Biomimetic skeletal muscle material, method of making and use thereof

Abstract

The application provides a kind of bionic skeletal muscle material and its preparation method and application, it is related to biomaterial field.The bionic skeletal muscle material includes muscle bundle simulation unit;Muscle bundle simulation unit includes first hollow tube with nanofiber membrane as tube wall and first cell-loaded gel filled in first hollow tube;Nanofiber membrane is the oriented nanofiber membrane prepared by electrospinning method.The bionic skeletal muscle material alleviates the problem that the existing technology lacks to construct large-size vascular networked skeletal muscle tissue engineering construction, so that muscle tissue cannot realize normal physiological function.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, and in particular to a biomimetic skeletal muscle material, its preparation method, and its application. Background Technology

[0002] Skeletal muscle, a common type of muscle tissue, accounts for approximately 40% of the total body weight. Muscle tissue performs basic functions such as daily movement, maintaining balance, and respiration through the force generated by the process of relaxation and contraction. Simultaneously, skeletal muscle plays a crucial role in energy storage, thermoregulation, and basal energy metabolism. Skeletal muscle is a fibrous tissue with a complex, multi-level structure, composed of neatly arranged bundles of multinucleated muscle fibers. These bundles are enclosed by the epimysium, forming muscle tissue. Connective tissue surrounding the muscle tissue is interconnected, allowing blood vessels and nerves to enter along the connective tissue membranes, facilitating substance exchange and enabling skeletal muscle movement and contraction. Skeletal muscle is a highly vascularized and neurally networked tissue. The spaces between the connective tissue membranes are filled with oxygen and nutrients, facilitating energy exchange and maintaining the normal physiological functions of muscle fibers. Skeletal muscle contraction and movement are regulated by motor input through neuromuscular junctions (NMJs), which are specialized synapses between the presynaptic motor neuron terminal and the motor endplate of the postsynaptic muscle fiber. When a nerve impulse reaches the NMJ, it causes acetylcholine-containing vesicles to be released from the axon terminal and bind to ACh receptors (AChRs) on the muscle fiber membrane, generating an action potential. This action potential leads to the release of a large number of calcium ions into the muscle fiber. The calcium ions interact with shielding proteins on actin and myosin filaments, resulting in muscle contraction. Causes of skeletal muscle injury are very common, including muscle contusions from exercise, muscle tears from accidents, and muscle damage from most surgical procedures. In addition, skeletal muscle atrophy caused by disease and aging due to age can also lead to irreversible damage to skeletal muscle tissue. For minor injuries, skeletal muscle has a relatively good regenerative capacity, but severe injuries such as large volume muscle loss (VML) lead to the loss of its inherent regenerative capacity, resulting in irreversible fibrosis and overall loss of muscle function. Currently, effectively treating skeletal muscle wounds and promoting the regeneration of damaged muscle tissue remains a significant clinical challenge. Muscle injury healing is a dynamic and complex physiological process involving three overlapping yet distinct phases: inflammation, repair, and remodeling. These three healing phases involve interactions between various cell types and their products, bioactive factors, and the extracellular matrix (ECM). However, severe injury can lead to physiological imbalances, causing the healing process to halt at one of these phases, resulting in impaired cellular function at the site of injury and hindering normal healing.

[0003] The above brief explanation of muscle anatomy and physiology highlights the importance of the layered fibromuscular structure and its integration with other tissues (including the vascular and nervous systems) for normal tissue function. This structure needs to be maintained and reproduced after injury through the natural repair of skeletal muscle or through targeted regeneration using various treatment methods. Currently, the clinical treatment for VML involves transplanting autologous tissue to the defect area for functional reconstruction. However, this method is limited by insufficient tissue sources and can lead to complications such as wound infection and necrosis. Therefore, seeking a tissue engineering therapy to actively promote muscle regeneration is of great significance.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a biomimetic skeletal muscle material and its preparation method, so as to alleviate the problem that the existing technology lacks the ability to construct large-scale vascular network skeletal muscle tissue engineering constructs, which leads to the inability of muscle tissue to achieve normal physiological functions.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] In a first aspect, a biomimetic skeletal muscle material is provided, the biomimetic skeletal muscle material comprising a muscle bundle simulation unit; the muscle bundle simulation unit comprising a first hollow tube with a nanofiber membrane as the tube wall and a first cell-carrying gel filled in the first hollow tube; the nanofiber membrane is an oriented nanofiber membrane prepared by electrospinning.

[0008] In an optional embodiment, the biomimetic skeletal muscle material further comprises a skeletal muscle simulation unit, which includes a second hollow tube with the nanofiber membrane as the tube wall and a plurality of muscle bundle simulation units filled in the second hollow tube; the plurality of muscle bundle simulation units are arranged in a bundle-like manner along the extension direction of the tube in the second hollow tube; the gap between the second hollow tube and the first hollow tube is filled with a second cell-carrying gel; the cells in the second cell-carrying gel include endothelialized stem cells.

[0009] In an optional embodiment, the biomimetic skeletal muscle material comprises a plurality of skeletal muscle simulation units arranged in a bundle.

[0010] In an optional embodiment, the nanofiber membrane constituting each hollow tube is independently selected from PLGA-gelatin nanofiber membrane or PCL-PCL nanofiber membrane.

[0011] The PLGA-gelatin nanofiber membrane is prepared by simultaneously spinning PLGA electrospinning solution and gelatin electrospinning solution through spinnerets and receiving them on the same receiving device.

[0012] The PCL-PCL nanofiber membrane is prepared by simultaneously spinning two sets of PCL electrospinning solutions through spinnerets and receiving them onto the same receiving device. One set of PCL electrospinning solution spins micron-sized fibers, and the other set of PCL electrospinning solution spins nano-sized fibers. The PCL-PCL nanofiber membrane is a micron-nano composite membrane.

[0013] In an optional embodiment, the gels in the first cell-carrying gel and the second cell-carrying gel are each independently hydrogels.

[0014] Secondly, a method for preparing the biomimetic skeletal muscle material of the first aspect is also provided, comprising filling a first hollow tube with a nanofiber membrane as the tube wall with a first cell-carrying gel.

