技术领域technical field
本发明针对组织工程和再生医学领域，利用高分子有序纤维和细菌纤维素进行人工血管的制备，并创造性的将高分子有序纤维和细菌纤维素分别培养血管内皮细胞和平滑肌细胞，探究特定细胞的细胞外基质对自身再接种的作用。通过脱细胞技术得到血管内皮细胞和平滑肌细胞生存所依赖的细胞外基质复合的有序纤维和细菌纤维素，以期在体外构建不同的血管细胞微环境，有利于血管细胞渗透并为不同血管细胞提供特异性粘附结合位点，促进血管细胞表型转化，恢复血管细胞功能，让再生的血管接近受体自身的活组织。Aiming at the fields of tissue engineering and regenerative medicine, the present invention utilizes ordered polymer fibers and bacterial cellulose to prepare artificial blood vessels, and creatively cultures vascular endothelial cells and smooth muscle cells with ordered polymer fibers and bacterial cellulose, to explore specific The role of the extracellular matrix of cells on self-reseeding. The ordered fibers and bacterial cellulose compounded by the extracellular matrix that vascular endothelial cells and smooth muscle cells rely on for the survival of vascular endothelial cells and smooth muscle cells are obtained by decellularization technology, in order to construct different vascular cell microenvironments in vitro, which is conducive to vascular cell penetration and provides different vascular cells. Specifically adhere to the binding site, promote the transformation of vascular cell phenotype, restore the function of vascular cells, and make the regenerated blood vessels close to the living tissue of the recipient itself.
背景技术Background technique
临床对安全有效的血管修复材料的需求是巨大的。心血管疾病，仍然是世界上患者发病率和死亡率的主要原因。在创伤性损伤，肿瘤手术或器官移植期间受损血管的修复和重建，以及用于血液透析动静脉(AV)导管的创建通常需要使用血管替代物。毋庸置疑自体动脉和静脉是血管重建的首选材料，然而在同一患者体内收获和重新利用血管通常受到患者病理生理状况的限制。因此，在过去的四十年中，已经在数百万患者中植入了由膨体聚四氟乙烯(ePTFE)或聚对苯二甲酸乙二醇酯(Dacron)为主要材料的人工合成的血管移植物，但是它们的使用伴随着感染，血栓形成等风险。自体血管和合成血管移植物的许多生物替代物，包括同种异体和异种血管和组织，已被美国食品和药物管理局评估和批准用于人类植入。然而，它们的潜力受到可用性，成本，加工技术和临床结果的限制。尽管许多替代物具有与天然血管组织相匹配甚至超过其的机械强度，但其基质的结构和组成不利于宿主细胞的适当再增殖。因此，这些同种异体和异种组织永远不会充分重塑到宿主自身的活组织中。There is a huge clinical need for safe and effective vascular repair materials. Cardiovascular disease remains the leading cause of patient morbidity and mortality worldwide. The repair and reconstruction of damaged blood vessels during traumatic injury, tumor surgery or organ transplantation, and the creation of arteriovenous (AV) catheters for hemodialysis often require the use of vascular substitutes. There is no doubt that autologous arteries and veins are the materials of choice for vascular reconstruction, however, harvesting and repurposing vessels in the same patient is usually limited by the patient's pathophysiological conditions. Therefore, over the past forty years, millions of patients have been implanted with expanded polytetrafluoroethylene (ePTFE) or polyethylene terephthalate (Dacron) as the main material of synthetic implants. Vascular grafts, but their use is accompanied by risks of infection, thrombosis, etc. Many biological alternatives to autologous and synthetic vascular grafts, including allogeneic and xenogeneic vessels and tissues, have been evaluated and approved by the U.S. Food and Drug Administration for human implantation. However, their potential is limited by availability, cost, processing techniques and clinical outcomes. Although many substitutes have mechanical strengths that match or even exceed native vascular tissue, the structure and composition of their matrices are not conducive to proper repopulation of host cells. Consequently, these allogeneic and xenogeneic tissues are never sufficiently remodeled into the host's own living tissue.
作为一种拥有复杂纤维状的蛋白质和多糖的三维网格结构，细胞外基质在人体的大多数组织和器官中具有高度调节的功能，更重要的是具有组织特异性的成分并能够提供动态信号刺激，调节细胞行为。细胞外基质(ECM)作为研究的热点，不仅局限于天然ECM的开发，研究者还利用ECM的独特生物特性与生物材料相结合，开发仿生ECM材料应用于组织工程和再生医学。As a three-dimensional network structure of complex fibrous proteins and polysaccharides, the extracellular matrix has highly regulated functions in most tissues and organs of the human body, and more importantly, has tissue-specific components and can provide dynamic signals Stimulates, regulates cell behavior. As a research hotspot, extracellular matrix (ECM) is not limited to the development of natural ECM. Researchers also use the unique biological characteristics of ECM to combine with biomaterials to develop bionic ECM materials for tissue engineering and regenerative medicine.
