技术领域Technical field
本发明涉及生物组织工程领域，具体涉及一种可电响应的功能性神经血管化工程肌肉及其制备方法。The 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.
背景技术Background technique
骨骼肌是人体内的重要器官，约占总质量的40％。它由结缔组织、血管和平行排列的肌纤维束组成，并且其独特的收缩功能依赖于运动神经元的神经支配。天然骨骼肌本身具有较强的自我适应和再生能力，但当其受到外伤、疾病或肿瘤切除等外界因素，造成大面积的创伤(超过总体积的20％)时，就会导致瘢痕组织取代受损区域，阻碍肌肉的再生过程，从而导致容积性肌肉缺损。这种未经治疗的容积性肌肉缺损会引起持续性的肌肉萎缩，以及一系列的功能障碍等问题。Skeletal muscle is an important organ in the human body, accounting for approximately 40% of the total mass. It is composed of connective tissue, blood vessels, and muscle fiber bundles arranged in parallel, and its unique contractile function relies on innervation by motor neurons. Natural skeletal muscle itself has strong self-adaptation and regeneration capabilities, but when it is exposed to external factors such as trauma, disease, or tumor resection, causing large-area trauma (more than 20% of the total volume), scar tissue will replace the affected muscle. Damage areas, hindering the muscle regeneration process, resulting in volumetric muscle defects. This untreated volumetric muscle defect can cause persistent muscle atrophy and a series of functional impairments.
长期以来，自体组织移植一直是容积性肌肉缺损的一线治疗方法，但这种侵入性的治疗方式不仅损害供区组织，还增加了术后感染的风险。由此看来，肌肉组织工程的发展具有很好的前景，为这类广泛肌肉缺损疾病的治疗带来了希望。目前的工程策略主要集中在肌肉的组织结构重建上，用不同的技术构建定向的肌肉结构从而达到仿生的目标。广泛使用的生物支架虽然可以实现宏观定向的肌肉结构，但它仍不足以调节单个肌纤维的微观结构，也可能存在生物相容性不佳的问题。此外，仅仅植入这种定向的工程肌肉只能填充肌肉体积，无法实现功能化的修复。为此，构建具有天然骨骼肌结构和功能特；征的工程肌肉组织，在实现组织修复的基础上，同时实现功能化修复具有重要意义。Autologous tissue transplantation has long been the first-line treatment for volumetric muscle defects, but this invasive treatment not only damages the donor site tissue but also increases the risk of postoperative infection. From this point of view, the development of muscle tissue engineering has good prospects and brings hope for the treatment of such extensive muscle defect diseases. Current engineering strategies mainly focus on the reconstruction of muscle tissue structure, using different technologies to build directional muscle structures to achieve bionic goals. Although widely used bioscaffolds can achieve macroscopically oriented muscle structures, they are still insufficient to regulate the microstructure of individual muscle fibers and may suffer from poor biocompatibility. In addition, merely implanting such oriented engineered muscles can only fill the muscle volume and cannot achieve functional repair. To this end, it is of great significance to construct engineered muscle tissue with the structural and functional characteristics of natural skeletal muscle to achieve functional repair on the basis of tissue repair.
然而这种功能化的实现不仅要求工程肌纤维的定向排列，更重要的是有效的神经支配以促进信号转导过程。电刺激作为一种常见的物理治疗方式，可有效调节神经传导和肌肉收缩，这可能是肌肉再生过程中实现功能化的关键。此外，良好的血流灌注对维持肌肉存活和延长功能也至关重要，是神经化肌肉再生的基础。目前，重建一个同时具有神经支配和血管化的功能性组织仍然是一个巨大的挑战，这也是近年来组织工程没有像预期的那样迅速发展的重要原因。However, the realization of this functionalization requires not only the directional arrangement of engineered muscle fibers, but more importantly, effective innervation to promote the signal transduction process. As a common physical therapy modality, electrical stimulation 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 critical to maintaining muscle survival and prolonged function, and is the basis for regeneration of neuralized muscles. Currently, reconstructing a functional tissue with both innervation and vascularization remains a huge challenge, which is an important reason why tissue engineering has not developed as rapidly as expected in recent years.
发明内容Contents of the invention
本发明的目的在于提供一种可电响应的功能性神经血管化工程肌肉，以及盖工程肌肉的制备方法。The object of the present invention is to provide an electrically responsive functional neurovascularized engineered muscle and a method for preparing the engineered muscle.
为解决上述问题，本发明所采用的技术方案如下：In order to solve the above problems, the technical solutions adopted by the present invention are as follows:
一种可电响应的功能性神经血管化工程肌肉，所述工程肌肉包括第一电极层、第二电极层，所述第一电极层与第二电极层之间还设置有神经肌肉层和血管层。An electrically responsive functional neurovascularized engineered muscle. The engineered muscle includes a first electrode layer and a second electrode layer. A neuromuscular layer and blood vessels are also provided between the first electrode layer and the second electrode layer. layer.
本发明中，进一步优选的方案为，所述神经肌肉层为两层，分别为第一神经肌肉层和第二神经肌肉层，所述血管层设置在第一神经肌肉层和第二神经肌肉层之间。In the present invention, a further preferred solution is that the neuromuscular layer is composed of two layers, namely a first neuromuscular layer and a second neuromuscular layer, and the blood vessel layer is provided on the first neuromuscular layer and the second neuromuscular layer. between.
本发明中，进一步优选的方案为，所述工程肌肉还包括用于承载神经肌肉层的第一水凝胶层、以及用于承载血管层的第二水凝胶层。In the present invention, a further preferred solution is that the engineered muscle further includes a first hydrogel layer for carrying the neuromuscular layer and a second hydrogel layer for carrying the vascular layer.
本发明中，进一步优选的方案为，所述第一电极层和第二电极层均为柔性电极，所述柔性电极包括网状聚酯纤维支架、以及附着于所述聚酯纤维支架上的导电墨水层。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 scaffold and a conductive electrode attached to the polyester fiber scaffold. Ink layer.
本发明中，进一步优选的方案为，所述柔性电极的厚度为40-60μm，所述聚酯纤维支架上的单纤维直径为25-60μm。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 stent is 25-60 μm.
本发明还提供一种如前所述工程肌肉制备方法，包括如下步骤：The present invention also provides an engineering muscle preparation method as described above, which includes the following steps:
S1：光控定向细胞片的制备：分别构建用于制备神经肌肉层的光控定向神经细胞片、以及用于制备血管层光控定向血管细胞片；S1: Preparation of light-controlled oriented cell sheets: construct light-controlled oriented nerve cell sheets for preparing neuromuscular layer and light-controlled oriented vascular cell sheets for preparing vascular layer;
S2：神经肌肉层和血管层的制备：利用光控定向技术将神经细胞和肌肉细胞的混合物铺展在光控定向神经细胞片上，并进行培养，得到神经肌肉细胞层；利用光控定向技术将内皮细胞铺展在光控定向血管细胞片上并进行培养，得到血管层；S2: Preparation of neuromuscular layer and vascular layer: Use light-controlled orientation technology to spread the mixture of nerve cells and muscle cells on the light-controlled nerve cell sheet, and culture it to obtain the neuromuscular cell layer; use light-controlled orientation technology to spread the endothelium The cells are spread on the light-controlled vascular cell sheet and cultured to obtain the vascular layer;
S3：肌肉组装：取第一电极层、第二电极层，将步骤S2制得的神经肌肉细胞层、血管层设置于第一电极层、第二电极层之间，即得工程肌肉。S3: Muscle assembly: Take the first electrode layer and the second electrode layer, and place the neuromuscular cell layer and blood vessel layer prepared in step S2 between the first electrode layer and the second electrode layer to obtain the engineered muscle.
