Small-diameter artificial blood vessel with both pro-endothelization and anti-coagulation functions and preparation method thereof

Abstract

The application discloses a small-diameter artificial blood vessel with the functions of promoting endothelialization and resisting coagulation and a preparation method thereof, and belongs to the technical field of biological bionic manufacturing. The method of the application adopts electrostatic spinning technology, prepares a gelatin sacrifice layer through a small-diameter rotating core shaft, prepares oriented fibers through a large-diameter rotating core shaft, and then forms a bionic oriented inner membrane by winding along the axial direction, and then electrostatically spins a C solution on the outer surface of the same core shaft to form a porous middle membrane layer, and after the sacrifice layer is removed, the heparinization is realized through a dopamine-heparin composite solution treatment, so that the technical problems of insufficient endothelialization and poor blood resistance of the small-diameter artificial blood vessel in the prior art are fundamentally overcome.

Description

Technical Field

[0001] This invention belongs to the field of biomimetic manufacturing technology, specifically relating to a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation, and its preparation method. Background Technology

[0002] Cardiovascular disease (CVD) is a group of diseases related to the heart and blood vessels, with coronary artery disease, cerebrovascular disease, and peripheral artery disease being the most prominent. Globally, more than 17 million people die from CVD each year, and this number continues to rise. CVD is widely recognized as the leading cause of death worldwide.

[0003] CVD often leads to vascular stenosis or occlusion, requiring surgical vascular reconstruction. Currently, grafts used clinically for vascular reconstruction include autologous vessels, allogeneic vessels, xenogeneic vessels, and artificial vessels. Among these, autologous vessels are the gold standard in clinical treatment, but their application is limited due to the limited availability of donors. Allogeneic and xenogeneic vessels are prone to immune rejection, further restricting their widespread use. In recent years, artificial vessels have received increasing attention. Artificial vessels made from materials such as polytetrafluoroethylene and polyester have been used clinically as substitutes for medium and large-diameter vessels. However, for small-diameter artificial vessels (less than 6 mm), the patency rate after transplantation is low, mainly due to the following problems: lack of an endothelialization-promoting microenvironment, strong surface bioinertness, and a high risk of thrombosis.

[0004] To fabricate biomimetic small-diameter artificial blood vessels, their structure and material design must closely mimic natural blood vessels. Natural blood vessels have a complex structure, consisting of three layers: the intima, media, and adventitia. The intima is composed of endothelial cells and extracellular matrix, with the cells aligned parallel to the vessel axis. The media contains multiple layers of spindle-shaped smooth muscle cells and extracellular matrix. The adventitia is mainly composed of fibroblasts and extracellular matrix. Studies have shown that the axial alignment of the extracellular matrix in the intima significantly promotes endothelialization after small-diameter artificial blood vessel transplantation, while the media is crucial for maintaining the vessel's mechanical strength, elasticity, and physiological responsiveness. From a functional perspective, the intima and media together constitute the key structural layers for achieving the functions of natural blood vessels: the intima primarily promotes endothelialization and prevents thrombosis, while the media supports the overall vascular structure. Therefore, small-diameter artificial blood vessels that simultaneously integrate the structural and functional characteristics of both the intima and media have become a key research focus in this field.

[0005] Electrospinning is a commonly used technique for fabricating small-diameter artificial blood vessels. It can construct structures with high specific surface area, high porosity, and interconnected fiber networks, thus highly mimicking the topology and composition of the natural vascular extracellular matrix, which is beneficial for promoting cell adhesion, growth, proliferation, and differentiation. However, currently, most small-diameter blood vessels fabricated using electrospinning technology employ disordered porous structures, resulting in insufficient endothelialization and poor anticoagulant properties. Therefore, how to achieve rapid endothelialization of small-diameter artificial blood vessels and improve their anticoagulant performance has become a pressing technical challenge in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a small-diameter artificial blood vessel that combines endothelialization and anticoagulation properties, and its preparation method, in order to solve the technical problems of poor endothelialization and anticoagulation performance in existing methods for preparing small-diameter artificial blood vessels.

[0007] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a method for preparing small-diameter artificial blood vessels that promote endothelialization and have anticoagulant properties, employing electrospinning technology and including the following steps: S1: A small-diameter rotating mandrel is used to receive electrospinning solution A, and a fiber film is formed on the surface of the mandrel as a sacrificial layer; S2: A large-diameter rotating mandrel is used to receive electrospinning solution B to form oriented fibers; S3: The oriented fibers obtained in step S2 are wound axially around the surface of the fiber membrane obtained in step S1 to form an intima layer of small-diameter blood vessels, which is used to promote rapid endothelialization. S4: Electrospinning is performed on the outer surface of the structure obtained in step S3 using solution C to form a porous middle membrane layer. Then, the sacrificial layer is removed to obtain a small-diameter blood vessel. S5: The small-diameter blood vessel obtained in step S4 is immersed in a dopamine-heparin composite solution, and heparinization is achieved by shaking to form a polydopamine-heparin composite coating, thus obtaining the small-diameter artificial blood vessel.

