技术领域technical field
本发明属于组织工程领域，具体涉及一种仿自然血管的三层人造血管支架及其制备方法，所述的三层人造血管支架的组成形式为MNM的三层结构，M层为生物相容性较好材料，N层为力学性质提供层。The invention belongs to the field of tissue engineering, and specifically relates to a three-layer artificial vascular stent imitating natural blood vessels and a preparation method thereof. The composition of the three-layer artificial vascular stent is a three-layer structure of MNM, and the M layer is biocompatible The better material, the N layer provides the layer for mechanical properties.
背景技术Background technique
随着社会的发展，各种血管疾病已经成为危害人类健康的第一大杀手，比如血管瘤、血管破损、心血管疾病、动脉粥样硬化等。而目前治疗血管疾病的主要方法为血管移植，血管移植的最佳来源是自体，但是自体血管有限，因此人造血管的需求越来越旺盛，大孔径的人造血管已经用于临床，但是小口径人造血管由于其远期通畅率低，易堵塞等一直没有成功。一个理想的血管移植支架需要满足的条件有：无毒不致癌、生物相容性较好、容易加工制备、廉价等。至今为止，人们提出了一系列的方法和材料来制备性能优异的血管支架，如热致相分离、溶剂铸造微粒浸出技术、水凝胶、分子自组装等方法以及丝素和胶原等材料。但这些方法设备复杂，操作不方便，且所得血管支架的结构、形貌、组成不能得到控制，即很难大规模的制备各种不同尺寸的人造血管支架。更重要的是这些方法和材料大部分是单一功能，比如有的人造血管其生物相容性较好，但是其机械性质却较差，有的机械性质较好，但是其生物相容性却较低等(参考文献1：Zuwei Ma,Masaya Kotaki,Ryuji Inai,andSeeram Ramakrishna.Potential of Nanofiber Matrix as Tissue-EngineeringScaffolds.Tissue Eng.2005,11(1-2):101-109；参考文献2：Chen R,Huang C,Ke QF,HeCL,Wang HS,Mo XM.Preparation and characterization of coaxial electrospunthermoplastic polyurethane/collagen compound nanofibers for tissueengineering applications.Colloid Surf B Biointerfaces2010；79:315-325)。因此，寻找和自然血管相媲美的人造血管成为研究的热点，近几年来，一些科研工作者在这方面做出了一些努力，比如做出了双层结构的人造小口径血管，其达到了较好的生物性质和良好的机械性质。但是大部分人造血管很难具备生物相容性好而且力学性质也优异，所以制备一种同时具有较好的生物相容性并且力学性质也优异的人造血管迫在眉睫。With the development of society, various vascular diseases have become the number one killer of human health, such as hemangioma, blood vessel damage, cardiovascular disease, atherosclerosis, etc. At present, the main method of treating vascular diseases is blood vessel transplantation. The best source of blood vessel transplantation is autologous, but autologous blood vessels are limited, so the demand for artificial blood vessels is increasing. Large-diameter artificial blood vessels have been used in clinical practice, but small-caliber artificial blood vessels Blood vessels have not been successful due to their low long-term patency rate and easy blockage. An ideal vascular graft stent needs to meet the following conditions: non-toxic and non-carcinogenic, good biocompatibility, easy to process and prepare, and cheap. So far, a series of methods and materials have been proposed to prepare vascular stents with excellent performance, such as thermally induced phase separation, solvent casting particle leaching technology, hydrogel, molecular self-assembly and other methods, as well as materials such as silk fibroin and collagen. However, these methods have complicated equipment and inconvenient operation, and the structure, shape and composition of the obtained vascular stent cannot be controlled, that is, it is difficult to prepare artificial vascular stents of various sizes on a large scale. More importantly, most of these methods and materials have a single function. For example, some artificial blood vessels have better biocompatibility, but their mechanical properties are poor, and some have better mechanical properties, but their biocompatibility is poorer. Low (Reference 1: Zuwei Ma, Masaya Kotaki, Ryuji Inai, and Seeram Ramakrishna. Potential of Nanofiber Matrix as Tissue-Engineering Scaffolds. Tissue Eng. 2005,11(1-2):101-109; Reference 2: Chen R , Huang C, Ke QF, HeCL, Wang HS, Mo XM. Preparation and characterization of coaxial electrospunthermoplastic polyurethane/collagen compound nanofibers for tissue engineering applications. Colloid Surf B Biointerfaces 2010; 79:315-325). Therefore, searching for artificial blood vessels comparable to natural blood vessels has become a research focus. Good biological properties and good mechanical properties. However, most artificial blood vessels are difficult to have good biocompatibility and excellent mechanical properties, so it is imminent to prepare an artificial blood vessel with good biocompatibility and excellent mechanical properties.