本发明中，进一步优选的方案为，所述步骤S1具体为：分别在两基板的表面上制备纳米点，然后分别利用紫外光对两基板上的纳米点进行第一图案化修饰、第二图案化修饰，所述第一图案为神经细胞层的细胞定向排列图案、所述第二图案为血管层细胞的定向排列图案，即得光控定向神经细胞片和光控定向血管细胞片。In the present invention, a further preferred solution is that step S1 is specifically: preparing nanodots on the surfaces of two substrates, and then using ultraviolet light to perform first patterning modification and second patterning on the nanodots on the two substrates. Chemical modification, the first pattern is the directional arrangement pattern of cells in the nerve cell layer, and the second pattern is the directional arrangement pattern of cells in the blood vessel layer, that is, a light-controlled oriented nerve cell sheet and a light-controlled oriented vascular cell sheet are obtained.
本发明中，进一步优选的方案为，所述步骤S2中神经细胞和肌肉细胞混合物中：肌肉细胞：神经细胞数量比例＝100：1。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 the number of muscle cells to nerve cells is 100:1.
本发明中，进一步优选的方案为，所述步骤S3中，将步骤S2中制得的神经肌肉细胞层通过第一水凝胶层从光控定向神经细胞片上转移、通过第二水凝胶层将血管层从光控定向血管细胞片上转移，然后再与第一电极、第二电极进行组装。In the present invention, a further preferred solution is that in step S3, the neuromuscular cell layer prepared in step S2 is transferred from the light-controlled oriented neural cell sheet through the first hydrogel layer and passed through the second hydrogel layer. The vascular layer is transferred from the light-controlled vascular cell sheet to the vascular cell sheet, and then assembled with the first electrode and the second electrode.
本发明中，进一步优选的方案为，将光可固化的水凝胶分别滴加至光控定向神经细胞片、光控定向血管细胞片上，待水凝胶均匀铺展在各细胞片上后，在紫外光下照射55-65s，分别形成第一水凝胶层和第二水凝胶层，然后将承载有神经肌肉细胞层的第一水凝胶层以及承载有血管细胞层的第二水凝胶层从各细胞片上取下，然后再与第一电极、第二电极进行堆叠组装，即得工程肌肉。In the present invention, a further preferred solution is to drop the light-curable hydrogel onto the light-controlled oriented neural cell sheet and the light-controlled oriented vascular cell sheet respectively, and after the hydrogel is evenly spread on each cell sheet, the UV gel is Irradiate under light for 55-65 seconds to form a first hydrogel layer and a second hydrogel layer respectively, and then place the first hydrogel layer carrying the neuromuscular cell layer and the second hydrogel carrying the vascular cell layer The layers are removed from each cell sheet, and then stacked and assembled with the first electrode and the second electrode to obtain the engineered muscle.
本发明中，进一步优选的方案为，所述步骤S3中的第一电极层、第二电极层均为柔性电极，所述柔性电极包括网状聚酯纤维支架和导电墨水层，所述柔性电极通过如下步骤制得：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 scaffold and a conductive ink layer. Prepared by the following steps:
S31：网状聚酯纤维支架制备：绘制支架的网状结构图形，然后通过熔融近场直写技术，依据网状结构图形打印出网状聚酯纤维支架；S31: Preparation of mesh polyester fiber stent: draw the mesh structure pattern of the stent, and then use melted near-field direct writing technology to print the mesh polyester fiber stent based on the mesh structure pattern;
S32：聚酯纤维亲水性处理：采用多巴胺的原位聚合方式，对网状聚酯纤维支架表面进行疏水性处理以改善其疏水性；S32: Hydrophilic treatment of polyester fiber: Using in-situ polymerization of dopamine, hydrophobic treatment is performed on the surface of the meshed polyester fiber scaffold to improve its hydrophobicity;
S33：导电墨水层涂覆：将碳纳米管液态导电墨水，将导电墨水涂覆至经S2处理后的网状聚酯纤维支架上，然后进行干燥，重复如上涂覆及干燥步骤多次，即得。S33: Conductive ink layer coating: Apply the carbon nanotube liquid conductive ink to the mesh polyester fiber stent processed by S2, then dry it, and repeat the above coating and drying steps several times, that is, have to.
与现有技术相比，本发明具有如下优点：本发明的工程肌肉，具有良好的电响应性；并可以基于良好的电相应性，在结合物理电刺激治疗方面(如相关的医疗器械)等可进行应用、并可拓展出更多的功能性应用；此外，该工程肌肉具有良好的生物相容性，并且结构标准，能够实现工业化生产，具有良好的产业及临床应用前景。Compared with the existing technology, the present invention has the following advantages: the engineered muscle of the present invention has good electrical responsiveness; and can be combined with physical electrical stimulation treatment (such as related medical equipment) based on the good electrical responsiveness. It can be applied and expanded into more functional applications; in addition, the engineered muscle has good biocompatibility, standard structure, can achieve industrial production, and has good industrial and clinical application prospects.
下面结合说明书附图和具体实施方式对本发明进行详细说明。The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments of the description.
附图说明Description of the drawings
图1为实施例1中的工程肌肉的层状结构示意图；Figure 1 is a schematic diagram of the layered structure of the engineered muscle in Example 1;
图2为实施例1的工程肌肉的应用示意图；Figure 2 is a schematic diagram of the application of the engineered muscle in Embodiment 1;
其中，1、第一电极层；2、第二电极层；3、第一神经肌肉层；4、第二神经肌肉层；5、血管层；6、第一水凝胶层；7、第二水凝胶层。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.
具体实施方式Detailed ways
下面，结合附图以及具体的实施方式对本发明做进一步的描述，需要说明的是，在不相冲突的前提下，一下描述的各实施例之间或各技术特征之间可以任意组合成新的实施例。除特殊说明的之外，具体实施方式中的实施例与实验例，其中的设备和试剂材料均从市场途径获得，具体的实施例是示范性的，仅用于解释本申请，而不能理解作为本申请保护范围的限制。Below, the present invention will be further described with reference to the accompanying drawings and specific implementations. It should be noted that, on the premise that there is no conflict, each embodiment or each technical feature described below can be arbitrarily combined to form a new implementation. example. Unless otherwise specified, the examples and experimental examples in the specific implementation mode, in which the equipment and reagent materials are all obtained from the market, are exemplary and are only used to explain the present application and cannot be understood as Limitations on the scope of protection of this application.
一种可电响应的功能性神经血管化工程肌肉，所述工程肌肉包括第一电极层、第二电极层，所述第一电极层与第二电极层之间还设置有神经肌肉层和血管层。An electrically responsive functional neurovascularized engineered muscle. The engineered muscle includes a first electrode layer and a second electrode layer. A neuromuscular layer and blood vessels are also provided between the first electrode layer and the second electrode layer. layer.