[0015] In an optional embodiment, the preparation method further includes preparing a nanofiber membrane by electrospinning; the nanofiber membrane includes a PLGA-gelatin nanofiber membrane and / or a PCL-PCL nanofiber membrane;

[0016] The method for preparing the PLGA-gelatin nanofiber membrane includes preparing PLGA electrospinning solution and gelatin electrospinning solution respectively, receiving the two electrospinning solutions from the same rotating roller, and obtaining the PLGA-gelatin nanofiber membrane after drying.

[0017] The method for preparing the PCL-PCL nanofiber membrane includes preparing two sets of PCL electrospinning solutions, using the same rotating roller to receive the spinnerets from the two sets of PCL electrospinning solutions, and drying them to obtain the PCL-PCL nanofiber membrane; wherein one set of PCL electrospinning solutions is used to spin micron-sized fibers, and the other set of PCL electrospinning solutions is used to spin nano-sized fibers.

[0018] In an optional embodiment, the preparation method further includes fixing a plurality of muscle bundle simulation units into a bundle shape, wrapping them with a nanofiber membrane, and then filling the gap between the second hollow tube and the first hollow tube with a second cell-carrying gel to obtain the skeletal muscle simulation unit.

[0019] Thirdly, the application of the above-mentioned biomimetic skeletal muscle material or the preparation method of the above-mentioned biomimetic skeletal muscle material in the preparation of skeletal muscle repair products is provided.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention discloses a biomimetic skeletal muscle material with a vascular network, which mimics the components and physiological structure of natural tissues by combining an oriented nanofiber membrane prepared by electrospinning with a cell-carrying gel. The fiber structure prepared by electrospinning has the greatest similarity to the connective tissue membrane of skeletal muscle, thus mimicking the fibrous structure of the connective tissue membrane; furthermore, the nanoscale properties of the nanofiber structure can promote the regulation of cell growth morphology, arrangement, and gene expression. The cell-carrying gel has the greatest similarity to the extracellular matrix, thus mimicking the function of the extracellular matrix to provide a microenvironment for cell growth. Injecting the cell-carrying gel into a hollow tubular nanofiber membrane yields a muscle bundle simulation unit composed of a gel-nanofiber composite material, which functions as a skeletal muscle bundle. Multiple muscle bundle simulation units can be further combined, and the gaps filled with endothelialized stem cells to simulate a vascular network, resulting in a bundle-like skeletal muscle simulation unit that effectively mimics skeletal muscle.

[0022] The biomimetic skeletal muscle material provided by this invention has the following advantages: 1. The nanofiber membrane used as a cell scaffold in this invention has sufficient mechanical strength and elasticity, which can guide tissue growth in a suitable direction and has ideal biomedical functions; 2. It has good biocompatibility, few side effects, and a suitable degradation rate. When the damaged tissue regenerates to a certain extent, the cell scaffold can be completely degraded and absorbed by the organism, avoiding secondary damage to the organism; 3. In vitro and in vivo studies show that it can promote the repair of damaged skeletal muscle sites and has good clinical application prospects. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is a flowchart illustrating the preparation process of the biomimetic skeletal muscle material provided in Embodiment 1 of the present invention.

[0025] Figure 2a The morphology of the PLGA-gelatin nanofiber membrane under a scanning electron microscope in Example 1 of this invention;

[0026] Figure 2b The morphology of the heparin-gelatin-PLGA-gelatin nanofiber membrane under a scanning electron microscope in Example 1 of this invention;

[0027] Figure 2cThe morphology of the hydrogel GelMA combined with the nanofiber membrane in the muscle bundle simulation unit of Embodiment 1 of the present invention is shown under a scanning electron microscope.

[0028] Figure 2d The overall morphology of the tubular support under a scanning electron microscope in Embodiment 1 of the present invention;

[0029] Figure 2e The morphology of the PCL-PCL nanofiber film prepared in Example 2 of this invention is shown under an electron microscope.

[0030] Figure 3a The infrared spectrum of the heparin-gelatin-PLGA-gelatin nanofiber membrane in Example 1 of this invention;

[0031] Figure 3b The 1H NMR spectrum of the heparin-gelatin-PLGA-gelatin nanofiber membrane in Example 1 of this invention;

[0032] Figure 4 The results of cell proliferation detection in the biomimetic skeletal muscle material provided in Embodiment 2 of the present invention. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] In this document, "prepared from" and "comprising" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0035] In this document, when a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0036] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0037] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions. Similarly, all technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0038] Unless otherwise specified, the steps in this article can be performed sequentially or not.

[0039] In this document, relevant terms such as “first” and “second” may be used alone to distinguish one entity or behavior from another, rather than necessarily requiring or implying any actual such relationship or order between these entities or behaviors.

[0040] In this document, the terms “about” or “approximately” are applied to all numerical values, whether or not explicitly indicated. These terms generally refer to a range of numbers that a person skilled in the art would consider equivalent to the enumerated numerical values ​​(i.e., having the same function or result). In many examples, these terms may include numbers around the most recent significant digit.

[0041] Skeletal muscle tissue engineering is an emerging discipline in regenerative medicine. It involves constructing muscle tissue in vitro and in vivo using a combination of biological scaffolds, cells, and growth factors to repair or regenerate skeletal muscle tissue lost due to traumatic injury, tumor resection, or muscle disease. After a long period of development, tissue engineering strategies applied to skeletal muscle injury have yielded many significant results, but the optimal methods for scaffold design and cell sourcing have not yet been fully explored. Ideal engineered skeletal muscle should possess the following characteristics: (1) Cells have the ability to differentiate into muscle fibers, cells are oriented and attached to the scaffold, and cells can combine and assemble to form muscle bundles. (2) The implanted scaffold has a certain tension, maintaining its original shape when implanted at the defect site. (3) The scaffold has good biocompatibility, providing a favorable microenvironment for injury healing in the damaged area. (4) It has a functional vascular network, effectively transporting nutrients and oxygen and removing metabolic waste, providing a normal physiological environment.