[0008] Further, in S1, the diameter of the small-diameter rotating mandrel is 1mm to 6mm; the A solution is a gelatin solution, and the solvent of the gelatin solution is trifluoroethanol or hexafluoroisopropanol.

[0009] Further, in S1, the conditions for electrospinning solution A are as follows: the concentration of electrospinning solution A is 5%~15% (w / v), the distance between the electrospinning needle tip and the collector is 15cm~20cm, the electrospinning voltage is 15kV~30kV, and the electrospinning speed is 0.5mL / h~2mL / h.

[0010] Further, in S2, the diameter of the large-diameter rotating mandrel is 7cm~10cm; the B solution is a composite material solution of polycaprolactone and gelatin, the mass ratio of polycaprolactone to gelatin is 8:1, and the solvent is trifluoroethanol or hexafluoroisopropanol; the conditions for electrospinning the B solution are: the total concentration of the B solution is 8%~15% (w / v), the distance between the electrospinning needle tip and the collector is 15cm~20cm, the electrospinning voltage is 15kV~25kV, and the electrospinning speed is 0.5mL / h~1.5mL / h.

[0011] Further, in S4, the C solution is a composite solution of polycaprolactone and polyurethane, wherein the mass ratio of polycaprolactone to polyurethane is 90:10 to 50:50, and the solvent is a mixed solvent of N,N-dimethylformamide and tetrahydrofuran, or a mixed solvent of N,N-dimethylformamide and chloroform.

[0012] Furthermore, in S4, the conditions for electrospinning solution C are as follows: the total concentration of solution C is 8%~15% (w / v), the distance between the electrospinning needle tip and the collector is 15cm~20cm, the electrospinning voltage is 15kV~25kV, and the electrospinning speed is 0.5mL / h~1mL / h.

[0013] Furthermore, in S4, the method for removing the sacrificial layer is as follows: the mandrel with the sacrificial layer is placed in water to dissolve the gelatin fiber membrane, thereby detaching the small-diameter blood vessels from the mandrel.

[0014] Further, in S5, the dopamine-heparin complex solution contains dopamine at a concentration of 1.5 mg / mL to 2.5 mg / mL, heparin at a concentration of 1 mg / mL to 3 mg / mL, and Tris-HCl buffer as the solvent.

[0015] Furthermore, in S5, the soaking time is 5h to 24h.

[0016] The present invention also discloses a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation, which is prepared by the above-described preparation method.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for preparing a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation. It employs electrospinning technology, using a small-diameter rotating mandrel to prepare a gelatin sacrificial layer, and then using a large-diameter rotating mandrel to prepare oriented fibers, which are then wound axially to form a biomimetic oriented intima. A porous middle membrane layer is then formed on the outer surface of the same mandrel using electrospinning with a C solution. After removing the sacrificial layer, heparinization is achieved through treatment with a dopamine-heparin composite solution. This method utilizes biomimetic design from both the physical structure and composition of natural blood vessels. The oriented fiber structure and the intima made from the vascular extracellular matrix material can simulate the axial cell arrangement of the natural vascular intima, thus facilitating rapid, complete, and functional endothelial layer formation. This promotes long-term patency of the small-diameter artificial blood vessel and overcomes the problems of poor anticoagulation performance caused by the strong biological inertness and lack of an endothelialization-promoting microenvironment of existing blood vessels. By electrospinning a C solution onto the outer surface of a small-diameter rotating mandrel to form a porous middle membrane, the middle membrane acquires adjustable mechanical properties and a porous structure. This small-diameter artificial blood vessel is less prone to rupture or excessive expansion when subjected to cyclic loads generated by blood pressure in the body, and maintains good structural integrity during tissue regeneration, thus solving the defects of easily collapsing or rupturing small-diameter artificial blood vessels prepared in existing technologies. Finally, a polydopamine-heparin composite coating is formed by soaking in a dopamine-heparin composite solution and then shaking it. The simultaneous polymerization and co-deposition of heparin and dopamine allows for the integrated formation of a stable and long-lasting heparin-modified layer, which promotes endothelial cell growth and inhibits excessive smooth muscle cell proliferation, significantly improving the anticoagulant effect of the small-diameter artificial blood vessel. Combining the synergistic effects of these three levels, this method fundamentally overcomes the technical difficulties of insufficient endothelialization and poor anticoagulant performance in existing small-diameter artificial blood vessels.