本发明的工程肌肉，将神经肌肉层和血管层设置在第一电极层与第二电极层之间，在人工肌肉组织的主体部分(血管层和神经肌肉层)外侧设置两个电极层，可以有效改善肌肉组织的导电性，达到电响应的目的；在面对电刺激时，可以实现精准的电流传送，能够加速肌肉功能性的恢复，在结合物理电刺激治疗方面(如相关的医疗器械)等可进行应用、并可拓展出更多的功能性应用。本发明的工程肌肉中，神经肌肉层和血管层并非来自人体或动物，而是通过生物工程技术对细胞培养获得；可以实现工业化、标准化生产，并且具有良好的生物相容性。In the engineered muscle of the present invention, the neuromuscular layer and the vascular layer are arranged between the first electrode layer and the second electrode layer, and two electrode layers are arranged outside the main part of the artificial muscle tissue (the vascular layer and the neuromuscular layer). Effectively improves the electrical conductivity of muscle tissue to achieve electrical response; when faced with electrical stimulation, it can achieve precise current transmission and accelerate the recovery of muscle function. In combination with physical electrical stimulation treatment (such as related medical equipment) etc. can be applied and more functional applications can be developed. In the engineered muscle of the present invention, the neuromuscular layer and vascular layer do not come from the human body or animals, but are obtained through cell culture through bioengineering technology; industrialization and standardized production can be achieved, and it has good biocompatibility.
本发明中，在第一电极层和第二电极层之间，神经肌肉层和血管层可以根据实际需求设置其组合和叠放组装顺序；为了进一步提升契合人体和后续治疗方面的需求，可以做这样的设置，所述神经肌肉层为两层，分别为第一神经肌肉层和第二神经肌肉层，所述血管层设置在第一神经肌肉层和第二神经肌肉层之间；这样的设置，血管层能够对神经肌肉层提供营养和带走代谢物，两个神经肌肉层亦能对血管层进行保护，并且响应的肌肉、血管也能够更好的实现收缩，整体更接近于天然骨骼肌，生物相容性更好。In the present invention, between the first electrode layer and the second electrode layer, the neuromuscular layer and the blood vessel layer can be combined and stacked in an assembly sequence according to actual needs; in order to further improve the fit to the needs of the human body and subsequent treatment, it is possible to In such an arrangement, the neuromuscular layer is composed of two layers, namely a first neuromuscular layer and a second neuromuscular layer, and the blood vessel layer is arranged between the first neuromuscular layer and the second neuromuscular layer; such an arrangement , the vascular layer can provide nutrients 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 achieve better contraction, making the overall body closer to natural skeletal muscles. , better biocompatibility.
为了便于工程肌肉的成型、转移以及后续的移植，可以做这样的设置：所述工程肌肉还包括用于承载神经肌肉层的第一水凝胶层、以及用于承载血管层的第二水凝胶层；这样，通过水凝胶层对神经肌肉层、血管层进行承载，可以更便于后续相关结构的组装、转移和移植，另外水凝胶的加入能够进一步提升肌肉组织的弹性模量，整体上与人体肌肉的物理性能更为接近；此外，水凝胶层的加入后，水凝胶可将电极层进行粘合、固化，可进一步提升工程肌肉整体的稳定性。In order to facilitate the shaping, transfer and subsequent transplantation of the engineered muscle, such an arrangement can be made: the engineered muscle further includes a first hydrogel layer for carrying the neuromuscular layer, and a second hydrogel layer for carrying the vascular layer. Glue layer; in this way, the neuromuscular layer and vascular layer are supported by the hydrogel layer, which can make it easier to assemble, transfer and transplant subsequent related structures. In addition, the addition of hydrogel can further improve the elastic modulus of muscle tissue, and the overall The physical properties are closer to those of human muscles; in addition, after the hydrogel layer is added, the hydrogel can bond and solidify the electrode layer, which can further improve the overall stability of the engineered muscle.
对于第一电极层和第二电极层，可以根据实际需求选择响应的电极材料；为了进一步提升工程肌肉的组织顺应性，第一电极层和第二电极层可以选用柔性电极；为了进一步提升组织顺应性、更好的顺应组织的动度，柔性电极的厚度可以为40-60μm，更进一步优选的为50μm。对于柔性电极，可以根据实际需求选择电极的结构和组成，如选用包括支架以及支架上的导电层结构的柔性电极，对于支架材质，可以根据需求进行选择，优选聚酯纤维材质，更进一步优选聚己内酯PCL材质，更进一步优选的为网状结构的聚己内酯PCL支架；对于网状聚己内酯PCL支架，由聚己内酯纤维交织成网形成，其纤维直径优选为25-60μm，这样尺寸纤维制成的支架生物顺应性更好，更佳的尺寸为μm；对于导电层，可以基于实际需求进行选择，为了进一步提升柔性电极的组织顺应性，可以选用导电墨水层进行涂覆并经干燥得到，导电墨水层优选碳纳米管导电墨水制成的导电墨水层；对于导电墨水层，可以选用一层、两层或者多层；对于导电墨水层，其导电率优选3.9-5S/m，优选4.27S/m，发明人发现，该导电率状态下的柔性电极，其电响应效果好。For the first electrode layer and the second electrode layer, the responsive electrode materials can be selected according to actual needs; in order to further improve the tissue compliance of the engineered muscles, flexible electrodes can be selected for the first electrode layer and the second electrode layer; in order to further improve the tissue compliance The thickness of the flexible electrode can be 40-60 μm, and more preferably 50 μm. For flexible electrodes, the structure and composition of the electrode 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. The material of the bracket can be selected according to needs. Polyester fiber material is preferred, and polyester fiber material is further preferred. The caprolactone PCL material is further preferably a polycaprolactone PCL stent with a mesh structure; for the mesh polycaprolactone PCL stent, it is formed by interweaving polycaprolactone fibers into a mesh, and the fiber diameter is preferably 25- 60 μm, the scaffold 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 used for coating Covered and obtained by drying, the conductive ink layer is preferably a conductive ink layer made of carbon nanotube conductive ink; for the conductive ink layer, one, two or multiple layers can be selected; for the conductive ink layer, its conductivity is preferably 3.9-5S /m, preferably 4.27S/m. The inventor found that the flexible electrode under this conductivity state has a good electrical response effect.
如前所述的可电响应的功能性神经血管化工程肌肉，其制备方法包括如下步骤：The preparation method of the electrically responsive functional neurovascularized engineered muscle as mentioned above includes the following steps:
S1：光控定向细胞片的制备：分别构建用于制备神经肌肉层的光控定向神经细胞片、以及用于制备血管层光控定向血管细胞片；S1: Preparation of light-controlled oriented cell sheets: construct light-controlled oriented nerve cell sheets for preparing neuromuscular layer and light-controlled oriented vascular cell sheets for preparing vascular layer;
S2：神经肌肉层和血管层的制备：利用光控定向技术将神经细胞和肌肉细胞的混合物铺展在光控定向神经细胞片上，并进行培养，得到神经肌肉细胞层；利用光控定向技术将内皮细胞铺展在光控定向血管细胞片上并进行培养，得到血管层；S2: Preparation of neuromuscular layer and vascular layer: Use light-controlled orientation technology to spread the mixture of nerve cells and muscle cells on the light-controlled nerve cell sheet, and culture it to obtain the neuromuscular cell layer; use light-controlled orientation technology to spread the endothelium The cells are spread on the light-controlled vascular cell sheet and cultured to obtain the vascular layer;
S3：肌肉组装：取第一电极层、第二电极层，将步骤S2制得的神经肌肉细胞层、血管层设置于第一电极层、第二电极层之间，即得工程肌肉。S3: Muscle assembly: Take the first electrode layer and the second electrode layer, and place the neuromuscular cell layer and blood vessel layer prepared in step S2 between the first electrode layer and the second electrode layer to obtain the engineered muscle.
