A functional neurovascularized engineered muscle capable of electrical response and its preparation method

Abstract

The present invention relates to an electrically responsive, functional neurovascularized engineered muscle, comprising a first electrode layer, a second electrode layer, and a neuromuscular layer and a vascular layer disposed between the first and second electrode layers. The present invention also relates to a method for preparing the engineered muscle. The engineered muscle of the present invention exhibits excellent electrical responsiveness and can be used for a wider range of functional applications based on this excellent electrical responsiveness. Furthermore, the engineered muscle exhibits excellent biocompatibility and a standardized structure, enabling industrial production and promising prospects for industrial and clinical applications.

Description

Technical Field

[0001] The present invention relates to the field of biological tissue engineering, and in particular to an electrically responsive functional neurovascularized engineered muscle and a preparation method thereof. Background Art

[0002] Skeletal muscle is a vital organ in the human body, accounting for approximately 40% of the total body mass. It is composed of connective tissue, blood vessels, and bundles of parallel muscle fibers, and its unique contractile function relies on innervation by motor neurons. Natural skeletal muscle possesses strong self-adaptation and regeneration capabilities. However, when it is damaged by external factors such as trauma, disease, or tumor resection, resulting in extensive trauma (over 20% of the total volume), scar tissue replaces the damaged area, hindering muscle regeneration and leading to volumetric muscle loss. This untreated volumetric muscle loss can lead to persistent muscle atrophy and a range of functional impairments.

[0003] Autologous tissue transplantation has long been the first-line treatment for volumetric muscle defects. However, this invasive approach not only damages the donor tissue but also increases the risk of postoperative infection. Therefore, the development of muscle tissue engineering holds great promise for the treatment of these extensive muscle defects. Current engineering strategies primarily focus on reconstructing muscle tissue structure, employing various techniques to construct targeted muscle structures to achieve biomimetic goals. While widely used bioscaffolds can achieve macroscopically targeted muscle structures, they are insufficient to regulate the microstructure of individual muscle fibers and may also suffer from poor biocompatibility. Furthermore, simply implanting such targeted engineered muscle only fills the muscle volume but fails to achieve functional restoration. Therefore, constructing engineered muscle tissue that embodies the structural and functional characteristics of native skeletal muscle, while simultaneously achieving both tissue and functional restoration, is of great significance.

[0004] However, the realization of this functionalization requires not only the directional arrangement of engineered muscle fibers, but more importantly, effective nerve innervation to promote the signal transduction process. Electrical stimulation, as a common physical therapy method, can effectively regulate nerve conduction and muscle contraction, which may be the key to achieving functionalization during muscle regeneration. In addition, good blood perfusion is also crucial to maintaining muscle survival and prolonging function, and is the basis for neuralized muscle regeneration. At present, reconstructing a functional tissue that is both innervated and vascularized remains a huge challenge, which is also an important reason why tissue engineering has not developed as rapidly as expected in recent years. Summary of the Invention

[0005] The purpose of the present invention is to provide a functional neurovascularized engineered muscle that is electrically responsive, and a method for preparing the engineered muscle.

[0006] In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

[0007] A functional neurovascularized engineered muscle capable of electrical response comprises a first electrode layer and a second electrode layer, wherein a neuromuscular layer and a vascular layer are provided between the first electrode layer and the second electrode layer.

[0008] In the present invention, a further preferred solution is that the neuromuscular layer consists of two layers, namely a first neuromuscular layer and a second neuromuscular layer, and the vascular layer is arranged between the first neuromuscular layer and the second neuromuscular layer.

[0009] In the present invention, a further preferred embodiment is that the engineered muscle further comprises a first hydrogel layer for supporting the neuromuscular layer, and a second hydrogel layer for supporting the vascular layer.

[0010] In the present invention, a further preferred solution is that both the first electrode layer and the second electrode layer are flexible electrodes, and the flexible electrodes include a mesh polyester fiber support and a conductive ink layer attached to the polyester fiber support.

[0011] In the present invention, a further preferred solution is that the thickness of the flexible electrode is 40-60 μm, and the diameter of the single fiber on the polyester fiber support is 25-60 μm.

[0012] The present invention also provides a method for preparing the engineered muscle as described above, comprising the following steps:

[0013] S1: Preparation of light-controlled oriented cell sheets: construct light-controlled oriented neural cell sheets for the neuromuscular layer and light-controlled oriented vascular cell sheets for the vascular layer;

[0014] S2: Preparation of the neuromuscular and vascular layers: Using photo-controlled directional technology, a mixture of nerve cells and muscle cells is spread on a photo-controlled directional nerve cell sheet and cultured to obtain a neuromuscular layer; using photo-controlled directional technology, endothelial cells are spread on a photo-controlled directional vascular cell sheet and cultured to obtain a vascular layer;

[0015] S3: Muscle assembly: Take the first electrode layer and the second electrode layer, and place the neuromuscular layer and blood vessel layer prepared in step S2 between the first electrode layer and the second electrode layer to obtain the engineered muscle.

