slowly add a stimulation solution to the culture dish. The stimulation solution consisted of 70 millimoles of KCl (in millimoles). Data was recorded using two-dimensional planar electrodes and pre-amplified by an RHD amplifier (Intan). The data acquisition frequency was 30 kHz, and bandpass filtering (300 Hz–6 kHz) was applied using the Open Ephys acquisition system.

[0155] After seven days of culture, DRG neurons can be guided to the metal thin-film electrode through the directional structural layer, forming an interface with the metal thin-film electrode. Then, the signals of the DRG neurons can be recorded through a neural signal recording device connected to the back end.

[0156] Figure 11 This image illustrates the spontaneous signals generated by DRG neurons cultured on two-dimensional planar electrodes without external stimulation. The top image shows the original signal, and the bottom image shows the signal after filtering and denoising. Furthermore, by performing AI feature comparison analysis on the filtered and denoised signal, the signal can be classified according to its features, such as... Figure 12 As shown, three different neural signals were recorded in the experiment.

[0157] To verify that the recorded signals were indeed from DRG neurons, the DRG neurons were stimulated with KCl solution, and the results were as follows: Figure 13 As shown in the figure, the signal recording results showed that the frequency of cell signal emission increased significantly after the addition of KCl solution, proving that the cell signals recorded by the regenerative neural electrode are effective.

[0158] 5.2 Three-dimensional tubular structure experiment

[0159] Since the electrode is ultimately intended for implantation, we use the three-dimensional tubular electrode from Example 1 (e.g., Figure 14 (As shown), DRG neurons were then cultured at the electrode port. The specific culture process was as follows: SD rats were euthanized and disinfected by immersion in 75% alcohol for 15 minutes; then, DRG neurons were removed from the intervertebral foramen of the rat vertebrae under a stereomicroscope and digested and separated using trypsin. The separated DRG neurons were seeded at the three-dimensional electrode port with 2.5 mL of DRG culture medium (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 [DMEM / F-12], 2 mM GlutalMAX-I, 1% penicillin-streptomycin, and 1% B-27 factor) and cultured at 37°C and 5% CO2 for 7 days (n=3).

[0160] After seven days of culture, guided by the regenerative neural electrode, DRG neurons successfully grew into the electrode and came into contact with the Au membrane, thus successfully recording the signals from the DRG neurons. The same method was used to record and analyze the DRG neuron signals, yielding results as follows: Figure 15 The two types of neural signals are shown. A DRG neuron cell signal recording device is used to record cell signals, specifically as follows... Figure 10 As shown.

[0161] Similarly, we applied KCl stimulation to the DRG neurons on the electrodes (using the same method as the stimulation procedure in the two-dimensional planar structure experiment), and the results were as follows. Figure 16As shown. Figure 16 The results showed that the frequency of cell signaling increased significantly after stimulation, consistent with expectations.

[0162] 6. Animal experiments

[0163] A 5mm section of the sciatic nerve was severed from the rat's sciatic nerve, and then a regenerative neural electrode was sutured to the severed site. Figure 17 As shown. Nerve tissue was sectioned 6 weeks post-surgery. The specific steps were as follows: the sciatic nerve segment from the surgical side was removed and immediately immersed in 10 times the sample volume of 4% paraformaldehyde, and fixed at 4°C for 24 hours. The sample was then dehydrated in sucrose, embedded in a composite gel at the optimal cutting temperature, and flash-frozen in liquid nitrogen. The nerve graft was sectioned longitudinally (10 μm), stained with NF-200, S-100, and DAPI, and observed using a confocal panoramic scanner (Pannoramic, 3D HISTECH, Hungary). The results are shown below. Figure 19 As shown.

[0164] Depend on Figure 19 It can be seen that this regenerative neural electrode can significantly promote the discretization of neural tissue, and has the basis for improving the accuracy of recording neural signals in terms of tissue structure.

[0165] Seven weeks post-surgery, electromyographic signals were successfully recorded in the affected muscles after stimulation of the proximal end of the transection site. Figure 18 As shown. By Figure 18 It can be seen that the 5mm truncated section in rats regenerated in 7 weeks, demonstrating a faster regeneration capacity compared to other types of regenerative neural electrodes.

[0166] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0167] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A regenerative neural electrode, characterized in that, The regenerative neural electrode includes: The base layer is derived from a biodegradable polylactic acid copolymer; An oriented structural layer, the oriented structural layer being adhered to the surface of the base layer, and the oriented structural layer being composed of interwoven fiber filaments; and A metal thin film electrode, wherein the metal thin film electrode utilizes a metal adhesion layer formed on the side of the orientation structure layer opposite to the base layer; A portion of the metal thin-film electrode extends in the direction of nerve growth and is parallel to the orientation of the directional structure layer; another portion of the metal thin-film electrode extends in a direction perpendicular to nerve growth and is perpendicular to the orientation of the directional structure layer.

2. The regenerative neural electrode according to claim 1, characterized in that, The regenerative neural electrode can be at least partially curled to form a curled structure.

3. The regenerative neural electrode according to claim 1, characterized in that, The base layer material includes polylactic acid-polytrimethylene carbonate copolymer.

4. The regenerative neural electrode according to any one of claims 1-3, characterized in that, The oriented structural layer is derived from biodegradable polymer materials.

5. The regenerative neural electrode according to claim 4, characterized in that, The material of the oriented structural layer includes polycaprolactone.

6. The regenerative neural electrode according to any one of claims 1-3, characterized in that, The metal thin-film electrode is derived from precious metal materials.

7. The regenerative neural electrode according to claim 6, characterized in that, The precious metal material is one or a combination of two or more of gold, silver, and platinum.

8. The regenerative neural electrode according to any one of claims 1-3, characterized in that, The metal adhesion layer is derived from a metal material with adhesive properties.

9. The regenerative neural electrode according to claim 8, characterized in that, The materials of the metal adhesion layer include titanium and / or chromium.

10. The regenerative neural electrode according to any one of claims 1-3, characterized in that, The thickness of the base layer is 100μm~300μm; and / or The thickness of the oriented structural layer is 20~40μm; and / or The thickness of the metal adhesion layer is 1~20nm; and / or The thickness of the metal thin film electrode is 50~300 nm.

11. A method for preparing a regenerative neural electrode according to any one of claims 1-10, characterized in that, This includes the steps of compositing a base layer, an oriented structural layer, and a metal thin-film electrode.

12. The preparation method according to claim 11, characterized in that, The preparation method includes the following steps: The oriented structural layer is prepared on one side of the base layer using electrospinning technology; A metal adhesion layer is formed on the side of the oriented structure layer opposite to the base layer using magnetron sputtering technology, and a metal thin film electrode is formed on the metal adhesion layer.

13. The preparation method according to claim 12, characterized in that, The regenerative neural electrode can be at least partially curled to form a curled structure.

14. Use of a regenerative neural electrode according to any one of claims 1-10 in the preparation of neural repair articles.

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