Preparation method of silk conical artificial blood vessel with anticoagulation / endothelialization promotion function

Abstract

The invention discloses a preparation method of a silk conical artificial blood vessel with anticoagulation / endothelialization promotion functions, which comprises the following steps: preparing an artificial blood vessel for intracavity isolation through silk and polyester yarns, and successfully constructing a phosphorylcholine / heparin bionic functional coating on the surface of the blood vessel by using dopamine hydrochloride and hexamethylenediamine. According to the method, phosphorylcholine / heparin is combined with surface coating modification, and a novel intracavitary isolated blood vessel with excellent biological functions and mechanical properties is constructed. The structure of the artificial blood vessel is more matched with the shape of the inner wall of the blood vessel, blood flow distribution and tissue regeneration are facilitated, the anticoagulant property and endothelialization ability of the artificial blood vessel are effectively improved through the phosphorylcholine / heparin coating, protein adsorption and platelet activation can be remarkably reduced, the blood coagulation cascade reaction can be blocked, endothelial cell adhesion and proliferation can be accelerated, and the risk of acute thrombus after operation can be reduced. The method provides a new technical path for design and functionalization of the conical artificial blood vessel in the intracavitary isolation operation, and has important clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials and relates to a method for preparing a silk tapered artificial blood vessel with anti-coagulation / endothelialization promoting functions. Background Art

[0002] Abdominal aortic aneurysm (AAS) is a common vascular disease characterized by a permanent, localized dilation of the abdominal aorta. While typically asymptomatic in the early stages, the aneurysm increases in size and the risk of rupture increases significantly as the disease progresses. Traditional treatment primarily involves revascularization, which involves resecting the diseased segment and implanting a graft or autologous blood vessel to restore vascular function. While this open procedure offers intuitive access to the lesion, it involves extensive tissue dissection, which can easily cause secondary trauma and requires rigorous microsuturing techniques. Furthermore, postoperative mismatched compliance of the anastomotic sites can lead to complications such as vascular embolism, lumen thickening, and atherosclerosis at the distal end of the vessel, compromising the long-term patency of the graft. Traditional AAS carries increased surgical risks, particularly in the elderly with multiple underlying medical conditions. In contrast, endovascular isolation involves a catheter-based approach, where a graft is delivered through the femoral artery to the affected area. It is then expanded and secured with a metal stent, isolating blood flow and repairing the lesion. This minimally invasive procedure effectively occludes the aneurysm while preserving the integrity of the surrounding tissue to the greatest extent possible. Its clinical advantages of "less trauma and faster recovery" significantly reduce the risk of postoperative complications. It is particularly suitable for elderly patients and significantly improves the treatment experience and long-term prognosis.

[0003] Currently, research on artificial blood vessels for endovascular isolation has largely focused on straight and round types. However, the human abdominal aorta physiologically has a taper of approximately 1 to 3 degrees, making it difficult for traditional artificial blood vessels to fully conform to the native vessels, potentially leading to poor blood flow and limited graft function. Furthermore, the axial velocity distribution of blood in conical and straight vessels differs significantly. The conical structure is more effective in regulating hemodynamics and altering shear stress, which is beneficial to the health and function of endothelial cells. Compared to straight and round artificial blood vessels, conical artificial blood vessels not only have better compliance and deformation resistance, but are also more adaptable to natural vascular deformation, improving stent stability and reducing complications. Therefore, research on conical artificial blood vessels is of great significance for better simulating the real vascular environment, optimizing the effectiveness of endovascular isolation treatment, and improving clinical efficacy. Furthermore, research on the functionalization of artificial blood vessels has yielded several advances. For example, artificial blood vessels coated with dopamine hydrochloride and heparin have demonstrated excellent cell affinity and anticoagulant properties, respectively. However, research on conical artificial blood vessels with anticoagulant and endothelial-promoting properties remains relatively scarce. The conical structure not only brings geometric and fluid dynamic complexity, but its surface functionalization also needs to take into account long-term anticoagulation and the regulation of endothelial cell adhesion, migration, and proliferation. The preparation of artificial blood vessels with these complex functions faces great challenges. In the early stage of artificial blood vessel implantation, due to the lack of a complete endothelial layer on the surface of the material, anticoagulant factors cannot be secreted. The vascular material is directly exposed to the blood environment and easily adsorbs plasma proteins, activating the coagulation cascade, which in turn leads to platelet aggregation and fibrin deposition, forming thrombi. These thrombi can cause stenosis or even occlusion of the lumen, hindering blood flow and causing serious complications such as ischemia and necrosis. In the later stage of implantation, if the endothelium is not fully repaired, it may also be accompanied by abnormal proliferation of smooth muscle cells, inducing intimal hyperplasia and restenosis. It is worth noting that endothelial cells can inhibit excessive smooth muscle proliferation. Therefore, achieving rapid and complete endothelialization is key to ensuring the long-term patency and stability of artificial blood vessels.