[0016] In the present invention, a further preferred scheme is that the step S1 is specifically: preparing nanodots on the surfaces of the two substrates respectively, and then using ultraviolet light to perform a first pattern modification and a second pattern modification on the nanodots on the two substrates respectively, wherein the first pattern is a directional arrangement pattern of cells in the nerve cell layer, and the second pattern is a directional arrangement pattern of cells in the vascular layer, thereby obtaining a light-controlled directional nerve cell sheet and a light-controlled directional vascular cell sheet.

[0017] In the present invention, a further preferred solution is that in the mixture of nerve cells and muscle cells in step S2, the ratio of muscle cells to nerve cells is 100:1.

[0018] In the present invention, a further preferred scheme is that in step S3, the neuromuscular layer prepared in step S2 is transferred from the photocontrolled oriented nerve cell sheet through the first hydrogel layer, and the vascular layer is transferred from the photocontrolled oriented vascular cell sheet through the second hydrogel layer, and then assembled with the first electrode and the second electrode.

[0019] In the present invention, a further preferred scheme is to drop the photocurable hydrogel onto the photocontrolled oriented neural cell sheet and the photocontrolled oriented vascular cell sheet respectively. After the hydrogel is evenly spread on each cell sheet, it is irradiated under ultraviolet light for 55-65s to form a first hydrogel layer and a second hydrogel layer respectively. Then, the first hydrogel layer carrying the neuromuscular layer and the second hydrogel layer carrying the vascular layer are removed from each cell sheet, and then stacked and assembled with the first electrode and the second electrode to obtain the engineered muscle.

[0020] In the present invention, a further preferred solution is that the first electrode layer and the second electrode layer in step S3 are both flexible electrodes, and the flexible electrodes include a mesh polyester fiber support and a conductive ink layer, and the flexible electrodes are prepared by the following steps:

[0021] S31: Preparation of mesh polyester fiber scaffold: Draw the mesh structure pattern of the scaffold, and then use the melt near-field direct writing technology to print the mesh polyester fiber scaffold according to the mesh structure pattern;

[0022] S32: Hydrophilic treatment of polyester fiber: The surface of the mesh polyester fiber scaffold is treated with hydrophilicity to improve its hydrophobicity by in-situ polymerization of dopamine;

[0023] S33: Conductive ink layer coating: apply carbon nanotube liquid conductive ink to the mesh polyester fiber support treated by S2, and then dry it. Repeat the above coating and drying steps multiple times to obtain the result.

[0024] Compared with the existing technology, the present invention has the following advantages: the engineered muscle of the present invention has good electrical responsiveness; and based on the good electrical responsiveness, it can be applied in combination with physical electrical stimulation therapy (such as related medical devices) and can expand more functional applications; in addition, the engineered muscle has good biocompatibility and a standard structure, which can be industrialized and has good industrial and clinical application prospects.

[0025] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the layered structure of the engineered muscle in Example 1;

[0027] Figure 2 This is a schematic diagram of the application of the engineered muscle of Example 1;

[0028] Among them, 1. first electrode layer; 2. second electrode layer; 3. first neuromuscular layer; 4. second neuromuscular layer; 5. blood vessel layer; 6. first hydrogel layer; 7. second hydrogel layer. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the devices and reagents in the embodiments and experimental examples in the specific embodiments are obtained from commercial sources. The specific embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the scope of protection of the present application.

[0030] A functional neurovascularized engineered muscle capable of electrical response comprises a first electrode layer and a second electrode layer, wherein a neuromuscular layer and a vascular layer are provided between the first electrode layer and the second electrode layer.

[0031] The engineered muscle of the present invention arranges the neuromuscular layer and the vascular layer between the first electrode layer and the second electrode layer, and arranges two electrode layers outside the main part of the artificial muscle tissue (vascular layer and neuromuscular layer), which can effectively improve the conductivity of the muscle tissue and achieve the purpose of electrical response; when facing electrical stimulation, accurate current transmission can be achieved, which can accelerate the recovery of muscle functionality. It can be applied in combination with physical electrical stimulation therapy (such as related medical devices) and can expand more functional applications. In the engineered muscle of the present invention, the neuromuscular layer and the vascular layer are not derived from the human body or animals, but are obtained by cell culture through bioengineering technology; industrialized and standardized production can be achieved, and it has good biocompatibility.