[0018] Furthermore, this method solves the technical problem of ensuring that the sacrificial layer can stably adhere to the mandrel and dissolve rapidly in water to facilitate vessel demolding in subsequent operations by limiting the diameter of the small-diameter rotating mandrel to 1mm~6mm, solution A to gelatin solution, and specifically defining the electrospinning conditions (concentration 5%~15%, distance 15~20cm, voltage 15~30kV, speed 0.5~2mL / h). This limitation ensures that the gelatin fiber membrane has appropriate thickness and porosity, which can provide temporary support for the electrospinning of the intima and media, and can completely dissolve in water without leaving harmful substances, thereby achieving non-destructive demolding of small-diameter vessels and avoiding mechanical damage to the vascular structure.

[0019] Furthermore, by limiting the diameter of the large-diameter rotating mandrel to 7cm~10cm, limiting solution B to a composite material of polycaprolactone and gelatin, and limiting the electrospinning conditions (concentration 8%~15%, distance 15~20cm, voltage 15~25kV, speed 0.5~1.5ml / h), the technical problem of how to prepare a biomimetic fiber endomembrane with an axially oriented structure was solved. This limitation allows the oriented fibers to guide the endothelial cells to oriented and proliferate along the blood flow direction after axial winding, thereby achieving rapid and complete formation of a functional endothelial layer and significantly improving the long-term patency rate of small-diameter artificial blood vessels.

[0020] Furthermore, by limiting solution C to a composite material of polycaprolactone and polyurethane, and specifying the mass ratio of the two to be 90:10~50:50, using a mixed solvent of DMF and THF or chloroform, and specifying the electrospinning conditions (concentration 8%~15%, distance 15~20cm, voltage 15~25kV, speed 0.5~1ml / h), the problem of the mechanical properties required for the medial layer of small-diameter artificial blood vessels was solved. In this specification, polycaprolactone provides biodegradability and basic mechanical strength, while polyurethane provides elasticity and flexibility. By adjusting the ratio of the two, the elastic modulus, tensile strength, and elongation at break of the medial layer can be precisely controlled, making the prepared artificial blood vessels less prone to rupture or excessive expansion when subjected to in vivo circulatory loads, and maintaining structural integrity during tissue regeneration.

[0021] Furthermore, by limiting the dopamine concentration to 1.5–2.5 mg / mL, the heparin concentration to 1–3 mg / mL, the solvent to Tris-HCl buffer, and the soaking time to 5–24 h, the technical problem of how to achieve a stable and long-lasting heparinized anticoagulant coating while maintaining the three-dimensional structure and bioactivity of blood vessels was solved. This limitation utilizes the property of dopamine to self-polymerize into polydopamine under weakly alkaline conditions, which can simultaneously co-deposit and fix heparin on the inner and outer surfaces of blood vessels to form a uniform and dense composite coating. Compared with physical adsorption or chemical cross-linking methods, this coating has a stronger bond and a more lasting heparin release. It can effectively inhibit platelet adhesion and thrombus formation, promote endothelial cell growth, and avoid adverse reactions caused by excessively high heparin concentrations.

[0022] Furthermore, by defining the method of removing the sacrificial layer as dissolving the gelatin fiber membrane by immersing the mandrel with the sacrificial layer in water, the technical problem of how to completely peel the blood vessel from the mandrel without damaging the oriented fiber structure of the intima and the porous structure of the media was solved. This method utilizes the excellent warm water solubility of gelatin (37℃-45℃), avoiding the tearing or deformation of the fragile oriented fiber intima caused by traditional mechanical peeling methods. At the same time, the water dissolution process is mild and non-toxic, leaving no organic solvent residues or causing material degradation. This ensures the accuracy of the fiber orientation of the intima layer of the small-diameter artificial blood vessel and the integrity of the porous structure of the media, providing an ideal substrate for the subsequent heparinization coating. Attached Figure Description

[0023] Figure 1 This is a flowchart of the preparation method of the small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation according to the present invention; Figure 2 This is a morphology diagram of the porous membrane layer prepared in Example 1 of the present invention; Figure 3 This is a morphology diagram of the oriented fibers in the inner membrane layer prepared in Example 1 of the present invention; Figure 4 This is an image of a small-diameter artificial blood vessel prepared in Example 1 of the present invention, after the intima and media layers are combined. Detailed Implementation