静电纺丝作为近年来新起的热门领域，是一种制备微纳米纤维的十分方便简洁方法，主要原理是利用高压静电场的作用来实现纺丝液的喷射，喷射过程中，溶剂挥发，纤维进一步拉伸细化最终获得目标纤维。在电纺丝过程中，通过调节和控制参数，如纺丝液的浓度、电压、针头、转速、纺丝距离等，纤维的结构、形貌和组成能得到有效的优化。通过替换前驱液的方法科研制备多层结构的人造血管，参见参考文献3：Park K.,Ju Y.M.,Son J.S.etal.Surface modification of biodegradable electrospun nanofiber scaffolds andtheir interaction with fibroblasts.Journal of Biomaterials Science:PolymerEdition,2007,4:369-382。As a new hot field in recent years, electrospinning is a very convenient and simple method for preparing micro-nano fibers. The main principle is to use the effect of high-voltage electrostatic field to realize the injection of spinning liquid. During the injection process, the solvent volatilizes and the fibers Further stretching refinement finally obtains the target fiber. During the electrospinning process, by adjusting and controlling parameters, such as the concentration of spinning solution, voltage, needle, rotating speed, spinning distance, etc., the structure, morphology and composition of fibers can be effectively optimized. Artificial blood vessels with multi-layer structure were prepared by replacing the precursor solution. See reference 3: Park K., Ju Y.M., Son J.S.etal. Surface modification of biodegradable electrospun nanofiber scaffolds and their interaction with fibroblasts. Journal of Biomaterials Science: PolymerEdition, 2007, 4:369-382.
发明内容Contents of the invention
本发明针对现有人工血管支架的不足，根据自然血管的三层结构及其相互协同的作用，提出了一种静电纺丝制备三层人造血管支架的方法，通过顺序静电纺丝的方法，本发明成功制备了三层人造血管支架，这种三层人造血管支架具有良好的生物相容性和良好的力学性质，有望在临床使用方面得到应用。Aiming at the deficiency of existing artificial vascular stents, the present invention proposes a method for preparing a three-layer artificial vascular stent by electrospinning according to the three-layer structure of natural blood vessels and their synergistic effects. Through the method of sequential electrospinning, the present invention The invention successfully prepared a three-layer artificial vascular stent, which has good biocompatibility and good mechanical properties, and is expected to be applied in clinical use.
本发明提供一种仿自然血管的三层人造血管支架及其制备方法，所制得的三层人造血管支架纤维分布均匀，所得的高生物相容性、高力学性质的三层人造血管支架在临床应用上有很大的前景。本发明所制备的高强度高弹性三层人造血管支架是由纳米纤维所构成的，纳米纤维直径为200nm～2μm。所述纳米纤维直径可通过调节纺丝溶液的浓度、纺丝电压、纺丝距离等电纺参数来控制。The invention provides a three-layer artificial vascular stent imitating natural blood vessels and a preparation method thereof. The fibers of the prepared three-layer artificial vascular stent are evenly distributed, and the obtained three-layer artificial vascular stent with high biocompatibility and high mechanical properties is There are great prospects for clinical application. The high-strength and high-elastic three-layer artificial vascular stent prepared by the invention is composed of nanofibers, and the diameter of the nanofibers is 200nm-2μm. The diameter of the nanofiber can be controlled by adjusting the electrospinning parameters such as the concentration of the spinning solution, the spinning voltage, and the spinning distance.
本发明所得到的仿自然血管的三层人造血管支架能用于组织工程、临床医学等领域。The three-layer artificial blood vessel stent imitating natural blood vessels obtained by the invention can be used in the fields of tissue engineering, clinical medicine and the like.