[0042] To achieve at least one of the above-mentioned technical effects, in a first aspect, the present invention provides a biomimetic skeletal muscle material comprising a muscle bundle simulation unit. The muscle bundle simulation unit comprises a first hollow tube with a nanofiber membrane as its wall and a first cell-carrying gel filled within the first hollow tube. The nanofiber membrane is an oriented nanofiber membrane prepared by electrospinning. The first hollow tube with the nanofiber membrane as its wall is used to simulate the connective tissue membrane of skeletal muscle. Injecting the first cell-carrying gel into it yields a cell-carrying gel-nanofiber membrane composite material, used to simulate the muscle fiber structure of skeletal muscle, i.e., the muscle bundle simulation unit. The imitation of natural tissue components and physiological structures is achieved by combining the oriented nanofiber membrane prepared by electrospinning with the cell-carrying gel.

[0043] In an optional embodiment, the biomimetic skeletal muscle material further comprises a skeletal muscle simulation unit. The skeletal muscle simulation unit includes a second hollow tube with a nanofiber membrane as its wall and a plurality of muscle bundle simulation units filling the second hollow tube. The plurality of muscle bundle simulation units are arranged in a bundle-like manner along the extension direction of the tube within the second hollow tube. A second cell-carrying gel is filled in the gap between the second hollow tube and the first hollow tube. The cells in the second cell-carrying gel include endothelialized stem cells, which form a mesh structure in the gap to simulate a vascular network. The biomimetic skeletal muscle material containing this skeletal muscle simulation unit in the hollow tube can simulate the structure of skeletal muscle. Compared with the prior art, it achieves myogenic formation while simultaneously constructing a three-dimensional vascular network. In an optional embodiment, the number of muscle bundle simulation units in each skeletal muscle simulation unit is 2, 3, 4, or 5, preferably 3.

[0044] In an optional embodiment, the biomimetic skeletal muscle material comprises a plurality of skeletal muscle simulation units arranged in a bundle.

[0045] In an optional embodiment, the biomimetic skeletal muscle material further comprises a third hollow tube with the nanofiber membrane as the tube wall and a plurality of skeletal muscle simulation units filled in the third hollow tube; the plurality of skeletal muscle simulation units are arranged in a bundle-like manner along the extension direction of the tube within the third hollow tube.

[0046] In an optional embodiment, the number of skeletal muscle simulation units in the biomimetic skeletal muscle material is 2, 3, 4 or 5, preferably 3.

[0047] In an optional embodiment, the nanofiber membrane constituting each hollow tube is independently selected from PLGA (polylactic acid-glycolic acid copolymer)-gelatin nanofiber membrane or PCL (polycaprolactone)-PCL (polycaprolactone) nanofiber membrane.

[0048] The PLGA-gelatin nanofiber membrane is a composite nanofiber membrane obtained by co-spinning two electrospinning solutions. Specifically, the PLGA electrospinning solution and the gelatin electrospinning solution are spun simultaneously through separate spinnerets and received on the same receiving device to prepare the nanofiber membrane. That is, the PLGA electrospinning solution is spun through one spinneret, and the gelatin electrospinning solution is spun through another spinneret, and the two are spun simultaneously and received on the same receiving device to achieve co-spinning, thus obtaining the PLGA-gelatin nanofiber membrane.

[0049] PCL-PCL nanofiber membranes are prepared by simultaneously spinning two sets of PCL electrospinning solutions through separate spinnerets and receiving them on the same receiving device. Specifically, one set of PCL electrospinning solution is spun through one spinneret, and the other set is spun through another spinneret; both sets are spun simultaneously and received on the same receiving device to achieve co-spinning, thus obtaining the PCL-PCL nanofiber membrane. Furthermore, one set of PCL electrospinning solution spins micron-sized fibers, and the other set spins nano-sized fibers; therefore, the PCL-PCL nanofiber membrane is a micron-nano composite membrane.

[0050] In an optional embodiment, the nanofiber membrane constituting each hollow tube is independently modified with gelatin and / or an anticoagulant. The modification refers to loading the gelatin and / or anticoagulant onto the nanofiber membrane via physical and / or chemical bonding. The chemical bonding includes, but is not limited to, bonding to the nanofiber membrane via chemical bonds and / or chemical groups. In an optional embodiment, the nanofiber membrane constituting each hollow tube is modified with both gelatin and an anticoagulant. In an optional embodiment, the anticoagulant includes heparin or a pharmaceutically acceptable salt thereof.

[0051] In an optional embodiment, the PLGA-gelatin nanofiber membrane is prepared using the preparation method described in any of the following embodiments.

[0052] In an optional embodiment, the PCL-PCL nanofiber membrane is prepared using the preparation method described in any of the following embodiments.

[0053] It is understood that the nanofiber membranes constituting each hollow tube can be the same or different. Optionally, the nanofiber membranes constituting the first and second hollow tubes can be the same, both selected from PLGA-gelatin nanofiber membranes, or both selected from PCL-PCL nanofiber membranes. Optionally, the nanofiber membranes constituting the first and second hollow tubes can be different, for example, but not limited to, the first hollow tube being composed of a PLGA-gelatin nanofiber membrane and the second hollow tube being composed of a PCL-PCL nanofiber membrane; or, the first hollow tube being composed of a PCL-PCL nanofiber membrane and the second hollow tube being composed of a PLGA-gelatin nanofiber membrane. Optionally, the nanofiber membranes constituting each hollow tube in the biomimetic skeletal muscle material can be the same, all using PLGA-gelatin nanofiber membranes or all using PCL-PCL nanofiber membranes.

[0054] In an optional embodiment, the nanofiber membranes constituting each hollow tube in the biomimetic skeletal muscle material are all made of PLGA-gelatin nanofiber membranes, and the PLGA-gelatin nanofiber membranes are modified with gelatin and an anticoagulant, wherein the anticoagulant is preferably sodium heparin.

[0055] In an optional embodiment, the hydrogels in the first and second cell-carrying gels are each independently a hydrogel. A hydrogel is a substance formed when natural or synthetic polymers are cross-linked by covalent, ionic, or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules to form a gel. The hydrogel may be selected from those acceptable in the art for use as a cell matrix, including but not limited to polysaccharides, polyacrylates, collagen, polyvinylpyrrolidone, or hyaluronic acid.

[0056] In an optional embodiment, the hydrogel includes an injectable hydrogel to facilitate filling the muscle bundle simulation unit and the skeletal muscle simulation unit with a cell-carrying gel.