[0024] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0025] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0026] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0027] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0028] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0029] like Figure 1 As shown, this invention provides a method for preparing small-diameter artificial blood vessels that combine endothelialization promotion and anticoagulation, comprising the following steps: S1: A fiber membrane formed by electrospinning solution A is received by a small-diameter rotating mandrel as a sacrificial layer so that it can be removed from the mandrel after the small-diameter blood vessels are prepared; S2: Replace the large-diameter rotating mandrel to receive the oriented fibers formed by electrospinning solution B; S3: The oriented fibers prepared in S2 are wound axially onto the surface of the fiber membrane prepared in S1 to serve as the intima of small-diameter blood vessels responsible for rapid intimal endothelialization. S4: Electrospinning solution C on the outer surface of the small-diameter rotating mandrel in S3 to obtain a porous membrane for small-diameter blood vessels. The entire mandrel is then placed in water to dissolve the fibrous membrane in S1 to obtain small-diameter blood vessels. S5: The small-diameter blood vessels obtained in S4 are immersed in a dopamine-heparin composite solution and placed on a shaker to form a polydopamine-heparin composite coating, thereby achieving heparinization of the small-diameter blood vessels and obtaining small-diameter artificial blood vessels that have both endothelialization and anticoagulation properties.

[0030] Preferably, in step S1, the diameter of the small-diameter rotating mandrel is 1mm to 6mm; the A solution is gelatin, wherein the solvent is trifluoroethanol or hexafluoroisopropanol. The electrospinning conditions for the A solution are: A solution concentration 5% to 15% (w / v), distance between the electrospinning needle tip and the collector 15cm to 20cm, electrospinning voltage 15kV to 30kV, and electrospinning speed 0.5mL / h to 2mL / h.

[0031] Preferably, the diameter of the large-diameter rotating mandrel in step S2 is 7cm to 10cm. The B solution is a composite material formed from polycaprolactone and gelatin, wherein the solvent is trifluoroethanol or hexafluoroisopropanol. The electrospinning conditions for the B solution are: total concentration of B solution 8% to 15% (w / v), distance between the electrospinning needle tip and the collector 15cm to 20cm, electrospinning voltage 15kV to 25kV, and electrospinning speed 0.5mL / h to 1.5mL / h.

[0032] Preferably, the C solution in step S4 is a composite material formed by polycaprolactone and polyurethane, wherein the ratio of polycaprolactone to polyurethane is 90:10 to 50:50, and the solvent is a mixed solvent formed by N,N-dimethylformamide and tetrahydrofuran or chloroform. The electrospinning conditions for the C solution are: total C solution concentration 8% to 15% (w / v), distance between the electrospinning needle tip and the collector 15cm to 20cm, electrospinning voltage 15kV to 25kV, and electrospinning speed 0.5mL / h to 1mL / h.

[0033] Preferably, in step S5, the dopamine concentration is 1.5 mg / mL to 2.5 mg / mL, the heparin concentration is 1 mg / mL to 3 mg / mL, the solvent of the composite solution is Tris-HCl buffer, and the soaking time is 5 h to 24 h.

[0034] This invention employs biomimetic design based on both the physical structure and composition of natural blood vessels. The intima of this invention, featuring an oriented fiber structure and extracellular matrix material mimicking blood vessels, facilitates rapid, complete, and functional endothelial formation, thereby promoting long-term patency of small-diameter artificial blood vessels.

[0035] Furthermore, the medial membrane possesses adjustable mechanical properties and a porous structure. This small-diameter artificial blood vessel is less prone to rupture or excessive expansion when subjected to cyclic loads generated by blood pressure within the body, and it can maintain good structural integrity during tissue regeneration.

[0036] Furthermore, a polydopamine-heparin composite coating is integrally formed by simultaneously polymerizing and co-depositing heparin and dopamine solutions. This composite coating enables small-diameter artificial blood vessels to efficiently obtain a stable and long-lasting heparin-modified layer, which can both promote the growth of endothelial cells and inhibit the excessive proliferation of smooth muscle cells, thereby significantly improving the anticoagulation effect of small-diameter artificial blood vessels.