[0004] Silk-based artificial blood vessels offer unique advantages over traditional synthetic materials. As a naturally renewable material, silk possesses excellent mechanical properties and tunable degradability. Rich in fibroin, silk promotes cell-specific adhesion and accelerates endothelialization, while inhibiting platelet adhesion and reducing the risk of thrombosis. Phosphorylcholine, a zwitterionic polymer, exhibits excellent hydration capacity due to its unique molecular structure, forming a dense hydration layer on the material surface, thereby strongly repelling nonspecific protein adsorption. The phosphorylcholine group mimics the structure of natural cell membranes, creating a biomimetic interface on the material surface. This significantly reduces plasma protein adsorption, inhibits platelet adhesion and activation, and enhances the material's anticoagulant properties. Furthermore, heparin, a biomolecule from the glycosaminoglycan family, is widely used as an anticoagulant in vascular therapy and effectively prevents acute thrombosis. By combining heparin with the material, excellent antithrombotic properties can be achieved, including reducing coagulation, complement, and blood cell activation, and inhibiting the adhesion of plasma to platelets and proteins involved in the coagulation cascade.

[0005] Therefore, it is of great significance to develop a silk tapered artificial blood vessel with anticoagulant / endothelialization promoting function. Summary of the Invention

[0006] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a silk tapered artificial blood vessel with anticoagulant / endothelialization promoting functions.

[0009] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing a silk tapered artificial blood vessel with anticoagulant / endothelialization promoting function, characterized by comprising:

[0010] Using silk and polyester as raw materials, an artificial blood vessel for intracavitary isolation is prepared through an integrated weaving process;

[0011] cleaning, degumming, and heat-setting the intraluminal isolation artificial blood vessel to obtain an artificial blood vessel sample;

[0012] adding a dopamine hydrochloride solution and a hexamethylenediamine solution into a Tris buffer solution to obtain a first mixed solution, immersing the artificial blood vessel sample in the first mixed solution, then removing the artificial blood vessel sample, washing, and drying the artificial blood vessel sample to obtain an ammonia-rich coating sample blood vessel sample;

[0013] Heparin sodium and poly (2-methacryloyloxyethyl phosphorylcholine) were dissolved in Tris buffer in sequence to obtain a second mixed solution. The ammonia-rich coated sample blood vessel sample was immersed in the second mixed solution. The ammonia-rich coated sample blood vessel sample was then removed, cleaned, and dried to obtain a silk cone-shaped artificial blood vessel with anticoagulant / endothelialization promoting function.

[0014] As a preferred embodiment of the preparation method of the present invention, the yarn fineness of the silk and polyester is 3.3 tex, and the plurality is 1 to 12f; the upper weave is any one or more combinations of plain weave, twill, satin weave, double plain weave, honeycomb weave, crepe weave or small jacquard weave; the warp density at the big end is 1220 to 2260 strands / (10 cm), the weft density at the big end is 1100 to 1600 strands / (10 cm), the warp density at the small end is 1860 to 2780 strands / (10 cm), and the weft density at the small end is 440 to 800 strands / (10 cm).

[0015] As a preferred embodiment of the preparation method of the present invention, the intracavitary isolation artificial blood vessel is cleaned, degummed, and heat-set, wherein the surface cleaning agent used for cleaning is a citric acid solution or an oxalic acid solution, the mass fraction of the surface cleaning agent is 1% to 5%, the cleaning time is 30 to 60 minutes, and the temperature is 70 to 80°C.

[0016] As a preferred embodiment of the preparation method of the present invention, the degumming agent used for degumming is a Na2CO3 solution, the mass fraction of the degumming agent is 0.01% to 0.05%, the degumming time is 30 to 60 minutes, and the degumming temperature is 80 to 100°C.

[0017] As a preferred embodiment of the preparation method of the present invention, the heat setting temperature is 100-150° C. and the time is 30-60 minutes.

[0018] As a preferred embodiment of the preparation method of the present invention, the concentration of the dopamine hydrochloride solution is 1-3 mg / mL, the concentration of the hexamethylenediamine solution is 0.5-2 mg / mL, the concentration of the Tris buffer is 1-3 mg / mL, and the immersion time is 12-48 hours.

[0019] As a preferred embodiment of the preparation method of the present invention, the cleaning and drying are performed to obtain an ammonia-rich coating sample blood vessel specimen, wherein the cleaning method is ultrasonic cleaning and the drying method is nitrogen drying.