[0057] In an optional embodiment, the hydrogel comprises a photocurable hydrogel. In an optional embodiment, the hydrogel comprises a methacrylic hydrogel. In an optional embodiment, the concentration of the methacrylic hydrogel is 5-15% wt, for example, but not limited to 5, 8, 10, 12, or 15% wt, or a concentration range between any two of the above points.

[0058] In an optional embodiment, the gels in the first cell-carrying gel and the second cell-carrying gel can be the same or different, and the differences include different hydrogel materials and / or different concentrations of hydrogels of the same material.

[0059] In an optional embodiment, when the biomimetic skeletal muscle material includes more than one muscle bundle simulation unit, the hydrogel filled in each muscle bundle simulation unit can be the same, or at least the hydrogel in some muscle bundle simulation units can be different from the hydrogel in the remaining muscle bundle simulation units. In an optional embodiment, the difference includes different hydrogel materials and / or different concentrations of hydrogels of the same material. In an optional embodiment, when the biomimetic skeletal muscle material includes more than one muscle bundle simulation unit, the hydrogel filled in each muscle bundle simulation unit is the same.

[0060] In an optional embodiment, the first cell-carrying gel and the second cell-carrying gel may also contain, independently, optional excipients, auxiliaries and / or active substances acceptable in the art, including but not limited to buffer components, crosslinking agents, photocuring agents and growth factors that promote cell growth.

[0061] In an optional embodiment, the contact surfaces of two adjacent hollow tubes are fixedly connected. The fixed connection can be any connection method acceptable in the art, and the present invention is not limited thereto. In an optional embodiment, the fixed connection is adhesive, and the adhesive used for adhesive bonding can be any adhesive acceptable in the art, and the present invention is not limited thereto.

[0062] In an optional implementation, the contact surface of two adjacent hollow tubes is the contact surface of the outer walls of the two hollow tubes, such as the contact surface of adjacent muscle bundle simulation units.

[0063] In an optional implementation, the contact surface of two adjacent hollow tubes is the outer wall of one hollow tube and the inner wall of the other hollow tube, such as the outer wall of the muscle bundle simulation unit and the inner wall of the second hollow tube.

[0064] In an optional embodiment, the biomimetic skeletal muscle material is composed of several skeletal muscle simulation units, and the nanofiber membrane constituting the three-dimensional scaffold is a PLGA-gelatin nanofiber membrane modified with gelatin and sodium heparin. The first cell-carrying gel filled in the biomimetic skeletal muscle material includes pluripotent stem cells and methacrylated hydrogel (GelMA).

[0065] PLGA-gelatin nanofiber membranes are used to simulate skeletal muscle connective tissue membranes. By organically combining nanofibers with an injectable cell-carrying gel, the advantages of synthetic polymer PLGA, natural polymer Gt (gelatin), heparin (Hep), and injectable gel GelMA in terms of composition and properties are complemented, mimicking the physiological composition of normal human skeletal muscle connective tissue. Furthermore, by combining the nanofiber membrane prepared by electrospinning with the structural characteristics of both the cell scaffold and the injectable gel, a biomimetic skeletal muscle tissue engineering scaffold with the original structure and function of skeletal muscle tissue is prepared, simulating the normal physiological structure of human skeletal muscle.

[0066] Secondly, a method for preparing the aforementioned biomimetic skeletal muscle material is provided, comprising filling a first cell-carrying gel into a first hollow tube with a nanofiber membrane as the tube wall.

[0067] In an optional embodiment, the nanofiber membrane is wound into shape using a mold to prepare the first hollow tube in the form of a hollow tube.

[0068] In an optional embodiment, the gels in the first cell-carrying gel and the second cell-carrying gel each independently include photocurable hydrogels, and the preparation method includes preparing a photocurable hydrogel containing a photoinitiator, filling it, and then irradiating it with ultraviolet light.

[0069] In an optional embodiment, the method further includes fixing several muscle bundle simulation units into a bundle shape, then wrapping them with a nanofiber membrane, injecting a hydrogel containing stem cells carrying endothelial differentiation (i.e., a second cell-carrying gel) into the gap between the first hollow tube and the second hollow tube, filling the gap, and then irradiating with ultraviolet light to obtain a skeletal muscle simulation unit.

[0070] In an optional embodiment, the fixing includes adhesive fixing.

[0071] In an optional embodiment, the nanofiber membrane constituting each first hollow tube and each second hollow tube is prepared independently using any one of the following embodiments.

[0072] In an alternative embodiment, rollers are used to receive the spinnerets of two electrospinning solutions to obtain continuous oriented nanofibers.

[0073] In an optional embodiment, the spinning conditions for each electrospinning solution are independent and include: a spinning ambient temperature of 15-25°C, a spinning ambient humidity of 20-40%, an electrostatic high voltage of 15-18kV between the metal spinneret and the receiving device, a receiving distance of 15-18cm, and a roller rotation speed of 1500-1600rpm.

[0074] In an optional embodiment, the spinning conditions for each electrospinning solution are independent, including: spinning ambient temperature of 20°C, spinning ambient humidity of 30%, positive voltage of 14kV, negative voltage of -1kV, receiving distance of 15cm, and roller receiving speed of 1570rpm.

[0075] In an optional embodiment, the PLGA-gelatin nanofiber membrane is prepared as follows: PLGA electrospinning solution and gelatin electrospinning solution are prepared separately, the two electrospinning solutions are received by the same rotating roller, and the membrane is obtained after drying.

[0076] In an optional embodiment, the concentration of PLGA in the PLGA electrospinning solution is 10-20%wt, for example, but not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20%wt, or any concentration range between any two of the above points, preferably about 15%wt.

[0077] In an optional embodiment, the solvent of the PLGA electrospinning solution includes hexafluoroisopropanol.

[0078] In an optional embodiment, the concentration of gelatin in the gelatin electrospinning solution is 10-20% wt, for example, but not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% wt, or any concentration range between any two of the above points, preferably about 10% wt.

[0079] In an optional embodiment, the solvent in the gelatin electrospinning solution includes hexafluoroisopropanol and acetic acid. Alternatively, the solvent in the gelatin electrospinning solution is hexafluoroisopropanol and acetic acid in a volume ratio of 1:1.