所述步骤S1可以具体为：分别在两基板的表面上制备纳米点，然后分别利用紫外光对两基板上的纳米点进行第一图案化修饰、第二图案化修饰，所述第一图案为神经细胞层的细胞定向排列图案、所述第二图案为血管层细胞的定向排列图案，即得光控定向神经细胞片和光控定向血管细胞片。The step S1 may be specifically: preparing nanodots on the surfaces of 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. The first pattern is The directional arrangement pattern of cells in the nerve cell layer and the second pattern are the directional arrangement pattern of cells in the blood vessel layer, that is, a light-controlled oriented nerve cell sheet and a light-controlled oriented vascular cell sheet are obtained.
对于基板，可以选择石英基板；对于纳米点，可以选用二氧化钛纳米点。For the substrate, you can choose quartz substrate; for nanodots, you can choose titanium dioxide nanodots.
对于图案化修饰，可以在步骤S1中进行，也可以设置在步骤S2的光控定向培养中进行；具体的图案化修饰可以选择这样进行，在基板上具有二氧化钛纳米点表面的一侧，外覆一个间距为29-32μm的平行图案的掩膜板，经过0.8-1.2小时的紫外光照后，即完成图案化修饰；更进一步的是，在紫外光照射后，将响应的细胞铺展基板的图案修饰侧，可实现细胞的定向排列。For patterned modification, it can be carried out in step S1, or it can be carried out in the light-controlled directional culture of step S2; the specific patterned modification can be carried out in this way, on the side of the substrate with the surface of titanium dioxide nanodots, covered with A mask with a parallel pattern with a spacing of 29-32 μm is patterned and modified after 0.8-1.2 hours of UV irradiation. Furthermore, after UV irradiation, the responding cells are spread on the pattern of the substrate. Side, the directional arrangement of cells can be achieved.
对于步骤S2中，神经细胞和肌肉细胞的混合物中响应细胞的比例，可以通过如下方式获得：选择不同比例的共培养系统，对神经、肌肉细胞进行共培养，设置不同共培养比例(1:0-500：1)，根据成肌率的高低进行选择；发明人通过大量实验，筛选出最佳共培养比例为：肌肉细胞：神经细胞数量比例＝100：1。In step S2, the proportion of responding cells in the mixture of nerve cells and muscle cells can be obtained in the following way: select co-culture systems with different ratios, co-culture nerve and muscle cells, and set different co-culture ratios (1:0 -500:1), selected based on the myogenesis rate; through extensive experiments, the inventor selected the optimal co-culture ratio as: muscle cells: nerve cells ratio = 100:1.
进一步优选的方案为，所述步骤S3中，将步骤S2中制得的神经肌肉细胞层通过第一水凝胶层从光控定向神经细胞片上转移、通过第二水凝胶层将血管层从光控定向血管细胞片上转移，然后再与第一电极、第二电极进行组装。对于水凝胶材质的选择，可以继续需求选择生物相容性好的水凝胶，进一步优选的为Gelma水凝胶。A further preferred solution is that in step S3, the neuromuscular cell 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 nerve cell sheet through the second hydrogel layer. Light-controlled directional transfer of vascular cells on the sheet, and then assembly with the first electrode and the second electrode. Regarding the choice of hydrogel material, you can continue to choose a hydrogel with good biocompatibility, and Gelma hydrogel is further preferred.
对于步骤S3中，进一步优选的方案为，将光可固化的水凝胶分别滴加至光控定向神经细胞片、光控定向血管细胞片上，待水凝胶均匀铺展在各细胞片上后，在紫外光下照射55-65s，分别形成第一水凝胶层和第二水凝胶层，然后将承载有神经肌肉细胞层的第一水凝胶层以及承载有血管细胞层的第二水凝胶层从各细胞片上取下，然后再与第一电极、第二电极进行堆叠组装，即得工程肌肉。For step S3, a further preferred solution is to drop the light-curable hydrogel onto the light-controlled oriented neural cell sheet and the light-controlled oriented vascular cell sheet respectively, and after the hydrogel is evenly spread on each cell sheet, Irradiate under ultraviolet light for 55-65 seconds to form a first hydrogel layer and a second hydrogel layer respectively. Then, the first hydrogel layer carrying the neuromuscular cell layer and the second hydrogel layer carrying the vascular cell layer are formed. The glue layer is removed from each cell sheet, and then stacked and assembled with the first electrode and the second electrode to obtain the engineered muscle.
本发明中，所述步骤S3中的第一电极层、第二电极层均可以选择为柔性电极，所述柔性电极包括网状聚酯纤维支架和导电墨水层，所述柔性电极通过如下步骤制得：In the present invention, both the first electrode layer and the second electrode layer in step S3 can be selected as flexible electrodes. The flexible electrodes include a mesh polyester fiber scaffold and a conductive ink layer. The flexible electrodes are manufactured by the following steps. have to:
S31：网状聚酯纤维支架制备：绘制支架的网状结构图形，然后通过熔融近场直写技术，依据网状结构图形打印出网状聚酯纤维支架；对于图形的绘制，可以通过AutoCAD等绘图软件进行绘制；对于网状的结构，可以为正弦形的网状结构；S31: Preparation of mesh polyester fiber stent: draw the mesh structure graphic of the stent, and then print out the mesh polyester fiber stent based on the mesh structure graphic through fused near-field direct writing technology; for drawing of the graphics, you can use AutoCAD, etc. Use drawing software to draw; for a mesh structure, it can be a sinusoidal mesh structure;
S32：聚酯纤维亲水性处理：采用多巴胺的原位聚合方式，对网状聚酯纤维支架表面进行疏水性处理以改善其疏水性(即提升亲水性能，以便于后续墨水的涂覆)；S32: Hydrophilic treatment of polyester fiber: Using in-situ polymerization of dopamine, the surface of the meshed polyester fiber scaffold is hydrophobically treated to improve its hydrophobicity (that is, to improve the hydrophilicity to facilitate subsequent ink coating) ;
S33：导电墨水层涂覆：将碳纳米管液态导电墨水，将导电墨水涂覆至经S2处理后的网状聚酯纤维支架上，然后进行干燥，重复如上涂覆及干燥步骤多次，即得；进一步的，可以将支架浸泡在墨水中以实现墨水的涂覆，浸泡可以使支架表面与墨水充分接触，浸泡的时间可以选择为12-18分钟，更有选为15分钟；浸泡后，将支架取出，去除多余的墨水，然后在32-37摄氏度的环境干燥25-35分钟，如在干燥箱中干燥，即得导电墨水层；为了实现良好的电响应性能，可以涂覆一定层数的导电墨水层(即对应好一定范围的导电率，如3.9-5S/m)，增加的导电墨水层可以选择上述的浸泡涂覆、干燥步骤实现制作。S33: Conductive ink layer coating: Apply the carbon nanotube liquid conductive ink to the mesh polyester fiber stent processed by S2, then dry it, and repeat the above coating and drying steps several times, that is, obtained; further, the stent can be soaked in ink to achieve ink coating. The soaking can make the surface of the stent fully contact with the ink. The soaking time can be selected as 12-18 minutes, or even 15 minutes; after soaking, Take out the stent, remove excess ink, and then dry it 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 layers can be applied The conductive ink layer (that is, corresponding to a certain range of conductivity, such as 3.9-5S/m), the additional conductive ink layer can be produced by selecting the above-mentioned immersion coating and drying steps.