[0020] As a preferred embodiment of the preparation method of the present invention, the concentration of heparin sodium is 1 to 3 mg / mL, the concentration of poly(2-methacryloyloxyethyl phosphorylcholine) is 1 to 3 mg / mL, the concentration of Tris buffer is 1 to 3 mg / mL, and the immersion time is 24 to 36 hours.

[0021] As a preferred embodiment of the preparation method of the present invention, the cleaning and drying are performed to obtain a silk conical artificial blood vessel with anticoagulant / endothelialization promoting function, wherein the cleaning method is ultrasonic cleaning with deionized water and the drying method is nitrogen drying.

[0022] Another object of the present invention is to overcome the deficiencies in the prior art and provide a silk tapered artificial blood vessel with anti-coagulation / endothelialization promoting functions.

[0023] Beneficial effects of the present invention:

[0024] (1) The prepared silk conical intracavitary isolation artificial blood vessels can better fit the inner wall of the blood vessel, improve sealing and stability, and meet the requirements of intracavitary therapy for vascular diseases;

[0025] (2) By degumming the silk vessels, sericin is effectively removed, significantly reducing the post-transplant inflammatory response and improving biosafety;

[0026] (3) Using the co-deposition technology of dopamine hydrochloride and hexamethylenediamine, an amino-rich coating is constructed on the surface of blood vessels, which significantly reduces the water contact angle and improves the hydrophilicity and wettability of the blood vessel surface;

[0027] (4) Using an amino coating prepared with dopamine hydrochloride / hexamethylenediamine, heparin / phosphorylcholine is covalently grafted onto the surface of blood vessels, giving the surface of blood vessels excellent anticoagulant and endothelialization functions, which can significantly reduce the adhesion of proteins and platelets, inhibit thrombosis, and promote endothelial cell proliferation and spreading, accelerate vascular endothelialization, and help maintain long-term vascular patency. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0029] Figure 1 This is a photo of a silk tapered artificial blood vessel with anticoagulant / endothelialization promoting function produced by the method for producing the same according to the present invention;

[0030] Figure 2 This is a comparison chart of the radial and longitudinal tensile strengths of the silk conical artificial blood vessel with anticoagulant / endothelialization-promoting functions prepared in Example 1 of the method for preparing the silk conical artificial blood vessel with anticoagulant / endothelialization-promoting functions of the present invention and commercial tubular artificial blood vessels;

[0031] Figure 3 This is a graph showing the change in water contact angle on the surface of the silk conical artificial blood vessel with anticoagulant / endothelialization promoting function prepared in Example 1 of the method for preparing the silk conical artificial blood vessel with anticoagulant / endothelialization promoting function of the present invention before and after coating with dopamine hydrochloride / hexamethylenediamine and heparin / phosphorylcholine;

[0032] Figure 4 This is a comparison chart of the fabric thrombin time of the silk tapered artificial blood vessel with anticoagulant / endothelialization function prepared in Example 1 of the method for preparing the silk tapered artificial blood vessel with anticoagulant / endothelialization function of the present invention before and after heparin / phosphorylcholine coating;

[0033] Figure 5 This is a graph showing the cell viability of human umbilical vein endothelial cells in a sample extract before and after dopamine hydrochloride / hexamethylenediamine coating and heparin / phosphorylcholine coating in Example 1 of the method for preparing a silk tapered artificial blood vessel with anticoagulant / endothelialization promoting function of the present invention;

[0034] Figure 6 This is a graph showing changes in radial tensile strength and probe burst strength of the silk conical artificial blood vessel with anticoagulant / endothelialization-promoting function prepared in Example 2 of the method for preparing the silk conical artificial blood vessel with anticoagulant / endothelialization-promoting function of the present invention before and after coating with dopamine hydrochloride / hexamethylenediamine and heparin / phosphorylcholine;

[0035] Figure 7The present invention provides a method for preparing a silk conical artificial blood vessel with anticoagulant / endothelialization promoting function. The graph shows the changes in the growth activity of human umbilical vein endothelial cells before and after coating with dopamine hydrochloride / hexamethylenediamine and heparin / phosphorylcholine in Example 2 of the silk conical artificial blood vessel with anticoagulant / endothelialization promoting function. (a) is unmodified and cultured for 24 hours; (a1) is unmodified and cultured for 72 hours; (b) is modified with dopamine hydrochloride / hexamethylenediamine coating and cultured for 24 hours; (b1) is modified with dopamine hydrochloride / hexamethylenediamine coating and cultured for 72 hours; (c) is modified with heparin / phosphorylcholine coating and cultured for 24 hours; and (c1) is modified with heparin / phosphorylcholine coating and cultured for 72 hours. DETAILED DESCRIPTION

[0036] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0037] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0038] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0039] The purpose of the present invention is to provide a method for preparing a silk tapered artificial blood vessel with anticoagulant / endothelialization promoting function, comprising the following steps:

[0040] Step 1: Using silk and polyester yarns as raw materials, a woven artificial blood vessel for intracavitary isolation is prepared through a trapezoidal steel reed and an integrated molding weaving process, wherein the yarn fineness of silk and polyester is 3.3 tex, the plurality is 1 to 12f, the upper weave is any one or more combinations of plain weave, twill, satin weave, double plain weave, honeycomb weave, crepe weave or small jacquard weave, the warp density at the big end is 1220 to 2260 strands / (10 cm), the weft density at the big end is 1100 to 1600 strands / (10 cm), the warp density at the small end is 1860 to 2780 strands / (10 cm), and the weft density at the small end is 440 to 800 strands / (10 cm).

[0041] Step 2: Clean the intracavitary isolation artificial blood vessel with a mass fraction of 1% to 5% citric acid solution or oxalic acid solution for 30 to 60 minutes at a washing temperature of 70 to 80°C, then immerse it in a mass fraction of 0.01% to 0.05% Na2CO3 solution for degumming, the degumming time is 30 to 60 minutes, and the degumming temperature is 80 to 100°C. Then, put the intracavitary isolation artificial blood vessel on a stainless steel tube with a diameter matching the prepared vascular stent, moisten it with distilled water, and place it in an oven for heat setting at a temperature of 100 to 150°C for 30 to 60 minutes to obtain an artificial blood vessel sample.

[0042] Step 3: Prepare a dopamine hydrochloride solution with a concentration of 1 to 3 mg / mL and a hexamethylenediamine solution with a concentration of 0.5 to 2 mg / mL, dissolve them in a 1 to 3 mg / mL Tris buffer, and then immerse the artificial blood vessel sample in the mixed solution for 12 to 48 hours. After the reaction is completed, ultrasonic cleaning is performed and nitrogen drying is performed to obtain an ammonia-rich coating sample blood vessel sample.

[0043] Step 4: 1-3 mg / mL heparin sodium and 1-3 mg / mL poly (2-methacryloyloxyethyl phosphorylcholine) are dissolved in 1-3 mg / mL Tris buffer in sequence, and then the ammonia-rich coated sample blood vessel specimen is immersed in the mixed solution for 24-36 hours. After the reaction is completed, it is ultrasonically cleaned with deionized water and dried with nitrogen to obtain a silk cone-shaped artificial blood vessel with anticoagulant / endothelialization function.

[0044] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, the technical solutions of the present invention are further described below with reference to specific embodiments. However, the present invention is not limited to the embodiments listed, but also includes any other known modifications within the scope of the claimed invention.

[0045] The term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it constitute a separate or selective embodiment that is mutually exclusive with other embodiments. It should also be noted that, in the present invention, the large end refers to the end with the relatively larger diameter of the tapered artificial blood vessel; the small end refers to the end with the relatively smaller diameter of the tapered artificial blood vessel.

[0046] Example 1

[0047] The following example demonstrates a method for preparing a tapered silk artificial blood vessel with anticoagulant / endothelialization promoting functions. The specific steps are as follows:

[0048] (1) Using medical polyester filament as warp yarn and raw silk as weft yarn, the warp density at the large end is 1220-2260 strands / (10 cm), the weft density at the large end is 1100-1600 strands / (10 cm), the warp density at the small end is 1860-2780 strands / (10 cm), the weft density at the small end is 440-800 strands / (10 cm), the warp and weft yarn fineness is 3.3 tex / 1f×3.3 tex / 12f, and the fabric structure is plain weave, an artificial blood vessel for intracavitary isolation is prepared by an integrated molding weaving process;

[0049] (2) The intracavitary isolation artificial blood vessel is placed in a citric acid solution for cleaning. The mass fraction of citric acid is 4%, the washing time is 40 minutes, and the washing temperature is 75°C. After washing, the artificial blood vessel is cleaned with deionized water to thoroughly remove the rust and residual oxalic acid adhering to the surface of the artificial blood vessel when the artificial blood vessel rubs against the machine during the weaving process. After cleaning, it is naturally air-dried. Since the weft yarn used in the weaving process is undegummed raw silk, but as an artificial blood vessel material, sericin must be removed to avoid inflammatory reaction after transplantation, the raw silk is degummed with sodium carbonate with a mass fraction of 0.01% at 100°C. A cycle of 20 minutes is a cycle. After each cycle is completed, it is scrubbed with distilled water at 60°C. A total of 3 cycles are processed. After completing the cleaning and degumming process, it is washed again with distilled water for several minutes. Then, the artificial blood vessel is heat-set. The artificial blood vessel is placed on a stainless steel tube with a diameter matching the prepared vascular stent, moistened with distilled water, and placed in an oven at 100°C for 60 minutes. It is dried and set to obtain an artificial blood vessel sample.