本发明所得到的一种仿自然血管的三层人造血管支架具有三层结构，具有高机械强度和良好生物相容性，所述的三层结构为MNM形式，M层代表最内层和最外层，生物相容性较好，N层代表中间层，为力学性质提供层。所述的制备方法包括以下步骤：A three-layer artificial vascular stent imitating a natural blood vessel obtained in the present invention has a three-layer structure with high mechanical strength and good biocompatibility. The three-layer structure is in the form of MNM, and the M layer represents the innermost layer and the outermost layer. The outer layer has better biocompatibility, and the N layer represents the middle layer, which provides the layer for mechanical properties. Described preparation method comprises the following steps:
第一步，静电纺丝溶液的配置；The first step, the configuration of the electrospinning solution;
称取生物相容性较好的聚合物A，在室温条件下溶于溶剂C中，充分搅拌至完全溶解，得到聚合物A的纺丝溶液，用于制备三层人造血管支架的内层和外层即M层。Weigh polymer A with better biocompatibility, dissolve it in solvent C at room temperature, and stir until it is completely dissolved to obtain a spinning solution of polymer A, which is used to prepare the inner layer of the three-layer artificial vascular stent and The outer layer is the M layer.
聚合物B在室温条件下搅拌溶解于溶剂C中，得到聚合物B的纺丝溶液，用于制备三层人造血管支架的中间层即N层。The polymer B is stirred and dissolved in the solvent C at room temperature to obtain a spinning solution of the polymer B, which is used to prepare the middle layer of the three-layer artificial vascular stent, that is, the N layer.
第二步，内层纤维层的制备；The second step, the preparation of the inner fiber layer;
将第一步中得到的聚合物A的纺丝溶液置于注射器(针头直径为0.5～1.2mm)中，固定在注射泵上，施加8～24kV的电压，接收距离为10～15cm，接收器转速为60～120r/min，电纺0.5～1h，在接收器外圆周制备得到内层纤维层。所述的接收器为带有不锈钢管的旋转电机，所述不锈钢管的直径为2～6mm。Put the spinning solution of polymer A obtained in the first step into a syringe (needle diameter of 0.5-1.2mm), fix it on the syringe pump, apply a voltage of 8-24kV, and the receiving distance is 10-15cm, the receiver The rotating speed is 60-120r/min, electrospinning is performed for 0.5-1h, and the inner fiber layer is prepared on the outer circumference of the receiver. The receiver is a rotary motor with a stainless steel tube with a diameter of 2-6mm.
第三步，中间纤维层制备；The third step, intermediate fiber layer preparation;
以所述的内层纤维层作为接收基底，继续制备中间纤维层，具体为：将第一步中得到的聚合物B的纺丝溶液置于直径为0.5～1.2mm的针头注射器中，然后将所述的针头注射器固定在注射泵上，施加12～25kV电压，接收距离为8cm～15cm，接收器转速在60～120r/min，电纺4～6h，得到中间纤维层。内层纤维层和中间层纤维层组成双层管状物。Using the inner fiber layer as the receiving base, continue to prepare the middle fiber layer, specifically: put the spinning solution of polymer B obtained in the first step into a needle syringe with a diameter of 0.5-1.2mm, and then put The needle syringe is fixed on the syringe pump, a voltage of 12-25kV is applied, the receiving distance is 8cm-15cm, the speed of the receiver is 60-120r/min, and the electrospinning is performed for 4-6 hours to obtain the intermediate fiber layer. The inner fiber layer and the middle layer fiber layer form a double-layer tubular product.
第四步，外层纤维层的制备；The 4th step, the preparation of outer fiber layer;
以第三步得到的中间纤维层为基底，继续制备外层纤维层，具体为：将第一步中聚合物A的纺丝溶液置于(0.5～1.2mm)针头注射器中，将注射器固定在注射泵上，施加12～25kV的电压，接收距离为10～15cm，接收器转速为60～120r/min，电纺0.5～1h，得到外层纤维层。Using the intermediate fiber layer obtained in the third step as the base, continue to prepare the outer fiber layer, specifically: put the spinning solution of polymer A in the first step in a syringe with a needle (0.5-1.2 mm), and fix the syringe on A voltage of 12-25 kV is applied to the syringe pump, the receiving distance is 10-15 cm, the rotational speed of the receiver is 60-120 r/min, and electrospinning is performed for 0.5-1 hour to obtain the outer fiber layer.