[0080] In an optional embodiment, the PLGA-gelatin nanofiber membrane is prepared as follows:

[0081] PLGA was weighed and dissolved in hexafluoroisopropanol, stirred until completely dissolved, to obtain a PLGA electrospinning solution with a mass fraction of approximately 15%. The prepared PLGA electrospinning solution was added to a syringe, the tip of which was connected to the electrospinning machine via a 20G flat-head metal spinneret. The syringe was then fixed to a syringe pump. The inner diameter of the metal spinneret was 1.05 mm. The distance between the metal receiving plate and the spinneret was adjusted to a receiving distance of 15 cm. During the spinning process, the electrostatic high voltage between the metal spinneret and the receiving plate was controlled at 15 kV, and the flow rate of the electrospinning solution was controlled at 1.2 mL / h using the syringe pump.

[0082] Gelatin was weighed and dissolved in a mixed solution of hexafluoroisopropanol and 5 mL of acetic acid (v / v = 1:1), and stirred until completely dissolved to obtain a gelatin electrospinning solution with a mass fraction of approximately 10%. The prepared gelatin electrospinning solution was added to a syringe, the tip of which was connected to the electrospinning machine via a 20G flat-head metal spinneret. The syringe was then fixed to a syringe pump. The inner diameter of the metal spinneret was 1.05 mm. The distance between the metal receiving plate and the spinneret was adjusted to a receiving distance of 15 cm. During the spinning process, the electrostatic high voltage between the metal spinneret and the receiving plate was controlled at 18 kV, and the flow rate of the gelatin electrospinning solution was controlled at 1.0 mL / h using the syringe pump.

[0083] Two gelatin electrospinning solutions were spun simultaneously for 3 hours. The resulting material was then removed and dried overnight in a fume hood to obtain a PLGA-gelatin nanofiber membrane.

[0084] In an optional embodiment, the PCL-PCL nanofiber membrane is prepared as follows: two sets of PCL electrospinning solutions are prepared, and the two sets of PCL electrospinning solutions are received and spun using the same rotating roller. After drying, a PCL-PCL nanofiber membrane is obtained. One set of PCL electrospinning solutions is used to spin micron-sized fibers, while the other set is used to spin nano-sized fibers, making the PCL-PCL nanofiber membrane a micron-nano composite membrane.

[0085] In an optional embodiment, the concentration of PCL in the PCL electrospinning solution is 10-15% wt, for example, but not limited to 10, 11, 12, 13, 14, or 15% wt, or any concentration range between any two of the above points. In an optional embodiment, one set of PCL electrospinning solutions has a PCL concentration of approximately 10%, and another set of PCL electrospinning solutions has a PCL concentration of approximately 12%.

[0086] In an optional embodiment, the solvent of the PCL electrospinning solution includes formic acid and acetic acid. In an optional embodiment, the volume ratio of formic acid to acetic acid is 7:3. Using a mixture of formic acid and acetic acid as a solvent, electrospinning fibers with nanometer-scale diameters can be obtained.

[0087] In an optional embodiment, the solvent of the PCL electrospinning solution includes dichloromethane and N,N-dimethylformamide. In an optional embodiment, the volume ratio of dichloromethane to N,N-dimethylformamide is 4:1. Using a mixture of dichloromethane and N,N-dimethylformamide as a solvent, electrospinning fibers with micron-scale diameters can be obtained.

[0088] In an optional embodiment, the PCL-PCL nanofiber membrane is prepared as follows:

[0089] PCL was weighed and dissolved in a mixed solution of formic acid and acetic acid (v / v = 7:3), and stirred until completely dissolved to obtain a PCL electrospinning solution with a mass fraction of approximately 10%. The prepared PCL electrospinning solution was added to a syringe, the tip of which was connected to the electrospinning machine via a 20G flat-head metal spinneret. The syringe was then fixed to a syringe pump. The inner diameter of the metal spinneret was 1.05 mm. The distance between the metal receiving plate and the spinneret was adjusted to a receiving distance of 15 cm. During the spinning process, the electrostatic high voltage between the metal spinneret and the receiving plate was controlled at 15 kV, and the flow rate of the PCL electrospinning solution was controlled at 1.0 mL / h using the syringe pump.

[0090] PCL was dissolved in a mixed solution of dichloromethane and N,N-dimethylformamide (v / v = 4:1) and stirred until completely dissolved to obtain a PCL electrospinning solution with a mass fraction of approximately 12%. The prepared PCL electrospinning solution was added to a syringe, the tip of which was connected to the electrospinning machine via a 20G flat-head metal spinneret. The syringe was then fixed to a syringe pump. The inner diameter of the metal spinneret was 1.05 mm. The distance between the metal receiving plate and the spinneret was adjusted to 18 cm. During the spinning process, the electrostatic high voltage between the metal spinneret and the receiving plate was controlled at 15 kV, and the flow rate of the PCL electrospinning solution was controlled at 1.0 mL / h using the syringe pump.

[0091] In an optional embodiment, the preparation method further includes modifying the nanofiber membrane with gelatin and / or an anticoagulant.

[0092] In an optional implementation, the anticoagulant includes heparin or a pharmaceutically acceptable salt thereof.

[0093] In an optional embodiment, the nanofiber membrane is treated with an alkaline solution before being modified with gelatin and / or heparin or a pharmaceutically acceptable salt thereof.

[0094] In an optional embodiment, the preparation method includes immersing the nanofiber membrane in a solution containing EDC / NHS buffer solution and gelatin, and drying it to obtain a gelatin-modified nanofiber membrane.

[0095] In an optional embodiment, the preparation method includes immersing a nanofiber membrane in a solution containing an EDC / NHS buffer solution and heparin or a pharmaceutically acceptable salt thereof, and drying it to obtain a nanofiber membrane modified with heparin or a pharmaceutically acceptable salt thereof.

[0096] In an optional embodiment, the preparation method includes immersing a nanofiber membrane in a solution containing an EDC / NHS buffer solution, gelatin, and heparin or pharmaceutically acceptable salts thereof, and drying it to obtain a nanofiber membrane modified with gelatin and heparin or pharmaceutically acceptable salts thereof.

[0097] Thirdly, the application of the aforementioned biomimetic skeletal muscle material, or the aforementioned method for preparing biomimetic skeletal muscle material, in the preparation of skeletal muscle repair products is also provided.