[0050] (3) 1 mg / mL dopamine hydrochloride and 0.5 mg / mL hexamethylenediamine were dissolved in 3 mg / mL Tris buffer, and the artificial blood vessel sample was immersed in the mixed solution and allowed to settle for 48 hours. After the reaction, the physically adsorbed polymer was removed by ultrasonic cleaning, and the sample was dried with nitrogen to obtain an ammonia-rich coating.

[0051] (4) The ammonia-rich coated blood vessel sample was immersed in a 1.2 mg / mL Tris buffer solution containing sodium heparin and poly(2-methacryloyloxyethyl phosphorylcholine) for 30 h, wherein the concentration of sodium heparin was 3 mg / mL and the concentration of poly(2-methacryloyloxyethyl phosphorylcholine) was 1 mg / mL. After the reaction, the ammonia-rich coated blood vessel sample was ultrasonically cleaned with deionized water and dried with nitrogen to obtain a silk conical artificial blood vessel with anticoagulant / endothelialization function.

[0052] See also Figure 1 , Figure 1This is a photo of a silk conical artificial blood vessel with anticoagulant / endothelialization promoting function produced by the method for producing the silk conical artificial blood vessel with anticoagulant / endothelialization promoting function described in the present invention. Figure 1 As shown in the figure, the silk conical artificial blood vessel is woven through integrated molding technology, with uniform warp yarn density, complete structure, and not easy to deform.

[0053] See also Figure 2 , Figure 2 This is a comparison chart of the radial and longitudinal tensile strength of the silk conical artificial blood vessel with anticoagulant / endothelialization function prepared in Example 1 of the preparation method of the present invention and the commercial tubular artificial blood vessel. Figure 2 As shown in the figure, the longitudinal tensile strength and radial tensile strength of the silk conical artificial blood vessels are better than those of commonly used commercial artificial blood vessels, which proves that the silk conical artificial blood vessels can significantly improve the mechanical properties of blood vessels.

[0054] See also Figure 3 , Figure 3 This is a graph showing the change in the water contact angle of the fabric surface of the silk conical artificial blood vessel with anticoagulant / endothelialization promoting function prepared in Example 1 of the method for preparing the silk conical artificial blood vessel with anticoagulant / endothelialization promoting function before and after coating with dopamine hydrochloride / hexamethylenediamine and heparin / phosphorylcholine. Figure 3 As shown in the figure, after modification with dopamine hydrochloride and hexamethylenediamine, the contact angle decreased slightly. After further grafting with heparin and phosphorylcholine, the water contact angle decreased significantly, indicating that the hydrophilicity of the vascular surface was significantly enhanced.

[0055] See also Figure 4 , Figure 4 This is a comparison chart of the fabric thrombin time of the silk conical artificial blood vessel with anticoagulant / endothelialization function prepared in Example 1 of the preparation method of the present invention before and after heparin / phosphorylcholine coating. Figure 4 As shown in the figure, the thrombin time of the artificial blood vessel increased by 18.78s before and after the heparin / phosphorylcholine coating, which shows that the artificial blood vessel has anti-coagulation function after the heparin / phosphorylcholine coating.

[0056] See also Figure 5 , Figure 5 This is a graph showing the cell viability of human umbilical vein endothelial cells in the sample extract before and after dopamine hydrochloride / hexamethylenediamine coating and heparin / phosphorylcholine coating in Example 1 of the method for preparing a silk conical artificial blood vessel with anticoagulant / endothelialization promoting function of the present invention. Figure 5As shown, the cell viability of all sample extracts incubated with the extracts was greater than 80%, indicating that the materials in each group had no significant cytotoxicity to the cells. Among them, the cell viability of the artificial blood vessel sample coated with heparin / phosphorylcholine reached 115±3.9% after 72 hours of culture, significantly better than that of the unmodified blood vessel sample. This indicates that the fabric grafted with heparin and phosphorylcholine can significantly promote the adhesion and proliferation of endothelial cells, demonstrating good cell compatibility.