[0037] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0038] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0039] Example 1 A method for preparing a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation includes the following steps: S1: A fiber membrane formed by electrospun gelatin solution is received by a rotating mandrel with a diameter of 2 mm, serving as a sacrificial layer to facilitate removal from the mandrel after the preparation of small-diameter blood vessels; wherein, the solvent of the gelatin solution is trifluoroethanol, the concentration of the gelatin solution is 10% (w / v), the distance between the electrospinning needle tip and the collector is 18 cm, the electrospinning voltage is 25 kV, and the electrospinning speed is 1 mL / h; S2: Replace the 8cm diameter rotating mandrel to receive the oriented fibers formed by electrospinning polycaprolactone and gelatin composite solution; wherein, the solvent of polycaprolactone and gelatin composite solution is trifluoroethanol, the solution concentration is 10% (w / v), the mass ratio of polycaprolactone to gelatin is 8:2, the distance between the electrospinning needle tip and the collector is 17cm, the electrospinning voltage is 16kV, and the electrospinning speed is 1mL / h; S3: The oriented fibers prepared in S2 are wound axially onto the surface of the fiber membrane prepared in S1 to serve as the intima of small-diameter blood vessels responsible for rapid intimal endothelialization. S4: Electrospinning a composite material of polycaprolactone and polyurethane on the outer surface of the small-diameter rotating mandrel in S3 yields a porous membrane for small-diameter blood vessels. The entire mandrel is then removed and placed in water to dissolve the fiber membrane from S1, thus obtaining small-diameter blood vessels. The solvent for the polycaprolactone and polyurethane composite solution is a mixture of N,N-dimethylformamide and tetrahydrofuran, with a solution concentration of 10% (w / v). The mass ratio of polycaprolactone to polyurethane is 90:10. The distance between the electrospinning needle tip and the collector is 17 cm. The electrospinning voltage is 20 kV, and the electrospinning speed is 0.7 mL / h. S5: The small-diameter blood vessels obtained in S4 are immersed in a dopamine-heparin composite solution and placed on a shaker to form a polydopamine-heparin composite coating, thereby achieving heparinization of the small-diameter blood vessels and obtaining small-diameter artificial blood vessels that have both endothelialization and anticoagulation properties; wherein, the concentration of dopamine is 2 mg / mL, the concentration of heparin is 1.5 mg / mL, the solvent in the dopamine-heparin composite solution is Tris-HCl buffer, and the immersion time is 24 h.

[0040] Example 2 A method for preparing a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation includes the following steps: S1: A fiber membrane formed by electrospun gelatin solution is received by a rotating mandrel with a diameter of 2 mm, serving as a sacrificial layer to facilitate removal from the mandrel after the preparation of small-diameter blood vessels; wherein, the solvent of the gelatin solution is trifluoroethanol, the concentration of the gelatin solution is 10% (w / v), the distance between the electrospinning needle tip and the collector is 18 cm, the electrospinning voltage is 25 kV, and the electrospinning speed is 1 mL / h; S2: Replace the 8cm diameter rotating mandrel to receive the oriented fibers formed by electrospinning polycaprolactone and gelatin composite solution; wherein, the solvent of polycaprolactone and gelatin composite solution is trifluoroethanol, the solution concentration is 10% (w / v), the mass ratio of polycaprolactone to gelatin is 8:2, the distance between the electrospinning needle tip and the collector is 17cm, the electrospinning voltage is 18kV, and the electrospinning speed is 1mL / h; S3: The oriented fibers prepared in S2 are wound axially onto the surface of the fiber membrane prepared in S1 to serve as the intima of small-diameter blood vessels responsible for rapid intimal endothelialization. S4: Electrospinning a composite material of polycaprolactone and polyurethane on the outer surface of the small-diameter rotating mandrel in S3 yields a porous membrane for small-diameter blood vessels. The entire mandrel is then removed and placed in water to dissolve the fiber membrane from S1, thus obtaining small-diameter blood vessels. The solvent for the polycaprolactone and polyurethane composite solution is a mixture of N,N-dimethylformamide and tetrahydrofuran, with a solution concentration of 10% (w / v). The mass ratio of polycaprolactone to polyurethane is 90:10. The distance between the electrospinning needle tip and the collector is 17 cm. The electrospinning voltage is 20 kV, and the electrospinning speed is 0.7 mL / h. S5: The small-diameter blood vessels obtained in S4 are immersed in a dopamine-heparin composite solution and placed on a shaker to form a polydopamine-heparin composite coating, thereby achieving heparinization of the small-diameter blood vessels and obtaining small-diameter artificial blood vessels that have both endothelialization and anticoagulation properties; wherein, the concentration of dopamine is 2 mg / mL, the concentration of heparin is 1.5 mg / mL, the solvent in the dopamine-heparin composite solution is Tris-HCl buffer, and the immersion time is 24 h.