[0098] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0099] Example 1

[0100] This embodiment provides a biomimetic skeletal muscle material, which is prepared according to the following steps:

[0101] 1. Weigh 1.35g of PLGA and dissolve it in 10mL of hexafluoroisopropanol. Stir at 25℃ for 6 hours until completely dissolved to obtain a PLGA electrospinning solution with a mass fraction of approximately 15%. Add the prepared PLGA electrospinning solution to a 5mL syringe. Connect the tip of the syringe to the electrospinning machine via a 20G flat-head metal spinneret and fix the syringe to the injection pump. The inner diameter of the metal spinneret is 1.05mm. Adjust the distance between the roller and the spinneret to determine a receiving distance of 15cm. Control the electrostatic high voltage between the metal spinneret and the roller during spinning to 15kV, and control the flow rate of the PLGA electrospinning solution to 1.2mL / h using the injection pump.

[0102] 2. Weigh 1.28g of gelatin and dissolve it in a mixed solution of 5mL hexafluoroisopropanol and 5mL acetic acid (v / v = 1:1). Stir at 25℃ for 6 hours until completely dissolved to obtain a gelatin electrospinning solution with a mass fraction of approximately 10%. Add the prepared gelatin electrospinning solution to a 5mL syringe, connecting the tip to the electrospinning machine via a 20G flat-head metal spinneret. Fix the syringe to the injection pump. The inner diameter of the metal spinneret is 1.05mm. Adjust the distance between the roller and the spinneret to determine a receiving distance of 15cm. Control the electrostatic high voltage between the metal spinneret and the roller during spinning to 18kV, and control the flow rate of the gelatin electrospinning solution to 1.0mL / h using the injection pump.

[0103] 3. Two electrospinning solutions, PLGA and gelatin, were used to start spinning simultaneously. After spinning for 3 hours, the electrospun fibers were collected using a high-speed (1570 rpm) roller wrapped with aluminum foil. The resulting material was removed and dried overnight in a fume hood to obtain the PLGA-gelatin nanofiber membrane.

[0104] 4. Immerse a suitable PLGA-gelatin nanofiber membrane in a 0.05 mol / L MES solution, maintaining the pH at approximately 5.5, for 1 hour. Dissolve EDC and NHS in the MES solution to prepare a 5 mg / mL EDC / NHS buffer solution, and add 1 mg / mL heparin sodium to the buffer solution. After a 30-minute pre-reaction, place the pre-soaked nanofiber membrane into the buffer solution and immerse it at room temperature (25°C) for 2 hours. After 2 hours, remove the PLGA-gelatin nanofiber membrane and gently rinse it three times with PBS aqueous solution to remove uncrosslinked gelatin and heparin components. Dry the prepared material overnight in a fume hood to obtain the heparin-gelatin-PLGA-gelatin nanofiber membrane.

[0105] 5. A hollow tubular cylindrical support was prepared by winding a heparin-gelatin-PLGA-gelatin nanofiber membrane through a mold (syringe needle) to form a hollow tubular support with an inner diameter of about 0.18 mm, which served as the first hollow tube.

[0106] 6. Dissolve GelMA powder in PBS solution, add photoinitiator LAP (lithium phenyl (2,4,6-trimethylbenzoyl)phosphate), to obtain a 5% (w / w) hydrogel solution. Filter this solution through a 0.2 μm sterile injection filter to obtain the hydrogel solution. Add pluripotent stem cells to the hydrogel to obtain a cell-loaded gel solution. Inject the solution into the hollow portion of the first hollow tube and irradiate with ultraviolet light for 10 s to obtain a muscle bundle mimicry unit.

[0107] 7. Adhere the muscle bundle simulation units to each other to construct a three-channel scaffold. The outermost layer is wrapped with the heparin-gelatin-PLGA-gelatin nanofiber membrane prepared in step 4. Inject cell-carrying hydrogel with endothelialization ability into the gap between the obtained scaffold and the fiber membrane. Irradiate with ultraviolet light for 10 seconds to obtain a biomimetic skeletal muscle material.

[0108] Example 2

[0109] This embodiment provides a biomimetic skeletal muscle material, which differs from Embodiment 1 only in that the mass fraction of GelMA is 10%.

[0110] Example 3

[0111] This embodiment provides a biomimetic skeletal muscle material, which differs from Embodiment 1 only in that the mass fraction of GelMA is 15%.

[0112] Example 4

[0113] 1. First, weigh 1.10 g of PCL (polycaprolactone) and dissolve it in a mixed solution of 7 mL formic acid and 3 mL acetic acid (v / v = 7:3). Stir at 25°C for 6 hours until completely dissolved to obtain a PCL electrospinning solution with a mass fraction of approximately 10%. Add the prepared PCL electrospinning solution to a 5 mL syringe, connecting the tip to the electrospinning machine via a 20G flat-head metal spinneret. Fix the syringe to the injection pump. The inner diameter of the metal spinneret is 1.05 mm. Adjust the distance between the metal receiving plate and the spinneret to a receiving distance of 15 cm. Control the electrostatic high voltage between the metal spinneret and the receiving plate at 15 kV during spinning, and control the flow rate of the PCL electrospinning solution at 1.0 mL / h using the injection pump.

[0114] 2. Weigh 1.36g of PCL and dissolve it in a mixed solution of 8mL dichloromethane and 2mL N,N-dimethylformamide (v / v = 4:1). Stir at 25℃ for 6 hours until completely dissolved to obtain a PCL electrospinning solution with a mass fraction of approximately 12%. Add the prepared PCL electrospinning solution to a 5mL syringe, connecting the tip to the electrospinning machine via a 20G flat-head metal spinneret. Fix the syringe to the injection pump. The inner diameter of the metal spinneret is 1.05mm. Adjust the distance between the metal receiving plate and the spinneret to a receiving distance of 18cm. Control the electrostatic high voltage between the metal spinneret and the receiving plate at 15kV during spinning, and control the flow rate of the PCL electrospinning solution at 1.0mL / h using the injection pump.

[0115] 3. Two PCL electrospinning solutions were used to start spinning simultaneously. After spinning for 3 hours, the electrospun fibers were collected using a high-speed (1570 rpm) roller wrapped with aluminum foil. The resulting material was removed and dried overnight in a fume hood to obtain a PCL-PCL nanofiber membrane.