本发明的工程肌肉制备方法，可实现标准化和工业化应用，具有良好的产业即临床应用前景。The engineering muscle preparation method of the present invention can achieve standardization and industrial application, and has good industrial and clinical application prospects.
实施例1Example 1
一种可电响应的功能性神经血管化工程肌肉，所述工程肌肉包括第一电极层1、第二电极层2，所述第一电极层与第二电极层之间还设置有神经肌肉层和血管层；所述神经肌肉层为两层，分别为第一神经肌肉层和第二神经肌肉层，所述血管层5设置在第一神经肌肉层3和第二神经肌肉层4之间；所述工程肌肉还包括用于承载神经肌肉层的第一水凝胶层6、以及用于承载血管层的第二水凝胶层7；所述第一电极层和第二电极层均为柔性电极，所述柔性电极包括网状聚酯纤维支架、以及附着于所述聚酯纤维支架上的导电墨水层；具体结构可结合图1进行理解；An electrically responsive functional neurovascularized engineered muscle. The engineered muscle includes a first electrode layer 1 and a second electrode layer 2. A neuromuscular layer is also provided between the first electrode layer and the second electrode layer. and a vascular layer; the neuromuscular layer is composed of 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 also includes a first hydrogel layer 6 for carrying the neuromuscular layer, and a second hydrogel layer 7 for carrying the blood vessel layer; both the first electrode layer and the second electrode layer are flexible. Electrode, the flexible electrode includes a mesh polyester fiber scaffold and a conductive ink layer attached to the polyester fiber scaffold; the specific structure can be understood in conjunction with Figure 1;
该工程肌肉通过如下步骤制得：The engineered muscle is produced through the following steps:
S1：光控定向细胞片的制备：分别构建用于制备神经肌肉层的光控定向神经细胞片、以及用于制备血管层光控定向血管细胞片；具体包括：S1: Preparation of light-controlled oriented cell sheets: Construct light-controlled oriented nerve cell sheets for preparing neuromuscular layer and light-controlled oriented vascular cell sheets for preparing vascular layer; specifically include:
1)二氧化钛纳米点的制备1) Preparation of titanium dioxide nanodots
A、前驱液制备：将5μL无水乙醇加入烧杯中，经磁力搅拌20分钟后，依次将680μL钛酸四丁酯、36μL去离子水、62μL乙酰丙酮加入，充分混合后加入0.4g聚乙烯吡咯烷酮，经磁力搅拌10分钟后，转移至容量瓶内，用无水乙醇定容，可得前驱液；A. Preparation of precursor solution: Add 5 μL of absolute ethanol into the beaker. After magnetic stirring for 20 minutes, add 680 μL of tetrabutyl titanate, 36 μL of deionized water, and 62 μL of acetylacetone. After thorough mixing, add 0.4g of polyvinylpyrrolidone. , after magnetic stirring for 10 minutes, transfer to a volumetric flask, and dilute to volume with absolute ethanol to obtain the precursor solution;
B、旋涂、烧结：将前驱液均匀旋涂于石英基板(10×10×1mm3)表面，将其至于马弗炉内500℃下热处理1小时，经高压灭菌后，将其置于孔板内用于后续细胞培养；B. Spin coating and sintering: Spin-coat the precursor liquid evenly on the surface of the quartz substrate (10×10×1mm3 ), heat it in a muffle furnace at 500°C for 1 hour, and sterilize it under high pressure before placing it in the The well plate is used for subsequent cell culture;
S2：神经肌肉层和血管层的制备：利用光控定向技术将神经细胞和肌肉细胞的混合物铺展在光控定向神经细胞片上，并进行培养，得到神经肌肉细胞层；利用光控定向技术将内皮细胞铺展在光控定向血管细胞片上并进行培养，得到血管层；具体可包括：S2: Preparation of neuromuscular layer and vascular layer: Use light-controlled orientation technology to spread the mixture of nerve cells and muscle cells on the light-controlled nerve cell sheet, and culture it to obtain the neuromuscular cell layer; use light-controlled orientation technology to spread the endothelium The cells are spread on the light-controlled vascular cell sheet and cultured to obtain the vascular layer; specifically, it may include:
光控细胞定向：使用紫外光源(254nm，300μW/cm2)通过由石英制成的掩膜板进行微图案修饰，并形成30μm宽度的Cr线(线/空间比为1:1)。经紫外辐照1小时后，立即用镊子转移样品至孔板内，加入细胞悬液，经培养12小时后，可实现细胞定向排列；Light-controlled cell orientation: Use an ultraviolet light source (254nm, 300μW/cm2 ) to perform micropattern modification through a mask made of quartz, and form Cr lines of 30μm width (line/space ratio of 1:1). After 1 hour of UV irradiation, immediately transfer the sample to the well plate with tweezers, add cell suspension, and after 12 hours of culture, the cells can be oriented and arranged;
定向神经肌肉层的构建：利用上述光控定向技术，将神经、肌肉细胞按照一定比例混匀后，铺展至基板表面。将孔板至培养箱内培养，经1天培养后，将生长培养基换为分化培养基。在分化培养基下继续培养7天，可获得定向的神经肌肉细胞层；Construction of oriented neuromuscular layer: Using the above-mentioned light-controlled directional technology, nerve and muscle cells are mixed in a certain proportion and spread to the surface of the substrate. Place the well plate into the incubator for culture. After 1 day of culture, replace the growth medium with differentiation medium. After continuing to culture in differentiation medium for 7 days, a oriented neuromuscular cell layer can be obtained;
定向血管层的构建：利用上述光控定向技术，将内皮细胞均匀铺展至基板表面，经培养7天后，细胞融合形成管腔状血管结构,获得定向血管层；Construction of directional vascular layer: Using the above-mentioned light-controlled directional technology, endothelial cells are evenly spread on the surface of the substrate. After 7 days of culture, the cells fuse to form a luminal vascular structure, and a directional vascular layer is obtained;
S3：肌肉组装：取第一电极层、第二电极层，将步骤S2制得的神经肌肉细胞层、血管层设置于第一电极层、第二电极层之间，即得工程肌肉；S3: Muscle assembly: Take the first electrode layer and the second electrode layer, and place the neuromuscular cell layer and blood vessel layer prepared in step S2 between the first electrode layer and the second electrode layer to obtain the engineered muscle;
其中，第一电极层和第二电极层的柔性电极通过如下步骤制得：Wherein, the flexible electrodes of the first electrode layer and the second electrode layer are made by the following steps:
A、PCL支架打印：采用AutoCAD设计了一种正弦型的网格结构，支架整体的形状呈正方形，长宽均为12mm，单个波形单元为正弦，周期为1mm，幅值为0.25mm。随后设置打印参数，使点胶喷嘴与玻璃板收集器之间的距离为2mm，打印速度为450mm/min，打印温度保持为110℃，层数为3。使用近场直写技术打印了纤维直径为50μm的支架，将玻璃板收集器浸泡到75％的酒精中，待PCL支架脱离后，用镊子取出支架放置到35℃的干燥箱中烘干30分钟；A. PCL bracket printing: A sinusoidal grid structure is designed using AutoCAD. The overall shape of the bracket is square, with a length and width of 12mm. A single waveform unit is sinusoidal, with a period of 1mm and an amplitude of 0.25mm. Then set the printing parameters so that the distance between the dispensing nozzle and the glass plate collector is 2mm, the printing speed is 450mm/min, the printing temperature is maintained at 110°C, and the number of layers is 3. A scaffold with a fiber diameter of 50 μm was printed using near-field direct writing technology. The glass plate collector was soaked in 75% alcohol. After the PCL scaffold was detached, the scaffold was taken out with tweezers and placed in a drying oven at 35°C for 30 minutes. ;