[0032] In the present invention, between the first electrode layer and the second electrode layer, the neuromuscular layer and the vascular layer can be arranged in their combination and stacking assembly order according to actual needs; in order to further improve the fit with the human body and subsequent treatment needs, such an arrangement can be made, the neuromuscular layer is two layers, namely the first neuromuscular layer and the second neuromuscular layer, and the vascular layer is arranged between the first neuromuscular layer and the second neuromuscular layer; with such an arrangement, the vascular layer can provide nutrition to the neuromuscular layer and take away metabolites, the two neuromuscular layers can also protect the vascular layer, and the responding muscles and blood vessels can also better achieve contraction, the overall structure is closer to natural skeletal muscle, and has better biocompatibility.

[0033] In order to facilitate the forming, transfer and subsequent transplantation of engineered muscles, the following arrangement can be made: the engineered muscle also includes a first hydrogel layer for supporting the neuromuscular layer, and a second hydrogel layer for supporting the vascular layer; in this way, the neuromuscular layer and the vascular layer are supported by the hydrogel layer, which can make the subsequent assembly, transfer and transplantation of related structures more convenient. In addition, the addition of hydrogel can further improve the elastic modulus of muscle tissue, which is closer to the physical properties of human muscle as a whole; in addition, after the addition of the hydrogel layer, the hydrogel can bond and solidify the electrode layer, which can further improve the overall stability of the engineered muscle.

[0034] For the first electrode layer and the second electrode layer, the corresponding electrode material can be selected according to actual needs; in order to further improve the tissue compliance of the engineered muscle, the first electrode layer and the second electrode layer can use flexible electrodes; in order to further improve the tissue compliance and better adapt to the mobility of the tissue, the thickness of the flexible electrode can be 40-60μm, and more preferably 50μm. For flexible electrodes, the structure and composition of the electrodes can be selected according to actual needs. For example, a flexible electrode including a bracket and a conductive layer structure on the bracket can be selected. For the bracket material, it can be selected according to needs, preferably polyester fiber material, further preferably polycaprolactone PCL material, and further preferably a mesh structure polycaprolactone PCL bracket; for the mesh polycaprolactone PCL bracket, it is formed by interweaving polycaprolactone fibers into a net, and its fiber diameter is preferably 25-60 μm. The bracket made of fibers of this size has better biocompliance, and the optimal size is μm; for the conductive layer, it can be selected based on actual needs. In order to further improve the tissue compliance of the flexible electrode, a conductive ink layer can be selected for coating and drying. The conductive ink layer is preferably a conductive ink layer made of carbon nanotube conductive ink; for the conductive ink layer, one layer, two layers or multiple layers can be selected; for the conductive ink layer, its conductivity is preferably 3.9-5S / m, preferably 4.27S / m. The inventors found that the flexible electrode in this conductivity state has a good electrical response effect.

[0035] The preparation method of the electrically responsive functional neurovascularized engineered muscle as described above comprises the following steps:

[0036] S1: Preparation of light-controlled oriented cell sheets: construct light-controlled oriented neural cell sheets for the neuromuscular layer and light-controlled oriented vascular cell sheets for the vascular layer;

[0037] S2: Preparation of the neuromuscular and vascular layers: Using photo-controlled directional technology, a mixture of nerve cells and muscle cells is spread on a photo-controlled directional nerve cell sheet and cultured to obtain a neuromuscular layer; using photo-controlled directional technology, endothelial cells are spread on a photo-controlled directional vascular cell sheet and cultured to obtain a vascular layer;

[0038] S3: Muscle assembly: Take the first electrode layer and the second electrode layer, and place the neuromuscular layer and blood vessel layer prepared in step S2 between the first electrode layer and the second electrode layer to obtain the engineered muscle.

[0039] The step S1 can be specifically as follows: preparing nanodots on the surfaces of the two substrates respectively, and then using ultraviolet light to perform a first pattern modification and a second pattern modification on the nanodots on the two substrates respectively, wherein the first pattern is a directional arrangement pattern of cells in the nerve cell layer, and the second pattern is a directional arrangement pattern of cells in the vascular layer, thereby obtaining a light-controlled directional nerve cell sheet and a light-controlled directional vascular cell sheet.

[0040] As for the substrate, a quartz substrate can be selected; as for the nanodots, titanium dioxide nanodots can be selected.