[0057] Example 2

[0058] The following example demonstrates a method for preparing a tapered silk artificial blood vessel with anticoagulant / endothelialization promoting functions. The specific steps are as follows:

[0059] (1) Using medical polyester filament as warp yarn and cooked silk as weft yarn, the warp density at the large end is 1220-2260 strands / (10 cm), the weft density at the large end is 1100-1600 strands / (10 cm), the warp density at the small end is 1860-2780 strands / (10 cm), the weft density at the small end is 440-800 strands / (10 cm), the warp and weft yarn fineness is 3.3 tex / 1f×3.3 tex / 12f, and the fabric structure is 3 / 1 twill, to prepare an artificial blood vessel for intracavitary isolation through an integrated molding weaving process;

[0060] (2) The artificial blood vessel for intraluminal isolation is placed in an oxalic acid solution for cleaning, with an oxalic acid mass fraction of 1%, a washing time of 60 minutes, and a washing temperature of 70°C. After washing, the artificial blood vessel is cleaned with deionized water to thoroughly remove the rust and residual oxalic acid adhering to the surface of the sample due to the friction between the artificial blood vessel and the machine during the weaving process. After cleaning, it is naturally air-dried and washed again with distilled water for several minutes. The artificial blood vessel is then subjected to thermal qualitative treatment. The artificial blood vessel is placed on a stainless steel tube with a diameter matching that of the prepared vascular stent, moistened with distilled water, and placed in an oven at 120°C for 40 minutes. It is dried and shaped to obtain an artificial blood vessel sample.

[0061] (3) 3 mg / mL dopamine hydrochloride and 1 mg / mL hexamethylenediamine were dissolved in 1.2 mg / mL Tris buffer. The artificial blood vessel sample was then immersed in the mixed solution for 36 hours of surface deposition. After the reaction was complete, the loose adsorbed material was removed using an ultrasonic cleaner and dried with nitrogen to obtain an ammonia-rich coating sample.

[0062] (4) The ammonia-enriched coated blood vessel sample was immersed in a Tris buffer system (3 mg / mL) containing 1.5 mg / mL heparin sodium and 3 mg / mL poly(2-methacryloyloxyethylphosphorylcholine) for 24 hours. After the reaction, the ammonia-enriched coated blood vessel sample was ultrasonically cleaned with deionized water and dried with nitrogen gas to obtain a silk conical artificial blood vessel with anticoagulant / endothelialization functions.

[0063] See also Figure 6 , Figure 6 This is a graph showing the changes in radial tensile strength and probe burst strength of the silk conical artificial blood vessel with anticoagulant / endothelialization function prepared in Example 2 of the method for preparing the silk conical artificial blood vessel with anticoagulant / endothelialization function described in the present invention before and after coating with dopamine hydrochloride / hexamethylenediamine and heparin / phosphorylcholine. Figure 6 As shown in the figure, the radial tensile strength and probe bursting strength of the sample did not change significantly after coating modification, which indicates that the coating treatment does not affect the mechanical properties of the blood vessel, thereby ensuring the mechanical stability of the artificial blood vessel in intraluminal isolation applications.

[0064] See also Figure 7 , Figure 7 This is a graph showing the changes in the growth activity of human umbilical vein endothelial cells in the silk conical artificial blood vessel with anticoagulant / endothelialization function prepared in Example 2 before and after coating with dopamine hydrochloride / hexamethylenediamine and heparin / phosphorylcholine. Figure 7 As shown, cells in all samples were well-developed, with clear cell outlines. In particular, the cell number in the vascular samples coated with phosphorylcholine / heparin increased significantly, and a large number of endothelial cells exhibited a highly diffused morphology, indicating that phosphorylcholine / heparin coating promoted endothelial cell growth and accelerated the endothelialization process.

[0065] Example 3

[0066] The following example demonstrates a method for preparing a tapered silk artificial blood vessel with anticoagulant / endothelialization promoting functions. The specific steps are as follows:

[0067] (1) Raw silk is used as warp yarn and cooked silk is used as weft yarn, the warp density at the large end is 1220 to 2260 strands / (10 cm), the weft density at the large end is 1100 to 1600 strands / (10 cm), the warp density at the small end is 1860 to 2780 strands / (10 cm), the weft density at the small end is 440 to 800 strands / (10 cm), the warp and weft yarn fineness is 3.3 tex / 1f × 3.3 tex / 12f, and the fabric structure is 2 / 2 twill, and an artificial blood vessel for intracavitary isolation is prepared by an integrated molding weaving process;

[0068] (2) The intracavitary isolation artificial blood vessel is placed in an oxalic acid solution for cleaning. The mass fraction of oxalic acid is 5%, the washing time is 30 minutes, and the washing temperature is 80°C. After washing, the artificial blood vessel is cleaned with deionized water to thoroughly remove the rust and residual oxalic acid adhering to the sample surface during the weaving process due to the friction between the artificial blood vessel and the machine. After cleaning, it is naturally air-dried. Since the warp yarn used in the weaving process is undegummed raw silk, but as an artificial blood vessel material, sericin must be removed to avoid inflammatory reactions after transplantation, the raw silk is degummed using a mass fraction of 0.05% Na2CO3 at 80°C. One cycle is 20 minutes. After each cycle, it is scrubbed under 60°C distilled water. A total of 2 cycles are processed. After completing the cleaning and degumming process, the artificial blood vessel is heat-set. The artificial blood vessel is placed on a stainless steel tube with a diameter matching the prepared vascular stent, moistened with distilled water, and placed in an oven at 150°C for 30 minutes. Drying and setting are performed to obtain an artificial blood vessel sample.