B、PCL支架改性：由于PCL支架表面光滑，高度疏水，液态墨水的导电组分无法有效地吸附在纤维表面。采用多巴胺的原位聚合对PCL支架表面作亲水化处理，取20mL的三羟甲基氨基甲烷盐酸盐Tris-HCl缓冲液，加入0.04g的盐酸多巴胺粉末配置成2mg/mL的交联剂溶液，然后将支架完全浸泡到溶液中，并放置在摇床上振荡12小时，使多巴胺在PCL支架表面原位聚合。随后取出交联后的PCL支架，用去离子水充分清洗3次，以除去支架表面未黏附的聚多巴胺颗粒，然后将支架放置在35℃的干燥箱中烘干1小时；B. PCL stent modification: Since the surface of the PCL stent is smooth and highly hydrophobic, the conductive component of the liquid ink cannot be effectively adsorbed on the fiber surface. Use in-situ polymerization of dopamine to hydrophilize the surface of the PCL stent. Take 20 mL of tris-hydroxymethylaminomethane hydrochloride Tris-HCl buffer and add 0.04 g of dopamine hydrochloride powder to prepare a 2 mg/mL cross-linking agent. solution, and then completely immerse the scaffold into the solution and place it on a shaker for 12 hours to polymerize dopamine in situ on the surface of the PCL scaffold. Then take out the cross-linked PCL stent, wash it thoroughly with deionized water three times to remove the polydopamine particles that are not adhered to the surface of the stent, and then place the stent in a drying oven at 35°C for 1 hour;
C、碳纳米管涂覆C. Carbon nanotube coating
a、碳纳米管液态导电墨水制备：将0.02g碳纳米纤维CNF粉末加入20mL去离子水中，在室温下搅拌1小时。待CNF完全溶解后向溶液中加入0.04g碳纳米管CNT粉末。充分搅拌混合物1小时，超声处理30分钟。之后，将超声处理完的混合物在2500rpm的转速下离心两次，每次十分钟，除去不分散的碳纳米管后，可得碳纳米管液态导电墨水；a. Preparation of carbon nanotube liquid conductive ink: Add 0.02g of carbon nanofiber CNF powder to 20 mL 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. The mixture was stirred thoroughly for 1 hour and sonicated for 30 minutes. After that, the ultrasonicated mixture is centrifuged twice at 2500 rpm for ten minutes each time. After removing the undispersed carbon nanotubes, the carbon nanotube liquid conductive ink can be obtained;
b、导电墨水组分的组装：将交联后的支架在墨水中浸泡15分钟，使支架表面与墨水充分接触。然后将支架取出，除去多余的墨水，放置到在35℃的干燥箱中烘干30分钟。然后再将涂覆一次的支架浸泡到墨水中1分钟，取出并除去多余的墨水，放置到在35℃的干燥箱中烘干10分钟。重复第二次的涂覆步骤，对支架进行层层涂覆,当支架的涂覆次数达到10次时，导电率达4.27S/m；。b. Assembly of conductive ink components: Soak the cross-linked stent in the ink for 15 minutes so that the surface of the stent is in full contact with the ink. Then take out the stent, remove excess ink, and place it in a drying oven at 35°C for 30 minutes. Then soak the once-coated stent into the ink for 1 minute, take it out and remove excess ink, and place it in a drying oven at 35°C for 10 minutes. Repeat the second coating step to coat the stent layer by layer. When the number of coatings on the stent reaches 10 times, the conductivity reaches 4.27S/m.
步骤S3中，相应的神经肌肉层、血管层转移以及与电极层的组装具体为：将可光固化的Gelma水凝胶滴加至细胞片表面，使液体充分均匀铺展后，将细胞片转移至紫外光下照射60秒，用刀片将固化的载细胞水凝胶片完整取下。重复以上步骤，将神经肌肉层、血管层完整取下，并按照神经肌肉层为上下层，血管层为中间层的顺序，堆叠形成复合水凝胶组织。将制备的柔性电极覆盖在组织的上下表面，使用水凝胶进一步粘合固化后，完成复合结构的构建。In step S3, the corresponding neuromuscular layer, vascular layer transfer and assembly with the electrode layer are as follows: drop the photocurable Gelma hydrogel onto the surface of the cell sheet, and then transfer the cell sheet to the cell sheet after the liquid is fully and evenly spread. Irradiate under UV light for 60 seconds, and use a blade to completely remove the solidified cell-laden hydrogel sheet. Repeat the above steps to completely remove the neuromuscular layer and vascular layer, and stack them to form a composite hydrogel tissue in the order that the neuromuscular layer is the upper and lower layers and the vascular layer is the middle layer. The prepared flexible electrodes are covered on the upper and lower surfaces of the tissue, and after further bonding and solidification using hydrogel, the construction of the composite structure is completed.
对应的，本实施例1获得的工程肌肉可以进行如下方式的使用(可结合图2进行理解)：根据肌肉缺损的形态、大小，将构建的工程肌肉组织进行适当修整后植入缺损区，于边缘处滴加少量的液态水凝胶，经紫外光照射固化，用于粘接工程组织与自体肌肉组织。完成植入、固定后，缝合皮肤。进一步的，将电极经皮肤放置于工程肌肉的两端，进行电刺激治疗，由于该工程肌肉良好的导电性，可将电流精准传输至植入区域，实现精准电刺激的目标。Correspondingly, the engineered muscle obtained in Example 1 can be used in the following manner (can be understood in conjunction with Figure 2): According to the shape and size of the muscle defect, the constructed engineered muscle tissue is appropriately trimmed and then implanted into the defect area. A small amount of liquid hydrogel is dropped on the edge and solidified by ultraviolet light, which is used to bond engineered tissue and autologous muscle tissue. After completion of implantation and fixation, the skin is sutured. Furthermore, electrodes are placed through the skin at both ends of the engineered muscle for electrical stimulation treatment. Due to the good electrical conductivity of the engineered muscle, current can be accurately transmitted to the implanted area to achieve the goal of precise electrical stimulation.
实验例1Experimental example 1
本实验例为依据实施例1获得的工程肌肉，进行的实验应用(小鼠胫前肌缺损模型)和相关工程肌肉性能的实验，具体为：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 experiments on related engineered muscle performance, specifically:
1、电响应的功能性神经血管化工程肌肉的制备：首先评估肌肉缺损的类型、大小、形态后，制备尺寸合适的工程化肌肉。通过光控定向技术构建定向的神经肌肉层以及血管层后，使用近场直写打印PCL支架后，经层层涂覆CNT导电墨水，构建出柔性电极。之后使用水凝胶对上述结构进行组装，构建出电响应的功能性神经血管化工程肌肉。1. Preparation of electrically responsive functional neurovascularized engineered muscles: After first assessing the type, size, and shape of the muscle defect, engineered muscles of appropriate size are prepared. After constructing the oriented neuromuscular layer and vascular layer through light-controlled directional technology, the PCL stent is printed using near-field direct writing, and then CNT conductive ink is coated layer by layer to construct a flexible electrode. The above structures were then assembled using hydrogels to construct electrically responsive functional neurovascularized engineered muscles.