[0041] Patterned modification can be performed in step S1 or in the light-controlled directional culture in step S2. Specifically, the patterned modification can be performed by covering one side of the substrate having a titanium dioxide nanodot surface with a mask having a parallel pattern with a spacing of 29-32 μm. After 0.8-1.2 hours of ultraviolet light exposure, the patterned modification is completed. Furthermore, after ultraviolet light exposure, the responding cells are spread on the patterned side of the substrate to achieve directional arrangement of the cells.

[0042] For step S2, the proportion of responsive cells in the mixture of nerve cells and muscle cells can be obtained by selecting co-culture systems with different ratios, co-culturing nerve and muscle cells, setting different co-culture ratios (1:0-500:1), and selecting according to the level of myogenesis; through a large number of experiments, the inventors screened out the optimal co-culture ratio: muscle cell: nerve cell number ratio = 100:1.

[0043] A further preferred embodiment is that in step S3, the neuromuscular layer prepared in step S2 is transferred from the photo-controlled oriented neural cell sheet via the first hydrogel layer, and the vascular layer is transferred from the photo-controlled oriented vascular cell sheet via the second hydrogel layer, and then assembled with the first and second electrodes. Regarding the choice of hydrogel material, a hydrogel with good biocompatibility can be selected, and Gelma hydrogel is more preferably selected.

[0044] For step S3, a further preferred scheme is to drop the photocurable hydrogel onto the photocontrolled oriented nerve cell sheet and the photocontrolled oriented vascular cell sheet respectively, and after the hydrogel is evenly spread on each cell sheet, irradiate under ultraviolet light for 55-65s to form a first hydrogel layer and a second hydrogel layer respectively, and then remove the first hydrogel layer carrying the neuromuscular layer and the second hydrogel layer carrying the vascular layer from each cell sheet, and then stack and assemble them with the first electrode and the second electrode to obtain the engineered muscle.

[0045] In the present invention, the first electrode layer and the second electrode layer in step S3 can both be flexible electrodes, and the flexible electrodes include a mesh polyester fiber support and a conductive ink layer. The flexible electrodes are prepared by the following steps:

[0046] S31: Preparation of a mesh polyester fiber scaffold: Draw a mesh structure pattern of the scaffold, and then print the mesh polyester fiber scaffold according to the mesh structure pattern using a near-field direct writing technique. The pattern can be drawn using drawing software such as AutoCAD. The mesh structure can be a sinusoidal mesh structure.

[0047] S32: Polyester fiber hydrophilic treatment: The surface of the mesh polyester fiber scaffold is treated with hydrophilicity by in-situ polymerization of dopamine to improve its hydrophobicity (i.e., enhance its hydrophilicity to facilitate subsequent ink coating);

[0048] S33: Conductive ink layer coating: carbon nanotube liquid conductive ink is coated on the mesh polyester fiber bracket treated by S2, and then dried. The above coating and drying steps are repeated several times to obtain the result; further, the bracket can be immersed in ink to achieve ink coating. The immersion can make the bracket surface fully contact with the ink. The immersion time can be selected as 12-18 minutes, and more preferably 15 minutes; after immersion, the bracket is taken out, the excess ink is removed, and then dried in an environment of 32-37 degrees Celsius for 25-35 minutes, such as drying in a drying oven, to obtain a conductive ink layer; in order to achieve good electrical response performance, a certain number of conductive ink layers can be coated (i.e. corresponding to a certain range of conductivity, such as 3.9-5S / m), and the additional conductive ink layer can be produced by the above-mentioned immersion coating and drying steps.

[0049] The engineered muscle preparation method of the present invention can be standardized and industrialized, and has good industrial and clinical application prospects.

[0050] Example 1

[0051] A functional neurovascularized engineered muscle capable of electrical response, the engineered muscle comprising a first electrode layer 1 and a second electrode layer 2, wherein a neuromuscular layer and a vascular layer are provided between the first electrode layer and the second electrode layer; the neuromuscular layer comprises two layers, namely a first neuromuscular layer and a second neuromuscular layer, and the vascular layer 5 is provided between the first neuromuscular layer 3 and the second neuromuscular layer 4; the engineered muscle further comprises a first hydrogel layer 6 for supporting the neuromuscular layer, and a second hydrogel layer 7 for supporting the vascular layer; the first electrode layer and the second electrode layer are both flexible electrodes, and the flexible electrodes comprise a mesh polyester fiber support and a conductive ink layer attached to the polyester fiber support; the specific structure can be combined with Figure 1 To understand;

[0052] The engineered muscle is prepared by the following steps:

[0053] S1: Preparation of light-controlled oriented cell sheets: Constructing light-controlled oriented neural cell sheets for the neuromuscular layer and light-controlled oriented vascular cell sheets for the vascular layer; specifically including:

[0054] 1) Preparation of titanium dioxide nanodots

[0055] A. Preparation of precursor solution: Add 5 μL of anhydrous ethanol to a beaker and stir magnetically for 20 minutes. Then, add 680 μL of tetrabutyl titanate, 36 μL of deionized water, and 62 μL of acetylacetone in that order. After thorough mixing, add 0.4 g of polyvinyl pyrrolidone. After magnetic stirring for 10 minutes, transfer the mixture to a volumetric flask and dilute to volume with anhydrous ethanol to obtain a precursor solution.