[0069] (3) Dissolve 2 mg / mL dopamine hydrochloride and 2 mg / mL hexamethylenediamine in 1 mg / mL Tris buffer, then immerse the artificial blood vessel sample in the mixed solution and allow to settle for 12 hours. After the reaction, ultrasonic cleaning is used to remove physically adsorbed polymers, and the sample is dried with nitrogen to obtain an ammonia-rich coating.

[0070] (4) Immersing the ammonia-rich coated sample blood vessel sample in 1 mg / mL Tris buffer containing sodium heparin and poly(2-methacryloyloxyethyl phosphorylcholine) for 36 hours, wherein the concentration of sodium heparin is 1 mg / mL and the concentration of poly(2-methacryloyloxyethyl phosphorylcholine) is 2 mg / mL. After the reaction is completed, the ammonia-rich coated sample blood vessel sample is ultrasonically cleaned with deionized water and dried with nitrogen to obtain a silk conical artificial blood vessel with anticoagulant / endothelialization function.

[0071] Comparative Example 1

[0072] The preparation method of Comparative Example 1 differs from the preparation method of Example 1 only in that step (4) is omitted.

[0073] Comparative Example 2

[0074] The preparation method of Comparative Example 2 differs from the preparation method of Example 1 only in that: step (4) is different, specifically: immersing the ammonia-rich coated sample blood vessel specimen in 1.2 mg / mL Tris buffer dissolved in heparin sodium for 24 hours, wherein the concentration of heparin sodium is 1 mg / mL. After the reaction is completed, the sample is ultrasonically cleaned with deionized water and dried with nitrogen to obtain a heparin-coated silk cone-shaped artificial blood vessel specimen.

[0075] Comparative Example 3

[0076] The preparation method of Comparative Example 3 differs from the preparation method of Example 1 only in that: step (4) is different, specifically: immersing the ammonia-rich coated sample blood vessel sample in 1.2 mg / mL Tris buffer dissolved with poly(2-methacryloyloxyethyl phosphorylcholine) for 24 hours, wherein the concentration of poly(2-methacryloyloxyethyl phosphorylcholine) is 2 mg / mL. After the reaction is completed, the sample is ultrasonically cleaned with deionized water and dried with nitrogen to obtain a phosphorylcholine-coated silk cone artificial blood vessel sample.

[0077] Performance Testing

[0078] The coating samples prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were tested, and the test items were as follows:

[0079] 1. Surface water contact angle: measured using a Kruss DSA 100 optical contact angle meter, with a sample size of 10 mm × 10 mm.

[0080] 2. Thrombin time: Sample 5mm×5mm, 100μL platelet-poor plasma, observe the time it takes for the plasma to coagulate.

[0081] 3. Hemolysis rate: Use a microplate reader to detect the absorbance at 545 nm. The sample is 10 mm × 10 mm and 200 μL of diluted anticoagulated rabbit blood is used.

[0082] 4. Surface protein adsorption: The BCA method was used to test the concentration of adsorbed protein at 562 nm. The sample was 10 mm × 10 mm, 1 mL of bovine serum albumin solution (concentration: 0.5 mg / mL), and 2 mL of sodium dodecyl sulfate solution.

[0083] 5. Cell viability of human umbilical vein endothelial cells in sample extracts: Cells were plated in a 96-well plate at a density of 3 × 103 cells / well. After staining with CCK8, fluorescence intensity was measured to characterize cell proliferation. Cell viability was observed after 24 hours of co-culture, 48 hours of co-culture, and 72 hours of co-culture.

[0084] The test results of the samples are shown in Table 1 below.

[0085] Table 1

[0086]

[0087]

[0088] As shown in Table 1, the addition of a heparin / phosphorylcholine coating effectively improves the hydrophilicity and blood compatibility of artificial blood vessels. Heparin can inhibit coagulation factors and enhance its anticoagulant effect by binding to antithrombin III. Phosphorylcholine, by mimicking the phospholipid structure of cell membranes, forms a biomimetic protective layer on the material surface, further inhibiting the adhesion of proteins involved in the coagulation cascade. Furthermore, the synergistic use of heparin and phosphorylcholine can enhance the cytocompatibility of artificial blood vessels. This is primarily because the coating significantly enhances the interaction between cells and the substrate, promotes the adhesion of endothelial cells, facilitates the formation of an endothelial layer after implantation, and improves clinical therapeutic efficacy.