发明内容Contents of the invention
本发明中，我们利用高分子有序纤维和细菌纤维素构建双层的人工血管。采用细菌纤维素作为人工血管的外层，用以增强损伤部位邻边血管细胞的募集和渗透。采用有序高分子纤维作为人工血管的内层，为血管内皮细胞提供粘附位点并引导其极性生长。同时我们还通过体外细胞培养，通过沉积的细胞特异性细胞外基质对有序纤维和细菌纤维素进行修饰。In the present invention, we use ordered polymer fibers and bacterial cellulose to construct double-layer artificial blood vessels. Bacterial cellulose is used as the outer layer of artificial blood vessels to enhance the recruitment and penetration of vascular cells adjacent to the injured site. Ordered polymer fibers are used as the inner layer of artificial blood vessels to provide adhesion sites for vascular endothelial cells and guide their polar growth. At the same time, we also modified ordered fibers and bacterial cellulose by depositing cell-specific extracellular matrix through in vitro cell culture.
由于设计的人工血管不仅在结构上与天然血管相似，并且修饰的细胞特异性细胞外基质含有大量信号分子，积极参与控制细胞的生长，极性，形态、迁移和代谢活动，可增强细胞的特异性迁移和选择性粘附，调控不同血管细胞在血管重建不同过程中的生理功能。Because the designed artificial blood vessels are not only structurally similar to natural blood vessels, but also the modified cell-specific extracellular matrix contains a large number of signaling molecules, which actively participate in the control of cell growth, polarity, morphology, migration and metabolic activities, which can enhance the specificity of cells. Sexual migration and selective adhesion, regulating the physiological functions of different vascular cells in different processes of vascular remodeling.
本发明制作方法及结构：Manufacturing method and structure of the present invention:
外层：以木醋杆菌生产的细菌纤维素为原料，其纤维直径小于100nm，具有与天然细胞外基质相似的结构。首先，将木醋杆菌生产的细菌纤维素水凝胶用清水进行清洗，然后用1％NaOH煮至溶液澄清最后用超纯水浸泡至弱碱性或中性。Outer layer: Bacterial cellulose produced by Acetobacter xylinum is used as raw material, and its fiber diameter is less than 100nm, which has a structure similar to natural extracellular matrix. First, wash the bacterial cellulose hydrogel produced by Acetobacter xylinum with clear water, then boil it with 1% NaOH until the solution is clear, and finally soak it with ultrapure water until it is weakly alkaline or neutral.
内层：以聚己内酯(PCL)等高分子材料为原材料，通过生物快速成型(3D打印)为有序纤维，为血管内皮细胞提供特异性粘附结合位点并诱导其极性生长。Inner layer: Polycaprolactone (PCL) and other polymer materials are used as raw materials to form ordered fibers through rapid biological prototyping (3D printing), providing specific adhesion and binding sites for vascular endothelial cells and inducing their polar growth.
与传统人工血管相比，本发明的创新点在于：Compared with traditional artificial blood vessels, the innovation of the present invention lies in:
1.本发明采用细菌纤维素为原料，不同于其他人造纳米结构材料，细菌纤维素不会阻碍细胞的渗透，并且因其高生物相容性，能够为细胞活动提供良好的生存环境。1. The present invention uses bacterial cellulose as a raw material. Unlike other artificial nanostructure materials, bacterial cellulose does not hinder the penetration of cells, and because of its high biocompatibility, it can provide a good living environment for cell activities.
2.本发明为多层结构，内层的有序纤维能够为血管内皮细胞提供结合位点并诱导其极性生长；外层细菌纤维素有利于血管内皮细胞渗透至内层，并为血管平滑肌细胞提供生存空间。2. The present invention has a multi-layer structure, and the ordered fibers of the inner layer can provide binding sites for vascular endothelial cells and induce their polar growth; the outer layer of bacterial cellulose is conducive to the penetration of vascular endothelial cells into the inner layer, and can provide vascular smooth muscle Cells provide living space.
3.本发明运用细胞特异性细胞外基质进行修饰，对自身细胞发挥特异性诱导和选择性粘附作用，调控细胞行为。3. The present invention modifies the cell-specific extracellular matrix to exert specific induction and selective adhesion to self-cells and regulate cell behavior.