[0056] B. Spin coating and sintering: Spin the precursor liquid evenly on the quartz substrate (10×10×1mm 3 ) surface, heat-treated it in a muffle furnace at 500°C for 1 hour, and after high-pressure sterilization, placed it in a well plate for subsequent cell culture;

[0057] S2: Preparation of neuromuscular layer and vascular layer: Using light-controlled directional technology, a mixture of nerve cells and muscle cells is spread on a light-controlled directional nerve cell sheet and cultured to obtain a neuromuscular layer; using light-controlled directional technology, endothelial cells are spread on a light-controlled directional vascular cell sheet and cultured to obtain a vascular layer; specifically, the process may include:

[0058] Photo-controlled cell orientation: using a UV light source (254 nm, 300 μW / cm 2) Micropatterning was performed using a quartz mask, forming 30 μm-wide Cr lines (line / space ratio of 1:1). After 1 hour of UV irradiation, the sample was immediately transferred to a well plate using tweezers, and a cell suspension was added. After 12 hours of incubation, oriented cell alignment was achieved.

[0059] Construction of a directional neuromuscular layer: Using the aforementioned light-controlled directional technology, nerve and muscle cells are mixed in a certain ratio and spread onto the substrate surface. The plate is placed in an incubator and cultured for one day. The growth medium is then replaced with differentiation medium. Culture in differentiation medium is continued for another seven days to obtain a directional neuromuscular layer.

[0060] Construction of a directional vascular layer: Using the aforementioned light-controlled directional technology, endothelial cells are evenly spread onto the substrate surface. After 7 days of culture, the cells fuse to form a tubular vascular structure, resulting in a directional vascular layer.

[0061] S3: Muscle assembly: Take the first electrode layer and the second electrode layer, and place the neuromuscular layer and vascular layer prepared in step S2 between the first electrode layer and the second electrode layer to obtain the engineered muscle;

[0062] The flexible electrodes of the first electrode layer and the second electrode layer are manufactured by the following steps:

[0063] A. PCL bracket printing: A sinusoidal grid structure was designed using AutoCAD. The overall shape of the bracket is square, with a length and width of 12 mm. The single waveform unit is sinusoidal, with a period of 1 mm and an amplitude of 0.25 mm. The printing parameters were then set so that the distance between the dispensing nozzle and the glass plate collector was 2 mm, the printing speed was 450 mm / min, the printing temperature was maintained at 110°C, and the number of layers was 3. A bracket with a fiber diameter of 50 μm was printed using near-field direct writing technology. The glass plate collector was immersed in 75% alcohol. After the PCL bracket was detached, the bracket was removed with tweezers and placed in a drying oven at 35°C for 30 minutes.

[0064] B. PCL scaffold modification: Since the surface of the PCL scaffold is smooth and highly hydrophobic, the conductive components of the liquid ink cannot be effectively adsorbed on the fiber surface. The surface of the PCL scaffold is hydrophilized by in-situ polymerization of dopamine. Take 20 mL of Tris-HCl buffer, add 0.04 g of dopamine hydrochloride powder to prepare a 2 mg / mL crosslinker solution, then completely immerse the scaffold in the solution and place it on a shaker for 12 hours to allow dopamine to polymerize in situ on the surface of the PCL scaffold. Then take out the cross-linked PCL scaffold and rinse it thoroughly with deionized water 3 times to remove the polydopamine particles that are not adhered to the surface of the scaffold. Then place the scaffold in a drying oven at 35°C to dry for 1 hour;

[0065] C. Carbon nanotube coating

[0066] a. Preparation of carbon nanotube liquid conductive ink: Add 0.02g of carbon nanofiber (CNF) powder to 20mL of deionized water and stir at room temperature for 1 hour. After the CNF is completely dissolved, add 0.04g of carbon nanotube (CNT) powder to the solution. Stir the mixture thoroughly for 1 hour and sonicate for 30 minutes. Then, centrifuge the sonicated mixture twice at 2500rpm for 10 minutes each to remove undispersed carbon nanotubes, resulting in a carbon nanotube liquid conductive ink.