2、工程肌肉的植入：将构建的工程肌肉进行适当修整后植入缺损区，检查对位情况后，于边缘处滴加少量的液态水凝胶，经紫外光照射固化，用于粘接工程组织与自体肌肉组织。完成植入、固定后，对表面皮肤进行缝合。2. Implantation of engineered muscles: After proper trimming, the constructed engineered muscles are implanted into the defect area. After checking the alignment, a small amount of liquid hydrogel is dropped at the edge and cured by UV irradiation for bonding. Engineered tissue versus autologous muscle tissue. After completion of implantation and fixation, the surface skin is sutured.
3、电刺激治疗辅助修复：两个表面刺激电极被放置在缺损区所对应的皮肤表面，对应至工程组织的两端。同时将电极连接到刺激器上，调整合适的刺激参数后进行电刺激，通过这种方式可使得电流精准传输至植入区域，实现精准治疗的目标。3. Electrical stimulation treatment-assisted repair: Two surface stimulation electrodes are placed on the skin surface corresponding to the defect area, corresponding to both ends of the engineered tissue. At the same time, connect the electrodes to the stimulator, adjust the appropriate stimulation parameters and then perform electrical stimulation. In this way, the current can be accurately transmitted to the implanted area to achieve the goal of precise treatment.
4、功能化评估：4. Functional evaluation:
1)步态学分析：采用CatWalk系统分析小鼠步态。首先，将小鼠置于暗室的玻璃板上，并允许其自由行走。来自荧光灯的光束在整个玻璃板上传播，光束可通过玻璃板完全反射。当爪接触玻璃板时，光束向下反射，形成了清晰明亮的爪印图像，用摄像机记录整个步行过程。使用CatWalk软件收集并详细分析所有数据。根据获得的数据进行评价：运行持续时间、运行平均速度、平均姿势、最大接触强度、爪印长度、爪印宽度、爪印面积、步长和摆动速度，来评价小鼠肌肉功能。根据测定数据分析，将小数的实验右后肢除以对照左后肢的爪印面积、强度可得出相对值，用于量化分析工程肌肉的功能化恢复效果。工程肌肉植入组爪印面积相对值可达0.68，爪印强度相对值可达0.76，均显著高于对照组。1) Gait analysis: Use CatWalk system to analyze mouse gait. First, place the mouse on a glass plate in a dark room and allow it to walk freely. A beam of light from a fluorescent lamp travels across the entire glass pane and is completely reflected by the glass pane. When the paw touches the glass plate, the light beam reflects downward, forming a clear and bright image of the paw print, and the entire walking process is recorded with a camera. All data is collected and analyzed in detail using CatWalk software. Mouse muscle function was evaluated based on the data obtained: running duration, average running speed, average posture, maximum contact intensity, paw print length, paw print width, paw print area, step length and swing speed. According to the analysis of the measurement data, the relative value can be obtained by dividing the decimal number of the experimental right hind limb by the paw print area and intensity of the control left hind limb, which can be used to quantitatively analyze the functional recovery effect of the engineered muscle. The relative value of the paw print area in the engineered muscle implant group can reach 0.68, and the relative value of the paw print intensity can reach 0.76, both of which are significantly higher than that of the control group.
2)肌肉电生理评估：小鼠经麻醉后，切开小腿部表面皮肤，暴露胫前肌。在胫前肌上端插入刺激电极，给予电流刺激；同时在胫前肌两端分别插入测量电极(相对于参考电极)产生电位差，开始记录神经肌肉系统活动时的生物电信号。记录到肌肉运动中产生的生物电，差分放大器监测到该信号后，经过放大，记录后得到肌电图形。计算肌电值积分：肌电图信号经整流滤波后1秒内曲线下面积的总和(肌电值积分用于分析肌肉在单位时间内的收缩特性)，用于评价肌肉的生理功能。经测定，工程肌肉植入组肌电值积分达0.299mv.s，对照组积分值仅为0.025mv.s，证实工程肌肉优越的功能化修复效果。2) Muscle electrophysiological assessment: After the mice were anesthetized, the surface skin of the calf was incised to expose the tibialis anterior muscle. Insert a stimulating electrode into the upper end of the tibialis anterior muscle to give current stimulation; at the same time, insert measurement electrodes (relative to the reference electrode) at both ends of the tibialis anterior muscle to generate a potential difference and start recording bioelectrical signals during neuromuscular system activity. The bioelectricity generated during muscle movement is recorded. After the differential amplifier detects the signal, it is amplified and the electromyographic pattern is obtained after recording. Calculate the myoelectric value integral: the sum of the area under the curve within 1 second after the electromyographic signal is rectified and filtered (myoelectric value integral is used to analyze the contraction characteristics of the muscle in unit time), and is used to evaluate the physiological function of the muscle. It was measured that the integrated myoelectric value of the engineered muscle implantation group reached 0.299mv.s, while the integrated value of the control group was only 0.025mv.s, confirming the superior functional repair effect of engineered muscles.
通过该实验例，亦可说明，本发明的部分有益效果可以部分例举(其余有益效果可结合说明书其他处内容)如下：Through this experimental example, it can also be illustrated that some of the beneficial effects of the present invention can be partially exemplified (the remaining beneficial effects can be combined with other contents in the specification) as follows:
1)由于人体内天然的骨骼肌由高度定向的平行排列的肌纤维束组成，其特异性的机械功能依赖于此。因此，实现定向排列是体外构建仿生肌肉组织的重要步骤。在目前的组织工程领域，构建定向的生物支架是目前常用的治疗策略。虽然这些材料可以实现宏观上的组织定向，但是仍然缺乏调节单一肌纤维微观结构的能力，还可能遭受生物相容性差等问题。通过上述光控细胞定向的技术，可以便捷地获得定向排列的细胞，并将其诱导成所需的组织结构。通过构建工程化的定向结构，可以模拟天然骨骼肌规则的形态，达到仿生的目的，对于肌肉组织再生、改建起到关键调控作用。这种无支架的策略具有良好的生物相容性，还保留了细胞外基质ECM信号，ECM信号可以调节细胞行为，诱导体内细胞迁移。此外，通过堆叠细胞片层可以组装形态可调的三维结构，可进一步诱导更多的肌纤维收缩。1) Since natural skeletal muscles in the human body are composed of highly oriented parallel-arranged muscle fiber bundles, their specific mechanical functions depend on them. Therefore, achieving directional alignment is an important step in constructing bionic muscle tissue in vitro. In the current field of tissue engineering, constructing directional biological scaffolds is a commonly used treatment strategy. Although these materials can achieve macroscopic tissue orientation, they still lack the ability to adjust the microstructure of single muscle fibers and may suffer from problems such as poor biocompatibility. Through the above-mentioned light-controlled cell orientation technology, directionally arranged cells can be easily obtained and induced into the desired tissue structure. By constructing engineered directional structures, the regular shape of natural skeletal muscles can be simulated to achieve bionic purposes, playing a key regulatory role in muscle tissue regeneration and reconstruction. This scaffold-free strategy has good biocompatibility and also preserves extracellular matrix ECM signals, which can regulate cell behavior and induce cell migration in vivo. In addition, by stacking cell sheets, a three-dimensional structure with adjustable morphology can be assembled, which can further induce more muscle fiber contraction.