第五步，脱管；The fifth step, take off the tube;
将具有三层纤维膜结构的接收器(管状结构)置于去离子水中半小时以上，在30℃烘箱中烘干，即得本发明的仿自然血管的三层人造血管支架。Place the receiver (tubular structure) with a three-layer fiber membrane structure in deionized water for more than half an hour, and dry it in an oven at 30° C. to obtain the three-layer artificial vascular stent imitating natural blood vessels of the present invention.
所述的聚合物A为生物相容性较好的胶原、明胶、聚乳酸、聚乳酸与聚己内酯混合物、壳聚糖、丝素或细菌纤维素等中的一种或两种以上。所述的聚合物B为力学性质较好的聚氨酯、聚己内酯、聚乳酸-羟基乙酸共聚物等中的一种或两种以上。The polymer A is one or more than two of collagen, gelatin, polylactic acid, mixture of polylactic acid and polycaprolactone, chitosan, silk fibroin or bacterial cellulose with good biocompatibility. The polymer B is one or more of polyurethane, polycaprolactone, polylactic acid-glycolic acid copolymer and the like with good mechanical properties.
所述的溶剂C为乙醇、丙酮、二氯甲烷、氯仿、氮氮二甲基甲酰胺(DMF)、水、四氢呋喃(THF)、三氯甲烷等中的一种或两种以上。The solvent C is one or more of ethanol, acetone, dichloromethane, chloroform, nitrogen dimethylformamide (DMF), water, tetrahydrofuran (THF), chloroform and the like.
所述的聚合物A的纺丝溶液的质量百分比浓度为8～15wt％，聚合物B的纺丝溶液的质量百分比浓度为10～15wt％。The mass percentage concentration of the spinning solution of the polymer A is 8-15 wt%, and the mass percentage concentration of the spinning solution of the polymer B is 10-15 wt%.
上述方法制备得到的三层人造血管支架为MNM形式，其内层M和外层M的厚度均为10～100μm，而中间层N的厚度为100～500μm。The three-layer artificial vascular stent prepared by the above method is in the form of MNM, the thickness of the inner layer M and the outer layer M are both 10-100 μm, and the thickness of the middle layer N is 100-500 μm.
所述的三层人造血管支架中纤维的直径为200nm～2μm。优选的，纤维的直径控制在600nm～1.5μm之间。The diameter of the fiber in the three-layer artificial blood vessel support is 200nm-2μm. Preferably, the diameter of the fiber is controlled between 600 nm and 1.5 μm.
本发明的三层人造血管支架的拉伸应力达到约30～70MPa，应变为0～600％。The tensile stress of the three-layer artificial blood vessel stent of the present invention reaches about 30-70 MPa, and the strain is 0-600%.
本发明以生物相容性聚合物材料为前驱体，通过静电纺丝制备内层和外层的纤维层，以力学性质较好的聚合物材料为前驱体，通过静电纺丝制备中间力学支撑层，在此基础上，使用逐步电纺法制备了仿自然血管的三层人造血管支架，构成所述三层人造血管支架的纤维表面光滑、形貌均匀、纤维直径分布范围较窄，且良好的生物性能和力学性能优异，并且三层结构MNM相互协同作用，弥补了原来单层结构的缺点。本发明所得的仿自然血管的三层人造血管支架可以大规模生产，有望用于实际的临床诊断中。The invention uses biocompatible polymer materials as precursors to prepare inner and outer fiber layers by electrospinning, and uses polymer materials with better mechanical properties as precursors to prepare intermediate mechanical support layers by electrospinning , on this basis, a three-layer artificial vascular scaffold imitating natural blood vessels was prepared by step-by-step electrospinning. The biological and mechanical properties are excellent, and the three-layer structure MNM interacts with each other to make up for the shortcomings of the original single-layer structure. The three-layer artificial vascular stent imitating natural blood vessels obtained in the present invention can be produced on a large scale, and is expected to be used in actual clinical diagnosis.