[0041] Example 3 A method for preparing a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation includes the following steps: S1: A fiber membrane formed by electrospun gelatin solution is received by a rotating mandrel with a diameter of 2 mm, serving as a sacrificial layer to facilitate removal from the mandrel after the preparation of small-diameter blood vessels; wherein, the solvent of the gelatin solution is trifluoroethanol, the concentration of the gelatin solution is 10% (w / v), the distance between the electrospinning needle tip and the collector is 18 cm, the electrospinning voltage is 25 kV, and the electrospinning speed is 1 mL / h; S2: Replace the 8cm diameter rotating mandrel to receive the oriented fibers formed by electrospinning a polycaprolactone and gelatin composite solution; wherein, the solvent of the polycaprolactone and gelatin composite solution is trifluoroethanol, the solution concentration is 10% (w / v), the mass ratio of polycaprolactone to gelatin is 8:2, the distance between the electrospinning needle tip and the collector is 17cm, the electrospinning voltage is 18kV, and the electrospinning speed is 1.5mL / h; S3: The oriented fibers prepared in S2 are wound axially onto the surface of the fiber membrane prepared in S1 to serve as the intima of small-diameter blood vessels responsible for rapid intimal endothelialization. S4: Electrospinning a composite material of polycaprolactone and polyurethane on the outer surface of the small-diameter rotating mandrel in S3 yields a porous membrane for small-diameter blood vessels. The entire mandrel is then removed and placed in water to dissolve the fiber membrane from S1, thus obtaining small-diameter blood vessels. The solvent for the polycaprolactone and polyurethane composite solution is a mixture of N,N-dimethylformamide and tetrahydrofuran, with a solution concentration of 10% (w / v). The mass ratio of polycaprolactone to polyurethane is 90:10. The distance between the electrospinning needle tip and the collector is 17 cm. The electrospinning voltage is 20 kV, and the electrospinning speed is 0.7 mL / h. S5: The small-diameter blood vessels obtained in S4 are immersed in a dopamine-heparin composite solution and placed on a shaker to form a polydopamine-heparin composite coating, thereby achieving heparinization of the small-diameter blood vessels and obtaining small-diameter artificial blood vessels that have both endothelialization and anticoagulation properties; wherein, the concentration of dopamine is 2 mg / mL, the concentration of heparin is 1.5 mg / mL, the solvent in the dopamine-heparin composite solution is Tris-HCl buffer, and the immersion time is 24 h.

[0042] Example 4 A method for preparing a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation includes the following steps: S1: A fiber membrane formed by electrospun gelatin solution is received by a rotating mandrel with a diameter of 2 mm, serving as a sacrificial layer to facilitate removal from the mandrel after the preparation of small-diameter blood vessels; wherein, the solvent of the gelatin solution is trifluoroethanol, the concentration of the gelatin solution is 10% (w / v), the distance between the electrospinning needle tip and the collector is 18 cm, the electrospinning voltage is 25 kV, and the electrospinning speed is 1 mL / h; S2: Replace the 8cm diameter rotating mandrel to receive the oriented fibers formed by electrospinning polycaprolactone and gelatin composite solution; wherein, the solvent of polycaprolactone and gelatin composite solution is trifluoroethanol, the solution concentration is 10% (w / v), the mass ratio of polycaprolactone to gelatin is 8:2, the distance between the electrospinning needle tip and the collector is 17cm, the electrospinning voltage is 18kV, and the electrospinning speed is 1mL / h; S3: The oriented fibers prepared in S2 are wound axially onto the surface of the fiber membrane prepared in S1 to serve as the intima of small-diameter blood vessels responsible for rapid intimal endothelialization. S4: Electrospinning a composite material of polycaprolactone and polyurethane on the outer surface of the small-diameter rotating mandrel in S3 yields a porous membrane for small-diameter blood vessels. The entire mandrel is then removed and placed in water to dissolve the fiber membrane from S1, thus obtaining small-diameter blood vessels. The solvent for the polycaprolactone and polyurethane composite solution is a mixture of N,N-dimethylformamide and tetrahydrofuran, with a solution concentration of 10% (w / v). The mass ratio of polycaprolactone to polyurethane is 75:25. The distance between the electrospinning needle tip and the collector is 17 cm. The electrospinning voltage is 20 kV, and the electrospinning speed is 0.7 mL / h. S5: The small-diameter blood vessels obtained in S4 are immersed in a dopamine-heparin composite solution and placed on a shaker to form a polydopamine-heparin composite coating, thereby achieving heparinization of the small-diameter blood vessels and obtaining small-diameter artificial blood vessels that have both endothelialization and anticoagulation properties; wherein, the concentration of dopamine is 2 mg / mL, the concentration of heparin is 1.5 mg / mL, the solvent in the dopamine-heparin composite solution is Tris-HCl buffer, and the immersion time is 24 h.