[0116] 4. Immerse a suitable size PCL-PCL nanofiber membrane in a 0.05 mol / L MES solution, maintaining the pH at approximately 5.5, for 1 hour. Dissolve EDC and NHS in the MES solution to prepare a 5 mg / mL EDC / NHS buffer solution, and add 1 mg / mL heparin sodium to the buffer solution, allowing for a pre-reaction of 30 minutes. Dissolve 20 mg of gelatin in 10 mL of deionized water to prepare a 2 mg / mL gelatin aqueous solution. Remove the soaked PCL-PCL nanofiber membrane and immerse it in the gelatin aqueous solution, adding the EDC / NHS buffer solution containing heparin sodium. Under aerobic conditions, soak at room temperature (25°C) for 2 hours. After 2 hours, remove the scaffold and gently rinse three times with PBS aqueous solution to remove any unattached gelatin components. Dry the prepared material overnight in a fume hood to obtain the heparin-gelatin-PCL-PCL nanofiber membrane.

[0117] 5. A hollow tubular cylindrical support was prepared by winding a heparin-gelatin-PCL-PCL nanofiber membrane through a mold (syringe needle) to form a hollow tubular support with an inner diameter of about 0.18 mm.

[0118] 6. Dissolve GelMA powder in PBS solution, add photoinitiator LAP to obtain a 5% (w / w) hydrogel solution. Filter this solution through a 0.2 μm sterile injection filter to obtain the hydrogel solution. Add pluripotent stem cells to the hydrogel to obtain a cell-loaded gel solution. Inject the solution into the hollow portion of the first hollow tube and irradiate with ultraviolet light for 10 s to obtain a muscle bundle mimic unit.

[0119] 7. Muscle bundle simulation units were bonded together to construct a three-channel scaffold. The outermost layer was wrapped with the heparin-gelatin-PCL-PCL nanofiber membrane prepared in step 4. Cell-carrying hydrogels with endothelialization capabilities were injected into the gaps between the scaffold and the fiber membrane. After irradiation with ultraviolet light for 10 seconds, a biomimetic skeletal muscle material was obtained. A schematic diagram of the structure is shown below. Figure 1 As shown.

[0120] Example 5

[0121] This embodiment provides a biomimetic skeletal muscle material, which differs from Embodiment 4 only in that the mass fraction of GelMA is 10%.

[0122] Example 6

[0123] This embodiment provides a biomimetic skeletal muscle material, which differs from Embodiment 4 only in that the mass fraction of GelMA is 15%.

[0124] Example 1

[0125] The microstructure of the heparin-gelatin-PLGA-gelatin nanofiber membrane and muscle bundle simulation unit prepared in Example 1 was observed using scanning electron microscopy (SEM). Before observation, the samples were sputter-coated with gold, and the fiber morphology was then observed under an accelerating voltage of 15 kV. The results are shown in [reference needed]. Figures 2a-2d .

[0126] The PCL-PCL nanofiber membrane prepared in Example 2 was observed using scanning electron microscopy (SEM). The results are shown in [link to SEM document]. Figure 2e .

[0127] Example 2

[0128] Mechanical property testing was conducted on the nanofiber membrane prepared in Example 1 using an electronic universal testing machine (n=5). The nanofiber membrane was cut into samples 60 mm long and 10 mm wide, and the thickness was measured using a thickness gauge. The tensile rate was 50 mm / min, and the gauge length was set to 3 cm. The tensile strength and elongation at break of the nanofiber membrane were obtained to determine if the strength exceeded 12 kPa, meeting the mechanical property standards for human skeletal muscle.

[0129] Table 1

[0130]

[0131] Example 3

[0132] Infrared spectroscopy and proton nuclear magnetic resonance spectroscopy were used to analyze the elemental composition of the heparin-gelatin-PLGA-gelatin nanofiber membrane prepared in Example 1, such as... Figure 3a and Figure 3b As shown.

[0133] Infrared spectroscopy analysis showed that the characteristic peaks of PLGA were located at 1752 cm⁻¹. -1 1180cm -1 1193cm -1 The peaks at which the stretching vibrations of the C=O double bond, the characteristic absorption peaks of the stretching vibrations of the COC ether bond, and the vibration peaks of the CO single bond appear are observed.

[0134] In the infrared spectrum of the PLGA / gelatin composite fiber membrane, not only the characteristic peaks of PLGA appeared, but also the three characteristic absorption peaks of gelatin: amide I stretching vibration peak, amide II bending vibration peak, and amide III stretching vibration peak (1657 cm⁻¹, 1535 cm⁻¹, 1093 cm⁻¹).

[0135] The infrared spectrum of PLGA / gelatin / heparin not only shows the characteristic peaks of PLGA and gelatin, but also shows peaks in the range of 3300–3480 cm⁻¹. -1 The image shows vibrational peaks for NH and CN. PLGA molecules do not contain NH and CN bonds, while the heparin coating of PLGA-gelatin-HEP is cross-linked onto PLGA through a reaction with gelatin and contains abundant NH and CN bonds.

[0136] Nuclear magnetic resonance spectroscopy showed that PLGA-gelatin-heparin 1 HNMR showed that peak A (5.2 ppm) was -CH in PLGA lactic acid, and peak B (1.6 ppm) was -NH2 on gelatin.