本发明提供的三层人造血管支架及其制备方法与现有技术中制备人造血管支架的方法相比，有益效果如下：Compared with the method for preparing artificial vascular stents in the prior art, the three-layer artificial vascular stent provided by the present invention and its preparation method have the following beneficial effects:
(1)与现有的单层血管相比，本发明的仿自然血管的三层人造血管支架不仅生物相容性较好，而且具有优异的力学性能。(1) Compared with the existing single-layer blood vessels, the three-layer artificial blood vessel stent imitating natural blood vessels of the present invention not only has better biocompatibility, but also has excellent mechanical properties.
(2)与现有的剥离法、热致相分离法、自组装法等相比，本发明采用的设备简单，操作方便，能够实现大规模的生产。(2) Compared with the existing exfoliation method, thermally induced phase separation method, self-assembly method, etc., the equipment adopted in the present invention is simple, easy to operate, and can realize large-scale production.
附图说明Description of drawings
图1本发明中仿自然血管的三层人造血管支架的结构示意图。Fig. 1 is a schematic structural view of a three-layer artificial vascular stent imitating a natural blood vessel in the present invention.
图2本发明制备得到的人造血管支架结构整体结构示意图。Fig. 2 is a schematic diagram of the overall structure of the artificial vascular stent prepared by the present invention.
图3本发明制备得到的人造血管支架的典型的应力-应变曲线。Fig. 3 is a typical stress-strain curve of the artificial vascular stent prepared by the present invention.
图4本发明制备得到的人造血管支架的内皮细胞增殖图。Fig. 4 is a graph of endothelial cell proliferation of the artificial vascular stent prepared in the present invention.
具体实施方式detailed description
下面结合附图和实施例对本发明进行详细说明。The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.
实施例1Example 1
第一步，聚合物A选取为聚己内酯(PCL,(C6H10O2)n)和聚乳酸(PLA,(C6H8O4)n)按质量比3:7混合后溶于混合溶剂中，配制成质量百分比浓度为10％纺丝液，即聚合物A的纺丝溶液，用于制备M层；所述混合溶剂中，丙酮和N，N-二甲基甲酰胺的质量比w/w＝1:5。In the first step, polymer A is selected as polycaprolactone (PCL, (C6 H10 O2 )n ) and polylactic acid (PLA, (C6 H8 O4 )n ) mixed in a mass ratio of 3:7 Dissolved in a mixed solvent, prepared into a spinning solution with a concentration of 10% by mass percentage, that is, the spinning solution of polymer A, used to prepare the M layer; in the mixed solvent, acetone and N,N-dimethylformamide The mass ratio w/w=1:5.
聚合物B选取为把PU-PCL(质量比3:1)溶于DMF和THF的混合溶剂中，配成质量百分比为10％的聚合物B的纺丝溶液，用于制备N层。所述的混合溶剂中DMF和THF的质量比为3:1。Polymer B is selected as dissolving PU-PCL (mass ratio 3:1) in a mixed solvent of DMF and THF to prepare a spinning solution of polymer B with a mass percentage of 10%, which is used to prepare the N layer. The mass ratio of DMF and THF in the mixed solvent is 3:1.
第二步，将聚合物A的纺丝溶液置于直径0.8mm针头的注射泵中，施加10～15kV的电压，在工作距离为10～15cm时进行静电纺丝1h，采用带有不锈钢管(直径为4mm)的旋转电机收集纤维，旋转电机转速为120r/min，得到生物相容性较好的并且力学也优异的内层纤维层。In the second step, the spinning solution of polymer A is placed in a syringe pump with a needle with a diameter of 0.8 mm, a voltage of 10-15 kV is applied, and electrospinning is carried out for 1 hour when the working distance is 10-15 cm. A rotating motor with a diameter of 4mm) collects the fibers, and the rotating speed of the rotating motor is 120r/min, so as to obtain an inner fiber layer with better biocompatibility and excellent mechanics.
第三步，在内层纤维层的基础上，将所述的聚合物B的纺丝溶液置于直径0.5mm针头的注射泵中，施加15kV的电压，工作距离为10cm时进行静电纺丝4h，旋转电机转速为120r/min，即可得到双层的纤维膜。In the third step, on the basis of the inner fiber layer, place the spinning solution of the polymer B in a syringe pump with a needle with a diameter of 0.5mm, apply a voltage of 15kV, and perform electrospinning for 4h when the working distance is 10cm , the rotation speed of the rotating motor is 120r/min, and a double-layer fiber membrane can be obtained.