4.本发明内层为有序纤维，通过电脑建模，可快速成型(3D打印)制造，并批量生产。4. The inner layer of the present invention is an ordered fiber, which can be manufactured by rapid prototyping (3D printing) through computer modeling and mass-produced.
5.本发明外层为细菌纤维素，通过培养木醋杆菌得到，方法简单成本低廉，亦可批量生产。5. The outer layer of the present invention is bacterial cellulose, which is obtained by culturing Acetobacter xylinum. The method is simple and low in cost, and can also be produced in batches.
附图说明Description of drawings
如图所示，as the picture shows,
图1为本发明设计的双层人工血管模型。外层为细菌纤维素，内层为高分子有序纤维。Fig. 1 is the double-layer artificial blood vessel model designed by the present invention. The outer layer is bacterial cellulose, and the inner layer is polymer ordered fiber.
图2为细菌纤维素的显微结构图，其纤维直径小于100nm。Figure 2 is a microstructure diagram of bacterial cellulose, the fiber diameter of which is less than 100nm.
图3为有序纤维显微结构图。Figure 3 is a microstructure diagram of ordered fibers.
具体实施方式Detailed ways
下面结合实施条例对本专利做进一步的说明。Below in conjunction with implementing regulation this patent is described further.
1)双层人工血管制作方法1) Manufacturing method of double-layer artificial blood vessel
外层：细菌纤维素。通过在醋酸菌培养基(#350)培养木醋杆菌得到。细菌纤维素膜先经清水冲洗，然后通过1％NaOH煮至溶液澄清，最后超纯水浸泡至弱碱性或中性。75％医用酒精浸泡10min，紫外照射1h进行灭菌。Outer layer: bacterial cellulose. Obtained by culturing Acetobacter xylinum in acetic acid bacteria medium (#350). The bacterial cellulose membrane is firstly rinsed with clean water, then boiled with 1% NaOH until the solution is clear, and finally soaked in ultrapure water until weakly alkaline or neutral. Soak in 75% medical alcohol for 10 minutes, and irradiate with ultraviolet light for 1 hour to sterilize.
内层：有序纤维丝。配制7％(v/v)聚己内酯(PCL)打印溶液，通过3D打印等快速成型手段得到聚己内酯有序纤维。75％医用酒精浸泡10min，紫外照射1h进行灭菌。Inner layer: ordered fiber filaments. A 7% (v/v) polycaprolactone (PCL) printing solution was prepared, and polycaprolactone ordered fibers were obtained by rapid prototyping means such as 3D printing. Soak in 75% medical alcohol for 10 minutes, and irradiate with ultraviolet light for 1 hour to sterilize.
2)体外细胞培养2) In vitro cell culture
取准备好的有序纤维和细菌纤维素分别培养血管内皮细胞和血管平滑肌细胞，通过沉积的细胞外基质对两种不同的支架材料进行修饰。The prepared ordered fibers and bacterial cellulose were used to culture vascular endothelial cells and vascular smooth muscle cells respectively, and the two different scaffold materials were modified by the deposited extracellular matrix.
3)双层人工血管的组装3) Assembly of double-layer artificial blood vessels
将细胞外基质修饰过的有序纤维和细菌纤维素先后缠绕于直径1.6mm的不锈钢管上，4℃自然干燥，制作内层为有序纤维，外层为细菌纤维素的双层人工血管模型。Ordered fibers modified with extracellular matrix and bacterial cellulose were wound successively on a stainless steel tube with a diameter of 1.6mm, and dried naturally at 4°C to make a double-layer artificial blood vessel model with ordered fibers on the inner layer and bacterial cellulose on the outer layer .
上述以较佳实例公开了本专利，然其并非用以限制本专利，凡采用等同替换或者等效替换方式所获得的技术方案，均落在本专利的保护范围之内。The above discloses this patent with preferred examples, but it is not intended to limit this patent. All technical solutions obtained by equivalent replacement or equivalent replacement methods fall within the protection scope of this patent.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910633942.6ACN110201236A (en) | 2019-07-15 | 2019-07-15 | A kind of novel artificial blood vessel |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910633942.6ACN110201236A (en) | 2019-07-15 | 2019-07-15 | A kind of novel artificial blood vessel |
| Publication Number | Publication Date |
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| CN110201236Atrue CN110201236A (en) | 2019-09-06 |
| Application Number | Title | Priority Date | Filing Date |
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| CN201910633942.6APendingCN110201236A (en) | 2019-07-15 | 2019-07-15 | A kind of novel artificial blood vessel |
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| CN (1) | CN110201236A (en) |
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| RJ01 | Rejection of invention patent application after publication | Application publication date:20190906 |