[0043] Example 5 A method for preparing a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation includes the following steps: S1: A fiber membrane formed by electrospun gelatin solution is received by a rotating mandrel with a diameter of 6 mm, serving as a sacrificial layer to facilitate removal from the mandrel after the preparation of small-diameter blood vessels; wherein, the solvent of the gelatin solution is trifluoroethanol, the gelatin solution concentration is 15% (w / v), the distance between the electrospinning needle tip and the collector is 15 cm, the electrospinning voltage is 30 kV, and the electrospinning speed is 2 mL / h; S2: Replace the 10cm diameter rotating mandrel to receive the oriented fibers formed by electrospinning a polycaprolactone and gelatin composite solution; wherein, the solvent of the polycaprolactone and gelatin composite solution is trifluoroethanol, the solution concentration is 15% (w / v), the mass ratio of polycaprolactone to gelatin is 8:2, the distance between the electrospinning needle tip and the collector is 15cm, the electrospinning voltage is 25kV, and the electrospinning speed is 0.5mL / h; S3: The oriented fibers prepared in S2 are wound axially onto the surface of the fiber membrane prepared in S1 to serve as the intima of small-diameter blood vessels responsible for rapid intimal endothelialization. S4: Electrospinning a composite material of polycaprolactone and polyurethane on the outer surface of the small-diameter rotating mandrel in S3 yields a porous membrane for small-diameter blood vessels. The entire mandrel is then removed and placed in water to dissolve the fiber membrane from S1, thus obtaining small-diameter blood vessels. The solvent for the polycaprolactone and polyurethane composite solution is a mixture of N,N-dimethylformamide and tetrahydrofuran, with a solution concentration of 15% (w / v). The mass ratio of polycaprolactone to polyurethane is 50:50. The distance between the electrospinning needle tip and the collector is 20 cm. The electrospinning voltage is 25 kV, and the electrospinning speed is 1 mL / h. S5: The small-diameter blood vessels obtained in S4 are immersed in a dopamine-heparin composite solution and placed on a shaker to form a polydopamine-heparin composite coating, thereby achieving heparinization of the small-diameter blood vessels and obtaining small-diameter artificial blood vessels that have both endothelialization and anticoagulation properties; wherein, the concentration of dopamine is 2.5 mg / mL, the concentration of heparin is 3 mg / mL, the solvent in the dopamine-heparin composite solution is Tris-HCl buffer, and the immersion time is 5 h.

[0044] Example 6 A method for preparing a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation includes the following steps: S1: A fiber membrane formed by electrospun gelatin solution is received by a rotating mandrel with a diameter of 1 mm, serving as a sacrificial layer to facilitate removal from the mandrel after the preparation of small-diameter blood vessels; wherein, the solvent of the gelatin solution is trifluoroethanol, the gelatin solution concentration is 5% (w / v), the distance between the electrospinning needle tip and the collector is 20 cm, the electrospinning voltage is 15 kV, and the electrospinning speed is 0.5 mL / h; S2: Replace the 7cm diameter rotating mandrel to receive the oriented fibers formed by electrospinning polycaprolactone and gelatin composite solution; wherein, the solvent of polycaprolactone and gelatin composite solution is trifluoroethanol, the solution concentration is 8% (w / v), the mass ratio of polycaprolactone to gelatin is 8:2, the distance between the electrospinning needle tip and the collector is 20cm, the electrospinning voltage is 15kV, and the electrospinning speed is 1mL / h; S3: The oriented fibers prepared in S2 are wound axially onto the surface of the fiber membrane prepared in S1 to serve as the intima of small-diameter blood vessels responsible for rapid intimal endothelialization. S4: Electrospinning a composite material of polycaprolactone and polyurethane on the outer surface of the small-diameter rotating mandrel in S3 yields a porous membrane for small-diameter blood vessels. The entire mandrel is then removed and placed in water to dissolve the fiber membrane from S1, thus obtaining small-diameter blood vessels. The solvent for the polycaprolactone and polyurethane composite solution is a mixture of N,N-dimethylformamide and tetrahydrofuran, with a solution concentration of 8% (w / v). The mass ratio of polycaprolactone to polyurethane is 90:10. The distance between the electrospinning needle tip and the collector is 15 cm. The electrospinning voltage is 15 kV, and the electrospinning speed is 0.5 mL / h. S5: The small-diameter blood vessels obtained in S4 are immersed in a dopamine-heparin composite solution and placed on a shaker to form a polydopamine-heparin composite coating, thereby achieving heparinization of the small-diameter blood vessels and obtaining small-diameter artificial blood vessels that have both endothelialization and anticoagulation properties; wherein, the concentration of dopamine is 1.5 mg / mL, the concentration of heparin is 1 mg / mL, the solvent in the dopamine-heparin composite solution is Tris-HCl buffer, and the immersion time is 12 h.

[0045] Figure 2 The image shows the morphology of the porous membrane layer prepared in Example 1 of the present invention. As can be seen from the image, the prepared electrospun fibers are uniform, defect-free, interwoven and exhibit a porous structure, which is beneficial to the growth and proliferation of smooth muscle cells. Figure 3 This is a morphology diagram of the oriented fibers of the inner membrane layer prepared in Example 1 of the present invention. It can be seen from the figure that the prepared electrospun fibers have good orientation, which is beneficial to the growth, directional arrangement, proliferation and endothelialization of endothelial cells. Figure 4The image shows a small-diameter artificial blood vessel prepared in Example 1 of this invention after the intima and media layers are combined. As can be seen from the image, the appearance is defect-free and it can be held with surgical forceps, which is beneficial for subsequent clinical operations.