第四步，将聚合物A的纺丝溶液置于直径0.9mm针头的注射泵中，施加15kV的电压，在工作距离为10cm时继续静电纺丝，静电纺丝时间为1h，旋转电机转速为120r/min，得到具有三层纤维膜结构的管状支架。In the fourth step, the spinning solution of polymer A is placed in a syringe pump with a needle diameter of 0.9 mm, a voltage of 15 kV is applied, and the electrospinning is continued when the working distance is 10 cm. The electrospinning time is 1 h, and the rotation speed of the rotating motor is 120r/min, a tubular scaffold with a three-layer fiber membrane structure was obtained.
第五步，最后将第四步中的带有三层纤维膜结构的管状支架的不锈钢管置于去离子水中浸泡2小时，进行脱管，然后30℃烘干，得到本发明的仿自然血管的三层人造血管支架。In the fifth step, finally place the stainless steel tube with the tubular stent of the three-layer fibrous membrane structure in the fourth step, soak it in deionized water for 2 hours, remove the tube, and then dry it at 30°C to obtain the natural blood vessel imitation tube of the present invention. Three-layer artificial vascular stent.
图1和图2所示，本实施例制备的仿自然血管的三层人造血管支架，最内层和最外层均为M层，中间层为N层，每层结构均由纳米纤维构成，纳米纤维直径为600nm～1.5μm。其中，最内层M层厚度为10μm，中间层N层厚度为100μm，最外层M层厚度为20μm。由图3所示的应力应变曲线图可知，本实施例制备得到的三层人造血管支架的纤维管拉伸应力强度为56MPa，拉伸应变600％。图4所示为内皮细胞在血管上的增殖荧光图，内皮细胞在内层上快速内皮化，大大减少血栓的形成，提高植入的成功率。As shown in Fig. 1 and Fig. 2, the three-layer artificial vascular stent imitating the natural blood vessel prepared in this embodiment, the innermost layer and the outermost layer are both M layers, the middle layer is N layer, and each layer structure is made of nanofibers, The diameter of the nanofiber is 600nm-1.5μm. Wherein, the innermost M layer has a thickness of 10 μm, the middle N layer has a thickness of 100 μm, and the outermost M layer has a thickness of 20 μm. From the stress-strain curve shown in FIG. 3 , it can be seen that the tensile stress strength of the fiber tube of the three-layer artificial vascular stent prepared in this embodiment is 56 MPa, and the tensile strain is 600%. Figure 4 shows the fluorescence image of the proliferation of endothelial cells on the blood vessels. The endothelial cells are rapidly endothelialized on the inner layer, which greatly reduces the formation of thrombus and improves the success rate of implantation.
实施例2Example 2
第一步，聚合物A选取为聚乳酸(PLA,(C6H8O4)n)，将聚合物A溶于混合溶剂中，配制成15wt％浓度的聚合物A的纺丝溶液，用于制备M层纤维膜；所述混合溶剂中丙酮和N，N-二甲基甲酰胺的质量比w/w＝1:1。In the first step, polymer A is selected as polylactic acid (PLA, (C6 H8 O4 )n ), and polymer A is dissolved in a mixed solvent to prepare a spinning solution of polymer A with a concentration of 15 wt%. To prepare M layers of fiber membranes; the mass ratio of acetone and N,N-dimethylformamide in the mixed solvent w/w=1:1.
聚合物B选取为纯的聚氨酯(PU,C28H44N2O11)，将聚合物B溶于DMF和THF中配成质量百分比为10％的聚合物B的纺丝溶液。其中，DMF和THF的质量比1:3。The polymer B is selected as pure polyurethane (PU, C28 H44 N2 O11 ), and the polymer B is dissolved in DMF and THF to prepare a spinning solution of the polymer B with a mass percentage of 10%. Wherein, the mass ratio of DMF and THF is 1:3.