2)神经支配能力：骨骼肌的功能化依赖于神经支配，目前工程化骨骼肌组织的有效神经整合仍然是重建广泛损伤肌肉和恢复肌肉功能的挑战。天然骨骼肌组织通过建立神经肌肉接点NMJ，受到周围神经系统支配，失神经的骨骼肌会失去收缩力并发生肌肉萎缩。在目前的骨骼肌组织工程领域，对于整合神经化的工程肌肉的研究仍鲜少涉及，其体内功能改善的效果也尚不明确。本发明通过神经肌肉共培养的方式，构建神经化肌肉组织，可形成有效的NMJ，使神经支配成为可能。2) Innervation capacity: The functionalization of skeletal muscle depends on innervation. Currently, effective neural integration of engineered skeletal muscle tissue remains a challenge to reconstruct extensively damaged muscles and restore muscle function. Natural skeletal muscle tissue is innervated by the peripheral nervous system through the establishment of the neuromuscular junction NMJ. Denervated skeletal muscles will lose contractility and undergo muscle atrophy. In the current field of skeletal muscle tissue engineering, research on integrated neuralized engineered muscles is still rarely involved, and its effect on improving in vivo function is still unclear. The present invention constructs neuralized muscle tissue through neuromuscular co-culture, which can form effective NMJ and make innervation possible.
3)血管再生能力：骨骼肌由于其高代谢量的特点，需要丰富的血运来为细胞、组织提供必需的氧气和营养物质。此外，良好的血液灌注对维持肌肉存活和延续功能也是必要的。通过预血管化的策略，构建体外工程结构维持血液灌注，可提高移植后植入细胞和组织的存活率。本发明通过该工程结构可实现血管化修复的目标，其特殊的多管腔状结构，模拟正常毛细血管网的形态。该结构在血管成熟度以及促肌肉分化方面具有显著优势。3) Vascular regeneration ability: Due to its high metabolic capacity, skeletal muscle requires abundant blood supply to provide cells and tissues with necessary oxygen and nutrients. In addition, good blood perfusion is necessary to maintain muscle survival and continued function. Through pre-vascularization strategies, constructing in vitro engineered structures to maintain blood perfusion can improve the survival rate of implanted cells and tissues after transplantation. The present invention can achieve the goal of vascularized repair through this engineering structure. Its special multi-lumen structure simulates the shape of a normal capillary network. This structure has significant advantages in vascular maturation and muscle differentiation.
4)辅助电疗：电刺激作为一种常见的物理治疗方式，适当的刺激可诱发肌肉运动或模拟正常的自主运动，以达到缓解肌肉痉挛，防止肌肉萎缩的作用。然而受限组织导电性不足的限制，使得电流刺激不能精准传达到目标组织，从而使得治疗效率低下。本发明构建的工程组织，利用柔性电极有效改善了导电性，可以将从皮肤电极施加的电流实现精准递送，提升该区域的肌肉功能。4) Auxiliary electrotherapy: Electrical stimulation is a common physical therapy method. Appropriate stimulation can induce muscle movement or simulate normal voluntary movement to relieve muscle spasm and prevent muscle atrophy. However, the limitation of insufficient electrical conductivity of restricted tissues prevents the electrical stimulation from being accurately conveyed to the target tissue, resulting in low treatment efficiency. The engineered tissue constructed by this invention uses flexible electrodes to effectively improve electrical conductivity, and can achieve precise delivery of current applied from skin electrodes, improving muscle function in this area.
本发明利用工程化的手段，在体外重建了包括肌肉、神经、血管在内的多层组织结构。其具有集成神经血管化肌肉的修复能力，同时可实现功能化肌肉再生。构建的工程化肌肉可模拟天然骨骼肌的解剖形态，形成高度定向的结构，可实现肌肉功能化的修复目标。此外，自带的柔性电极可用于辅助电刺激的物理治疗方式，进一步提升功能化。此外，本发明技术操作便捷、具备可重复性。工程化肌肉不仅拥有良好的生物相容性，还可根据不同类型的肌肉缺损设计对应的构建方式，实现个性化定制的目标。该发明从理论上可用于多种肌肉类型、尺寸的缺损，具备广泛的应用前景The present invention uses engineering means to reconstruct multi-layered tissue structures including muscles, nerves, and blood vessels in vitro. It has the ability to integrate neurovascularized muscle repair while achieving functional muscle regeneration. The constructed engineered muscles can simulate the anatomical shape of natural skeletal muscles and form a highly directional structure, which can achieve the goal of functional muscle repair. In addition, the built-in flexible electrodes can be used to assist physical therapy with electrical stimulation to further improve functionality. In addition, the technology of the present invention is easy to operate and repeatable. Engineered muscles not only have good biocompatibility, but can also be designed with corresponding construction methods according to different types of muscle defects to achieve personalized customization goals. This invention can theoretically be used for defects of various muscle types and sizes, and has broad application prospects.
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。The above-mentioned 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-substantive changes and substitutions made by those skilled in the art on the basis of the present invention fall within the scope of the present invention. Scope of protection claimed.
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| CN202310948119.0ACN116999622B (en) | 2023-07-28 | 2023-07-28 | A functional neurovascularized engineered muscle capable of electrical response and its preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20150125952A1 (en)* | 2012-04-04 | 2015-05-07 | University Of Washington Through Its Center For Commercialization | Systems and method for engineering muscle tissue |
| CN104921841A (en)* | 2015-04-10 | 2015-09-23 | 南开大学 | Method for manufacturing artificial blood vessels with double-layered structures and application of artificial blood vessels |
| CN110172126A (en)* | 2019-03-13 | 2019-08-27 | 浙江大学 | A kind of artificial-muscle drive module and preparation method thereof based on double-network hydrogel and dielectric elastomer |
| CN113288520A (en)* | 2021-04-06 | 2021-08-24 | 浙江大学 | Preparation method of magnesium-containing modified porous bioceramic tissue engineering bone with bionic periosteum |
| CN114214195A (en)* | 2021-12-15 | 2022-03-22 | 中国科学院大连化学物理研究所 | A mold for constructing large-scale vascularized muscle bundles in vitro and method of using the same |
| CN115890643A (en)* | 2022-12-15 | 2023-04-04 | 之江实验室 | Electrically-driven artificial muscle fiber with bidirectional linear strain and preparation method thereof |
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| US20150125952A1 (en)* | 2012-04-04 | 2015-05-07 | University Of Washington Through Its Center For Commercialization | Systems and method for engineering muscle tissue |
| CN104921841A (en)* | 2015-04-10 | 2015-09-23 | 南开大学 | Method for manufacturing artificial blood vessels with double-layered structures and application of artificial blood vessels |
| CN110172126A (en)* | 2019-03-13 | 2019-08-27 | 浙江大学 | A kind of artificial-muscle drive module and preparation method thereof based on double-network hydrogel and dielectric elastomer |
| CN113288520A (en)* | 2021-04-06 | 2021-08-24 | 浙江大学 | Preparation method of magnesium-containing modified porous bioceramic tissue engineering bone with bionic periosteum |
| CN114214195A (en)* | 2021-12-15 | 2022-03-22 | 中国科学院大连化学物理研究所 | A mold for constructing large-scale vascularized muscle bundles in vitro and method of using the same |
| CN115890643A (en)* | 2022-12-15 | 2023-04-04 | 之江实验室 | Electrically-driven artificial muscle fiber with bidirectional linear strain and preparation method thereof |
| CN116038665A (en)* | 2023-02-03 | 2023-05-02 | 东北电力大学 | Construction technology of flexible and variable stiffness artificial muscle device imitating multi-joint structure of elephant trunk |
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