[0067] b. Assembly of the conductive ink components: Soak the cross-linked stent in the ink for 15 minutes to ensure full contact between the stent surface and the ink. Remove the stent, remove any excess ink, and dry it in a 35°C drying oven for 30 minutes. Then, soak the coated stent in the ink for another minute, remove and remove any excess ink, and dry it in a 35°C drying oven for 10 minutes. Repeat the coating process for the second time, coating the stent layer by layer. After 10 coats, the conductivity reaches 4.27 S / m.

[0068] In step S3, the corresponding neuromuscular layer and vascular layer are transferred and assembled with the electrode layer as follows: light-curable Gelma hydrogel is dripped onto the surface of the cell sheet. After the liquid is fully and evenly spread, the cell sheet is transferred to ultraviolet light for 60 seconds, and the solidified cell-laden hydrogel sheet is completely removed with a blade. Repeat the above steps to completely remove the neuromuscular layer and vascular layer, and stack them in the order of the neuromuscular layer as the upper and lower layers and the vascular layer as the middle layer to form a composite hydrogel tissue. The prepared flexible electrodes are covered on the upper and lower surfaces of the tissue, and after further bonding and curing with the hydrogel, the construction of the composite structure is completed.

[0069] Correspondingly, the engineered muscle obtained in this Example 1 can be used in the following ways (can be combined with Figure 2 (To understand): According to the shape and size of the muscle defect, the constructed engineered muscle tissue is properly trimmed and implanted into the defect area. A small amount of liquid hydrogel is dripped at the edge and solidified by ultraviolet light to bond the engineered tissue to the autologous muscle tissue. After implantation and fixation, the skin is sutured. Furthermore, electrodes are placed through the skin at both ends of the engineered muscle for electrical stimulation therapy. Due to the good conductivity of the engineered muscle, the current can be accurately transmitted to the implanted area, achieving the goal of precise electrical stimulation.

[0070] Experimental Example 1

[0071] This experimental example is based on the experimental application of the engineered muscle obtained in Example 1 (mouse tibialis anterior muscle defect model) and the related engineering muscle performance experiments, specifically:

[0072] 1. Preparation of electrically responsive, functional neurovascularized engineered muscle: First, the type, size, and morphology of the muscle defect are assessed, and then engineered muscle of appropriate size is prepared. Using photo-controlled directional technology, the neuromuscular and vascular layers are constructed. A PCL scaffold is then printed using near-field direct writing. This scaffold is then coated layer by layer with CNT conductive ink to create flexible electrodes. This structure is then assembled using hydrogel to create electrically responsive, functional neurovascularized engineered muscle.

[0073] 2. Implantation of Engineered Muscle: After appropriate trimming, the engineered muscle is implanted into the defect. After checking for alignment, a small amount of liquid hydrogel is added to the edges. This is cured by UV light to bond the engineered tissue to the autologous muscle tissue. After implantation and fixation, the surface skin is sutured.

[0074] 3. Electrical stimulation therapy to assist repair: Two surface stimulation electrodes are placed on the skin surface corresponding to the defect area, corresponding to the ends of the engineered tissue. The electrodes are simultaneously connected to a stimulator, and electrical stimulation is performed after adjusting appropriate stimulation parameters. This method allows for precise current transmission to the implanted area, achieving the goal of precise treatment.

[0075] 4. Functional evaluation:

[0076] 1) Gait analysis: The CatWalk system was used to analyze mouse gait. First, the mice were placed on a glass plate in a dark room and allowed to walk freely. A light beam from a fluorescent lamp propagated across the entire glass plate and could be completely reflected by the plate. When the paw touched the glass plate, the light beam was reflected downward, forming a clear and bright paw print image. The entire walking process was recorded with a camera. All data were collected and analyzed in detail using CatWalk software. The obtained data were evaluated based on the following: running duration, average running speed, average posture, maximum contact intensity, paw print length, paw print width, paw print area, stride length, and swing speed to assess mouse muscle function. Based on the measurement data analysis, the paw print area and intensity of the experimental right hind limb were divided by the control left hind limb to obtain relative values, which were used to quantify the functional recovery effect of the engineered muscle. The relative values ​​of the paw print area and intensity of the engineered muscle implantation group reached 0.68, and the relative values ​​of the paw print intensity reached 0.76, both significantly higher than those of the control group.