第二步，将聚合物A的纺丝溶液置于配有直径1.0mm针头的注射泵中，施加15kV的电压在工作距离为15cm时进行静电纺丝，接收纤维的旋转电机转速为60r/min，得到力学性能优异且孔隙率80％内层纤维膜。In the second step, the spinning solution of polymer A is placed in a syringe pump equipped with a needle with a diameter of 1.0mm, and a voltage of 15kV is applied to carry out electrospinning when the working distance is 15cm, and the rotational speed of the rotating motor for receiving fibers is 60r/min , to obtain an inner layer fiber membrane with excellent mechanical properties and a porosity of 80%.
第三步，以内层为接收基底，将聚合物B的纺丝溶液置于直径1.2mm针头的注射泵中，施加12kV的电压，在工作距离为8cm时进行静电纺丝，采用不锈钢管的旋转电机收集纤维，旋转电机转速为60r/min，纺丝时间为6h，得到中间层纤维膜即N层。The third step is to use the inner layer as the receiving substrate, place the spinning solution of polymer B in a syringe pump with a needle diameter of 1.2mm, apply a voltage of 12kV, and perform electrospinning at a working distance of 8cm, using the rotation of a stainless steel tube The motor collects the fibers, the rotation speed of the rotating motor is 60r/min, and the spinning time is 6h to obtain the middle layer of fiber membrane, that is, the N layer.
第四步，将聚合物A的纺丝溶液置于直径0.8mm针头的注射泵中，施加15kV的电压，工作距离为10cm时进行静电纺丝，继续在已经得到中间层纤维膜上收集纤维，得到具有外层纤维膜的纤维管。纺丝过程中旋转电机的转速为60r/min，纺丝时间1h。The fourth step is to place the spinning solution of polymer A in a syringe pump with a needle diameter of 0.8mm, apply a voltage of 15kV, and perform electrospinning when the working distance is 10cm, and continue to collect fibers on the obtained intermediate fiber membrane. A fiber tube with an outer layer of fiber membrane is obtained. During the spinning process, the rotating speed of the rotating motor is 60r/min, and the spinning time is 1h.
第五步，把第四步得到的纤维管放入去离子水中浸泡3～5小时脱管，30℃烘干，得到目标三层人造血管支架。In the fifth step, soak the fiber tube obtained in the fourth step in deionized water for 3 to 5 hours, take off the tube, and dry at 30° C. to obtain the target three-layer artificial vascular stent.
上述制备方法制备得到的仿自然血管的三层人造血管支架，具有MNM形式的三层结构，其中，最内层的M层厚度为50μm，中间层N层的厚度为300μm，最外层M层的厚度为100μm。所述的三层人造血管支架为三层纤维膜结构，纤维的直径为700nm～2000nm之间。试验测试所得到的三层人造血管支架的拉伸应力为30MPa，应变达350％，力学性能优异，并且具有较好的生物相容性。内皮细胞在血管上的增殖荧光图显示，内皮细胞在内层上快速内皮化，大大减少血栓的形成，提高植入的成功率。The three-layer artificial vascular stent imitating natural blood vessels prepared by the above preparation method has a three-layer structure in the form of MNM, wherein the thickness of the innermost M layer is 50 μm, the thickness of the middle N layer is 300 μm, and the outermost M layer The thickness is 100 μm. The three-layer artificial blood vessel stent has a three-layer fibrous membrane structure, and the diameter of the fiber is between 700nm and 2000nm. The tensile stress of the obtained three-layer artificial vascular stent is 30MPa, the strain reaches 350%, and the mechanical properties are excellent, and it has good biocompatibility. Fluorescent images of endothelial cell proliferation on blood vessels show that endothelial cells rapidly endothelialize on the inner layer, greatly reducing thrombus formation and increasing the success rate of implantation.