[0089] In summary, the present invention discloses a method for preparing a silk conical artificial blood vessel with anticoagulant / endothelialization-promoting function. An artificial blood vessel for intraluminal isolation is prepared using silk and polyester yarns. Then, a phosphorylcholine / heparin biomimetic functional coating is successfully constructed on the blood vessel surface using dopamine hydrochloride and hexamethylenediamine. Finally, a silk conical artificial blood vessel with anticoagulant / endothelialization-promoting function is obtained. This method utilizes phosphorylcholine / heparin combined surface coating modification to construct a novel intraluminal isolation blood vessel with both excellent biological functions and mechanical properties. The structure of this artificial blood vessel better conforms to the morphology of the inner wall of the blood vessel, facilitating blood flow distribution and tissue regeneration. The phosphorylcholine / heparin coating effectively enhances its anticoagulant and endothelialization capabilities, significantly reducing protein adsorption and platelet activation, blocking the coagulation cascade reaction, and accelerating endothelial cell adhesion and proliferation, thereby reducing the risk of acute postoperative thrombosis. This method provides a new technical path for the design and functionalization of conical artificial blood vessels during intraluminal isolation surgery, and has important clinical application prospects.

[0090] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. A method for preparing a tapered silk artificial blood vessel with anticoagulant / endothelialization promoting functions, characterized by: include, Using silk and polyester as raw materials, an artificial blood vessel for intracavitary isolation is prepared through an integrated weaving process; cleaning, degumming, and heat-setting the intraluminal isolation artificial blood vessel to obtain an artificial blood vessel sample; adding a dopamine hydrochloride solution and a hexamethylenediamine solution into a Tris buffer solution to obtain a first mixed solution, immersing the artificial blood vessel sample in the first mixed solution, then removing the artificial blood vessel sample, washing, and drying the artificial blood vessel sample to obtain an ammonia-rich coating sample blood vessel sample; Heparin sodium and poly (2-methacryloyloxyethyl phosphorylcholine) were dissolved in Tris buffer in sequence to obtain a second mixed solution. The ammonia-rich coated sample blood vessel sample was immersed in the second mixed solution. The ammonia-rich coated sample blood vessel sample was then removed, cleaned, and dried to obtain a silk cone-shaped artificial blood vessel with anticoagulant / endothelialization promoting function.

2. The preparation method according to claim 1, wherein: The yarn fineness of the silk and polyester is both 3.3tex, and the plural number is 1 to 12f; the upper weave is any one or more combinations of plain weave, twill, satin weave, double plain weave, honeycomb weave, crepe weave or small jacquard weave; the warp density at the big end is 1220 to 2260 strands / (10cm), the weft density at the big end is 1100 to 1600 strands / (10cm), the warp density at the small end is 1860 to 2780 strands / (10cm), and the weft density at the small end is 440 to 800 strands / (10cm).

3. The preparation method according to claim 1, wherein: The intracavitary isolation artificial blood vessel is cleaned, degummed, and heat-set, wherein the surface cleaning agent used for cleaning is citric acid solution or oxalic acid solution, the mass fraction of the surface cleaning agent is 1% to 5%, the cleaning time is 30 to 60 minutes, and the temperature is 70 to 80°C.

4. The preparation method according to claim 1, wherein: The degumming agent used in the degumming is a Na2CO3 solution, the mass fraction of the degumming agent is 0.01% to 0.05%, the degumming time is 30 to 60 minutes, and the degumming temperature is 80 to 100°C.

5. The preparation method according to claim 1, wherein: The heat setting temperature is 100-150° C., and the time is 30-60 minutes.

6. The preparation method according to claim 1, wherein: The concentration of the dopamine hydrochloride solution is 1-3 mg / mL, the concentration of the hexamethylenediamine solution is 0.5-2 mg / mL, the concentration of the Tris buffer is 1-3 mg / mL, and the immersion time is 12-48 hours.

7. The preparation method according to claim 1, wherein: The cleaning and drying are performed to obtain an ammonia-rich coating sample blood vessel specimen, wherein the cleaning method is ultrasonic cleaning and the drying method is nitrogen drying.

8. The preparation method according to claim 1, wherein: The concentration of the heparin sodium is 1-3 mg / mL, the concentration of the poly(2-methacryloyloxyethyl phosphorylcholine) is 1-3 mg / mL, the concentration of the Tris buffer is 1-3 mg / mL, and the immersion time is 24-36 hours.

9. The preparation method according to claim 1, wherein: The cleaning and drying process obtains a silk conical artificial blood vessel with anticoagulation / endothelialization promoting function, wherein the cleaning method is ultrasonic cleaning with deionized water, and the drying method is nitrogen drying.

10. A silk tapered artificial blood vessel with anticoagulant / endothelialization promoting function obtained by the method according to any one of claims 1 to 9.

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