[0077] 2) Muscle electrophysiological assessment: After anesthetizing the mice, the skin on the surface of the calf was incised to expose the tibialis anterior muscle. A stimulating electrode was inserted at the upper end of the tibialis anterior muscle to deliver an electrical current. Simultaneously, measuring electrodes were inserted at both ends of the tibialis anterior muscle (relative to the reference electrode) to generate a potential difference, and the bioelectrical signals during neuromuscular activity were recorded. The bioelectricity generated by muscle movement was recorded, and the differential amplifier monitored the signal, amplified, and recorded to obtain an electromyographic graph. The electromyographic integral was calculated: the sum of the areas under the curve within 1 second of the rectified and filtered electromyographic signal (the electromyographic integral is used to analyze the contractile characteristics of the muscle per unit time) was used to evaluate the physiological function of the muscle. The electromyographic integral in the engineered muscle implant group reached 0.299 mv.s, while the integral in the control group was only 0.025 mv.s, confirming the superior functional repair effect of the engineered muscle.

[0078] Through this experimental example, it can also be illustrated that some of the beneficial effects of the present invention can be partially listed as follows (the remaining beneficial effects can be combined with other contents in the specification):

[0079] 1) Because natural skeletal muscle in the human body is composed of highly oriented, parallel bundles of muscle fibers, its specific mechanical functions rely on this orientation. Therefore, achieving oriented orientation is a key step in constructing biomimetic muscle tissue in vitro. In the current field of tissue engineering, constructing oriented bioscaffolds is a commonly used therapeutic strategy. Although these materials can achieve macroscopic tissue orientation, they still lack the ability to regulate the microstructure of individual muscle fibers and may also suffer from problems such as poor biocompatibility. The above-mentioned light-controlled cell orientation technology can easily obtain oriented cells and induce them to form the desired tissue structure. By constructing an engineered oriented structure, the regular morphology of natural skeletal muscle can be simulated to achieve the purpose of biomimetic, playing a key regulatory role in muscle tissue regeneration and reconstruction. This scaffold-free strategy has good biocompatibility and also retains extracellular matrix (ECM) signals, which can regulate cell behavior and induce cell migration in vivo. In addition, by stacking cell sheets, morphologically adjustable three-dimensional structures can be assembled, which can further induce the contraction of more muscle fibers.

[0080] 2) Innervation ability: The functionality of skeletal muscle depends on innervation. Currently, effective neural integration of engineered skeletal muscle tissue remains a challenge for rebuilding extensively damaged muscles and restoring muscle function. Natural skeletal muscle tissue is innervated by the peripheral nervous system by establishing neuromuscular junctions (NMJs). Denervated skeletal muscle loses its contractile force and suffers from muscle atrophy. In the current field of skeletal muscle tissue engineering, research on integrated neuralized engineered muscles is still rare, and the effect of its in vivo functional improvement is still unclear. The present invention constructs neuralized muscle tissue through neuromuscular co-culture, which can form effective NMJs and make innervation possible.

[0081] 3) Vascular regeneration ability: Due to its high metabolic rate, skeletal muscle requires abundant blood supply to provide cells and tissues with necessary oxygen and nutrients. In addition, good blood perfusion is also necessary to maintain muscle survival and continuation of function. Through the pre-vascularization strategy, an in vitro engineering structure is constructed to maintain blood perfusion, which can improve the survival rate of implanted cells and tissues after transplantation. The present invention can achieve the goal of vascular repair through this engineering structure, and its special multi-lumen structure simulates the morphology of a normal capillary network. This structure has significant advantages in vascular maturity and promoting muscle differentiation.

[0082] 4) Adjunctive electrotherapy: Electrical stimulation is a common physical therapy modality. Appropriate stimulation can induce muscle movement or simulate normal voluntary movement to relieve muscle spasms and prevent muscle atrophy. However, due to the limited conductivity of the tissue, the current stimulation cannot be accurately transmitted to the target tissue, resulting in low treatment efficiency. The engineered tissue constructed by the present invention effectively improves conductivity using flexible electrodes, allowing the current applied from the skin electrodes to be accurately delivered, thereby improving muscle function in the area.

[0083] The present invention uses engineering methods to reconstruct a multi-layer tissue structure including muscles, nerves, and blood vessels in vitro. It has the ability to repair integrated neurovascularized muscles and can achieve functional muscle regeneration. The constructed engineered muscle can simulate the anatomical morphology of natural skeletal muscle to form a highly directional structure, which can achieve the goal of repairing muscle functionalization. In addition, the built-in flexible electrodes can be used to assist in physical therapy with electrical stimulation to further enhance functionality. In addition, the technology of the present invention is easy to operate and has repeatability. Engineered muscles not only have good biocompatibility, but can also be designed according to different types of muscle defects to achieve the goal of personalized customization. Theoretically, this invention can be used for defects of various muscle types and sizes, and has broad application prospects.