实施例3Example 3
第一步，聚合物A选取为丝素，将聚合物A溶于乙醇中配成质量百分比8％的聚合物A的纺丝溶液；In the first step, the polymer A is selected as silk fibroin, and the polymer A is dissolved in ethanol to form a spinning solution of 8% by mass of the polymer A;
聚合物B选取为聚己内酯(PCL,(C6H10O2)n)、聚乳酸-羟基乙酸共聚物(PLGA，75:25)的混合物，其中，PCL与PLGA的质量比为3:7。将聚合物B溶于混合溶剂中，配制成质量百分比15％聚合物B的纺丝溶液。所述的混合溶剂中丙酮和N，N-二甲基甲酰胺质量百分比w/w＝1:4。Polymer B is selected as a mixture of polycaprolactone (PCL, (C6 H10 O2 )n ) and polylactic acid-glycolic acid copolymer (PLGA, 75:25), wherein the mass ratio of PCL to PLGA is 3 :7. Polymer B was dissolved in a mixed solvent to prepare a spinning solution with 15% by mass of polymer B. The mass percentage w/w of acetone and N,N-dimethylformamide in the mixed solvent is 1:4.
第二步，将聚合物A的纺丝溶液置于配有直径1mm针头的注射泵中，施加10kV的电压，在工作距离为15cm时进行静电纺丝，纺丝时间0.5h，采用带有不锈钢管的旋转电机收集纤维，制备三层血管支架的内层纤维层。所述旋转电机转速60r/min。In the second step, the spinning solution of polymer A is placed in a syringe pump equipped with a needle with a diameter of 1 mm, and a voltage of 10 kV is applied to perform electrospinning at a working distance of 15 cm. The spinning time is 0.5 h. The rotating motor of the tube collects the fibers to prepare the inner fibrous layer of the three-layer vascular stent. The rotational speed of the rotary motor is 60r/min.
第三步，在内层的基础上，继续制备一层力学性质较好的中间层，将聚合物B的纺丝溶液至于直径1.0mm针头的注射泵中，施加25kV的电压在工作距离为15cm时进行静电纺丝，纺丝时间6h，使用带有不锈钢管的的旋转电机接收纤维，旋转电机转速为60r/min，得到中间层纤维层。In the third step, on the basis of the inner layer, continue to prepare an intermediate layer with better mechanical properties, put the spinning solution of polymer B in a syringe pump with a needle with a diameter of 1.0mm, and apply a voltage of 25kV at a working distance of 15cm Electrospinning was carried out for 6 hours, and a rotating motor with a stainless steel tube was used to receive the fibers, and the rotating speed of the rotating motor was 60 r/min to obtain the middle fiber layer.
第四步，重复第二步，在中间层纤维层表面制备最外层丝素纤维层。The fourth step is to repeat the second step to prepare the outermost silk fibroin fiber layer on the surface of the middle fiber layer.
第五步，把第四步中得到的具有三层纤维膜的不锈钢管放入去离子水浸泡1小时脱管，30℃烘干，得到仿自然血管的三层人造血管支架。In the fifth step, the stainless steel tube with the three-layer fiber membrane obtained in the fourth step is soaked in deionized water for 1 hour to take off the tube, and dried at 30° C. to obtain a three-layer artificial vascular stent imitating a natural blood vessel.
所述的仿自然血管的三层人造血管支架具有三层纤维膜结构，纤维直径为200nm～1000nm。最内层厚度为100μm，中间层厚度为500μm，最外层厚度为100μm。The three-layer artificial vascular stent imitating natural blood vessels has a three-layer fiber membrane structure, and the fiber diameter is 200nm-1000nm. The thickness of the innermost layer was 100 μm, the thickness of the middle layer was 500 μm, and the thickness of the outermost layer was 100 μm.
所述目标三层人造血管支架的纤维管拉伸强度为70MPa，应变为400％，无细胞毒性，并具有良好的生物相容性。The tensile strength of the fiber tube of the target three-layer artificial vascular scaffold is 70 MPa, the strain is 400%, has no cytotoxicity, and has good biocompatibility.
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| CN201611113399.XACN106540327A (en) | 2016-12-06 | 2016-12-06 | A kind of three layers of artificial blood vessel bracket of imitative nature blood vessel and preparation method thereof |
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| CN201611113399.XACN106540327A (en) | 2016-12-06 | 2016-12-06 | A kind of three layers of artificial blood vessel bracket of imitative nature blood vessel and preparation method thereof |
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| CN201611113399.XAPendingCN106540327A (en) | 2016-12-06 | 2016-12-06 | A kind of three layers of artificial blood vessel bracket of imitative nature blood vessel and preparation method thereof |
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