[0046] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing a small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation, characterized in that, The electrospinning technology includes the following steps: S1: A small-diameter rotating mandrel is used to receive electrospinning solution A, and a fiber film is formed on the surface of the mandrel as a sacrificial layer; S2: A large-diameter rotating mandrel is used to receive electrospinning solution B to form oriented fibers; S3: The oriented fibers obtained in step S2 are wound axially around the surface of the fiber membrane obtained in step S1 to form an intima layer of small-diameter blood vessels, which is used to promote rapid endothelialization. S4: Electrospinning is performed on the outer surface of the structure obtained in step S3 using solution C to form a porous middle membrane layer. Then, the sacrificial layer is removed to obtain a small-diameter blood vessel. S5: The small-diameter blood vessel obtained in step S4 is immersed in a dopamine-heparin composite solution, and heparinization is achieved by shaking to form a polydopamine-heparin composite coating, thus obtaining the small-diameter artificial blood vessel.

2. The method for preparing a small-diameter artificial blood vessel with both endothelialization-promoting and anticoagulant properties according to claim 1, characterized in that, In S1, the diameter of the small-diameter rotating mandrel is 1mm to 6mm; the A solution is a gelatin solution, and the solvent of the gelatin solution is trifluoroethanol or hexafluoroisopropanol.

3. The method for preparing a small-diameter artificial blood vessel with both endothelialization-promoting and anticoagulant properties according to claim 1, characterized in that, In S1, the conditions for electrospinning solution A are as follows: the concentration of electrospinning solution A is 5%~15% (w / v), the distance between the electrospinning needle tip and the collector is 15cm~20cm, the electrospinning voltage is 15kV~30kV, and the electrospinning speed is 0.5mL / h~2mL / h.

4. The method for preparing a small-diameter artificial blood vessel with both endothelialization-promoting and anticoagulant properties according to claim 1, characterized in that, In S2, the diameter of the large-diameter rotating mandrel is 7cm~10cm; the B solution is a composite material solution of polycaprolactone and gelatin, the mass ratio of polycaprolactone to gelatin is 8:1, and the solvent is trifluoroethanol or hexafluoroisopropanol; the conditions for electrospinning the B solution are: the total concentration of the B solution is 8%~15% (w / v), the distance between the electrospinning needle tip and the collector is 15cm~20cm, the electrospinning voltage is 15kV~25kV, and the electrospinning speed is 0.5mL / h~1.5mL / h.

5. The method for preparing a small-diameter artificial blood vessel with both endothelialization-promoting and anticoagulant properties according to claim 1, characterized in that, In S4, solution C is a composite material solution of polycaprolactone and polyurethane, wherein the mass ratio of polycaprolactone to polyurethane is 90:10 to 50:50, and the solvent is a mixed solvent of N,N-dimethylformamide and tetrahydrofuran, or a mixed solvent of N,N-dimethylformamide and chloroform.

6. The method for preparing a small-diameter artificial blood vessel with both endothelialization-promoting and anticoagulant properties according to claim 1, characterized in that, In S4, the conditions for electrospinning solution C are: the total concentration of solution C is 8%~15% (w / v), the distance between the electrospinning needle tip and the collector is 15cm~20cm, the electrospinning voltage is 15kV~25kV, and the electrospinning speed is 0.5mL / h~1mL / h.

7. The method for preparing a small-diameter artificial blood vessel with both endothelialization-promoting and anticoagulant properties according to claim 1, characterized in that, In S4, the method for removing the sacrificial layer is as follows: the mandrel with the sacrificial layer is placed in water to dissolve the gelatin fiber membrane, thereby detaching the small-diameter blood vessels from the mandrel.

8. The method for preparing a small-diameter artificial blood vessel with both endothelialization-promoting and anticoagulant properties according to claim 1, characterized in that, In S5, the dopamine-heparin complex solution contains dopamine at a concentration of 1.5 mg / mL to 2.5 mg / mL, heparin at a concentration of 1 mg / mL to 3 mg / mL, and Tris-HCl buffer as the solvent.

9. The method for preparing a small-diameter artificial blood vessel with both endothelialization-promoting and anticoagulant properties according to claim 1, characterized in that, In S5, the soaking time is 5h~24h.

10. A small-diameter artificial blood vessel that combines endothelialization promotion and anticoagulation, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 9.

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