[0084] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A functional neurovascularized engineered muscle capable of electrical response, characterized in that: The engineered muscle comprises a first electrode layer and a second electrode layer, wherein a neuromuscular layer and a vascular layer are provided between the first electrode layer and the second electrode layer; The first electrode layer and the second electrode layer are both flexible electrodes, and the flexible electrodes include a mesh polyester fiber support and a conductive ink layer attached to the polyester fiber support; The neuromuscular layer is obtained by spreading a mixture of nerve cells and muscle cells on a light-controlled oriented nerve cell sheet using light-controlled oriented technology and culturing the mixture; The vascular layer is obtained by spreading endothelial cells on a light-controlled directional vascular cell sheet using light-controlled directional technology and culturing the cells.

2. The engineered muscle according to claim 1, characterized in that The neuromuscular layer consists of two layers, namely a first neuromuscular layer and a second neuromuscular layer, and the vascular layer is arranged between the first neuromuscular layer and the second neuromuscular layer.

3. The engineered muscle according to claim 1 or 2, characterized in that: The engineered muscle further includes a first hydrogel layer for supporting the neuromuscular layer and a second hydrogel layer for supporting the vascular layer.

4. The engineered muscle according to claim 1 or 2, characterized in that: The thickness of the flexible electrode is 40-60 μm, and the diameter of the single fiber on the polyester fiber support is 25-60 μm.

5. A method for preparing engineered muscle according to claim 1, characterized in that The steps include: S1: Preparation of light-controlled oriented cell sheets: construct light-controlled oriented neural cell sheets for the neuromuscular layer and light-controlled oriented vascular cell sheets for the vascular layer; S2: Preparation of the neuromuscular and vascular layers: Using photo-controlled directional technology, a mixture of nerve cells and muscle cells is spread on a photo-controlled directional nerve cell sheet and cultured to obtain a neuromuscular layer; using photo-controlled directional technology, endothelial cells are spread on a photo-controlled directional vascular cell sheet and cultured to obtain a vascular layer; S3: Muscle assembly: Take the first electrode layer and the second electrode layer, and place the neuromuscular layer and blood vessel layer prepared in step S2 between the first electrode layer and the second electrode layer to obtain the engineered muscle.

6. The method for preparing engineered muscle according to claim 5, characterized in that The step S1 specifically comprises: preparing nanodots on the surfaces of the two substrates respectively, and then using ultraviolet light to perform a first pattern modification and a second pattern modification on the nanodots on the two substrates respectively, wherein the first pattern is a directional arrangement pattern of cells in the neuromuscular layer, and the second pattern is a directional arrangement pattern of cells in the vascular layer, thereby obtaining a light-controlled directional nerve cell sheet and a light-controlled directional vascular cell sheet.

7. The method for preparing engineered muscle according to claim 5, characterized in that In the mixture of nerve cells and muscle cells in step S2, the ratio of muscle cells to nerve cells is 100:

1.

8. The method for preparing engineered muscle according to claim 5, characterized in that In step S3, the neuromuscular layer prepared in step S2 is transferred from the light-controlled oriented nerve cell sheet through the first hydrogel layer, and the vascular layer is transferred from the light-controlled oriented vascular cell sheet through the second hydrogel layer, and then assembled with the first electrode and the second electrode.

9. The method for preparing engineered muscle according to claim 8, characterized in that: The photocurable hydrogel is dropped onto the photocontrolled directional nerve cell sheet and the photocontrolled directional vascular cell sheet respectively. After the hydrogel is evenly spread on each cell sheet, it is irradiated under ultraviolet light for 55-65s to form the first hydrogel layer and the second hydrogel layer respectively. Then, the first hydrogel layer carrying the neuromuscular layer and the second hydrogel layer carrying the vascular layer are removed from each cell sheet, and then stacked and assembled with the first electrode and the second electrode to obtain the engineered muscle.

10. The method for preparing engineered muscle according to claim 5, characterized in that The first electrode layer and the second electrode layer in step S3 are both flexible electrodes, and the flexible electrodes include a mesh polyester fiber support and a conductive ink layer. The flexible electrodes are prepared by the following steps: S31: Preparation of mesh polyester fiber scaffold: Draw the mesh structure pattern of the scaffold, and then use the melt near-field direct writing technology to print the mesh polyester fiber scaffold according to the mesh structure pattern; S32: Hydrophilic treatment of polyester fiber: The surface of the mesh polyester fiber scaffold is treated with hydrophilicity to improve its hydrophobicity by in-situ polymerization of dopamine; S33: Conductive ink layer coating: apply carbon nanotube liquid conductive ink to the mesh polyester fiber support treated by S2, and then dry it. Repeat the above coating and drying steps multiple times to obtain the result.

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