[0137] Example of effect 4

[0138] Cell viability assays were performed using a cell counting kit-8 (CCK-8) to assess cell proliferation in the biomimetic skeletal muscle material prepared in Example 2. In well plates, absorbance values ​​of each solution were measured at 450 nm using a microplate reader on days 1, 3, 5, and 7 of cell-scaffold co-culture, and cell growth bar charts were plotted. Results are as follows: Figure 4 As shown, cell activity experiments demonstrated that the skeletal muscle scaffold prepared in Example 1 could regulate cell behavior and promote cell proliferation. Comparing the cell proliferation in the biomimetic skeletal muscle materials prepared in Examples 1-3, it was found that when the hydrogel concentration was between 5% and 10%, the cell proliferation effect improved with increasing hydrogel concentration. When the hydrogel concentration exceeded 10%, the effect on promoting cell proliferation was similar to that at a 10% concentration, and there was no longer a significant improvement.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomimetic skeletal muscle material, characterized in that, It includes a muscle bundle simulation unit; the muscle bundle simulation unit includes a first hollow tube with a nanofiber membrane as the tube wall and a first cell-carrying gel filled in the first hollow tube; the nanofiber membrane is an oriented nanofiber membrane prepared by electrospinning. The biomimetic skeletal muscle material further comprises a skeletal muscle simulation unit, which includes a second hollow tube with the nanofiber membrane as the tube wall and a plurality of muscle bundle simulation units filled in the second hollow tube; the plurality of muscle bundle simulation units are arranged in a bundle-like manner along the extension direction of the tube in the second hollow tube; the gap between the second hollow tube and the first hollow tube is filled with a second cell-carrying gel; the cells in the second cell-carrying gel include endothelialized stem cells; The number of muscle bundle simulation units in each skeletal muscle simulation unit is 2 to 5, each independently. The nanofiber membranes constituting each hollow tube are independently selected from PLGA-gelatin nanofiber membranes or PCL-PCL nanofiber membranes. The PLGA-gelatin nanofiber membrane is prepared by simultaneously spinning PLGA electrospinning solution and gelatin electrospinning solution through spinnerets and receiving them on the same receiving device. The PCL-PCL nanofiber membrane is prepared by two sets of PCL electrospinning solutions being spun simultaneously through spinnerets and received on the same receiving device. One set of PCL electrospinning solution is used to spin micron-scale fibers, and the other set of PCL electrospinning solution is used to spin nano-scale fibers. The PCL-PCL nanofiber membrane is a micron-nano composite membrane. The gels in the first cell-carrying gel and the second cell-carrying gel are each independently hydrogels.

2. The biomimetic skeletal muscle material according to claim 1, characterized in that, Each skeletal muscle simulation unit contains three independent muscle bundle simulation units.

3. The biomimetic skeletal muscle material according to claim 1, characterized in that, It includes several skeletal muscle simulation units arranged in a bundle.

4. The biomimetic skeletal muscle material according to any one of claims 1 to 3, characterized in that, The nanofiber membranes constituting each hollow tube are individually modified with gelatin and / or anticoagulants.

5. The biomimetic skeletal muscle material according to claim 4, characterized in that, The anticoagulant includes heparin or a pharmaceutically acceptable salt thereof.

6. The biomimetic skeletal muscle material according to any one of claims 1 to 3, characterized in that, The hydrogel includes a photocurable hydrogel.

7. The biomimetic skeletal muscle material according to any one of claims 1 to 3, characterized in that, The hydrogel includes methacrylic hydrogels.

8. The biomimetic skeletal muscle material according to claim 7, characterized in that, The concentration of the methacrylic hydrogel is 5~15%wt.

9. The biomimetic skeletal muscle material according to any one of claims 1 to 3, characterized in that, The cells in the first cell-loaded gel include pluripotent stem cells.

10. The biomimetic skeletal muscle material according to any one of claims 1 to 3, characterized in that, The contact surfaces of two adjacent hollow tubes are fixedly connected.

11. The biomimetic skeletal muscle material according to any one of claims 1 to 3, characterized in that, The contact surfaces of two adjacent hollow tubes are bonded together.

12. The biomimetic skeletal muscle material according to any one of claims 1 to 3, characterized in that, The inner diameter of each muscle bundle simulation unit is independently 0.15~0.2mm.

13. The method for preparing the biomimetic skeletal muscle material according to any one of claims 1 to 12, characterized in that, This includes filling a first cell-carrying gel into a first hollow tube with a nanofiber membrane as the tube wall.

14. The preparation method according to claim 13, characterized in that, It also includes the preparation of nanofiber membranes by electrospinning; the nanofiber membranes include PLGA-gelatin nanofiber membranes and / or PCL-PCL nanofiber membranes. The method for preparing the PLGA-gelatin nanofiber membrane includes preparing PLGA electrospinning solution and gelatin electrospinning solution respectively, receiving the two electrospinning solutions from the same rotating roller, and obtaining the PLGA-gelatin nanofiber membrane after drying. The method for preparing the PCL-PCL nanofiber membrane includes preparing two sets of PCL electrospinning solutions, using the same rotating roller to receive the spinnerets from the two sets of PCL electrospinning solutions, and drying them to obtain the PCL-PCL nanofiber membrane; wherein one set of PCL electrospinning solutions is used to spin micron-sized fibers, and the other set of PCL electrospinning solutions is used to spin nano-sized fibers.

15. The preparation method according to claim 14, characterized in that, The concentration of PLGA in the PLGA electrospinning solution is 10~20%wt, and hexafluoroisopropanol is used as the solvent.

16. The preparation method according to claim 14, characterized in that, The gelatin concentration in the gelatin electrospinning solution is 10-20%wt, and hexafluoroisopropanol and acetic acid are used as solvents.

17. The preparation method according to claim 14, characterized in that, The concentration of PCL in the two PCL electrospinning solutions was independently 10-15%wt; one PCL electrospinning solution used a mixture of formic acid and acetic acid as solvent, and the other PCL electrospinning solution used a mixture of dichloromethane and N,N-dimethylformamide as solvent.

18. The preparation method according to claim 14, characterized in that, The spinning conditions for each electrospinning solution are independent and include: spinning ambient temperature of 15~25℃, spinning ambient humidity of 20~40%, electrostatic high voltage between the metal spinneret and the receiving device of 15~18 kV, receiving distance of 15~18 cm, and roller speed of 1500~1600 rpm.

19. The preparation method according to claim 13, characterized in that, The preparation method further includes modifying the nanofiber membrane with gelatin and / or an anticoagulant.

20. The preparation method according to claim 13, characterized in that, It also includes fixing several muscle bundle simulation units into a bundle shape, wrapping them with a nanofiber membrane, and then filling the gap between the second hollow tube and the first hollow tube with a second cell-carrying gel to obtain the skeletal muscle simulation unit.

21. The preparation method according to claim 20, characterized in that, The fixing includes adhesive fixing.

22. The preparation method according to claim 13, characterized in that, The first hollow tube is prepared by winding the nanofiber membrane through a mold.

23. The application of the biomimetic skeletal muscle material according to any one of claims 1 to 12, or the preparation method of the biomimetic skeletal muscle material according to any one of claims 13 to 22, in the preparation of skeletal muscle repair products.

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