Artificial blood vessel for gradient modification of endothelialization-promoting active factors and preparation method and application of artificial blood vessel

Abstract

The invention relates to the technical field of biomedical materials, in particular to an artificial blood vessel for gradient modification of endothelialization promoting active factors and a preparation method and application of the artificial blood vessel. The artificial blood vessel subjected to electrostatic spinning is subjected to ammonolysis and uniform heparin coupling, then the artificial blood vessel is subjected to gradient modification in the mode that the artificial blood vessel rotates at a constant speed in the horizontal state and an active factor solution is infused in the middle, and then the artificial blood vessel with the middle active factor being high in concentration and the two ends being low in concentration is obtained. Near endothelial cells can be guided to migrate from two ends to the middle, so that rapid endothelialization of the artificial blood vessel is realized. The invention discloses an artificial blood vessel for gradient modification of endothelialization promoting active factors, inhibition of acute thrombus, promotion of endothelial cell migration and rapid endothelialization so as to improve the patency rate, the endothelialization rate and the regeneration capacity of the artificial blood vessel.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to an artificial blood vessel with gradient-modified endothelialization-promoting active factors, and a preparation method and application thereof. Background Art

[0002] Cardiovascular diseases (CVDs) are the diseases with the highest morbidity and mortality in the world. They have become the leading cause of death worldwide, surpassing other diseases, and pose a major threat to human health. Despite progress in prevention and treatment, the aging of the population, the global spread of smoking, and the increasing prevalence of obesity and diabetes ensure that the incidence of cardiovascular diseases will continue to rise in the coming decades.

[0003] Vascular transplantation is one of the effective means of treating cardiovascular diseases. The use of autologous bypass vessels, especially small-diameter veins or arteries (< 5 mm), for transplantation remains an important treatment for coronary artery disease. However, secondary site injuries and diseases that may coexist with autologous blood vessels limit its application. Although large-caliber artificial blood vessels have been successful in clinical applications, small-caliber (inner diameter < 6 mm) artificial blood vessels often have a low success rate within 5 years after surgery due to thrombosis (acute thrombosis), intimal hyperplasia and other problems, which makes it difficult to meet clinical needs.

[0004] The current view is that forming a complete endothelial layer on the surface of cardiovascular materials is an ideal treatment method. If endothelialization of artificial blood vessels is to be achieved, the artificial blood vessels and endothelial cells need to be treated differently. However, the above methods are not only cumbersome, time-consuming, easy to pollute, and expensive, but also may lead to the loss of cells and active substances or loss of function due to multi-step treatment.

[0005] Therefore, how to provide an artificial blood vessel modification method that is simple to handle and can achieve early protection against acute thrombosis and rapid endothelialization is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention discloses an artificial blood vessel with gradient modified endothelialization active factors and its preparation method and application. The artificial blood vessel is uniformly modified with heparin and gradient endothelialization active factors to improve the acute anticoagulant performance and endothelialization rate of the artificial blood vessel.

[0007] It should be noted that the present invention aims at the problem that small-caliber artificial blood vessels often fail to be transplanted due to thrombosis (acute thrombosis), intimal hyperplasia and other problems, and provides an artificial blood vessel with a gradient-modified endothelialization active factor to inhibit acute thrombosis, promote endothelial cell migration and rapid endothelialization, so as to improve the patency rate, endothelialization rate and regeneration ability of the artificial blood vessels.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The first technical purpose of the present invention is to provide an artificial blood vessel with gradient-modified endothelialization-promoting active factors, wherein the artificial blood vessel has uniform heparin modification, and the active factors are modified in a gradient manner; and the concentration of the active factors of the artificial blood vessel gradually decreases from the middle to both ends.

[0010] Optionally, the active factors include vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), C-type natriuretic peptide (CNP) or basic fibroblast growth factor (b-Fibroblast Growth Factor, b-FGF).

[0011] The second technical purpose of the present invention is to provide a method for preparing an artificial blood vessel with gradient-modified endothelialization-promoting active factors as described above, comprising the following steps:

[0012] The matrix material is dissolved in an organic solution; a matrix solution is formed after the polymer material is completely dissolved; the matrix solution is then prepared into a common artificial blood vessel by electrospinning, and then the active factors are modified on the artificial blood vessel by covalent cross-linking (aminolysis-cross-linking reaction) to obtain an artificial blood vessel with gradient-modified active factors.

[0013] Optionally, the aminolysis treatment is carried out using hexamethylenediamine, the concentration of which is 0.1M-1M, preferably 0.25M-0.75M, more preferably 0.5M; the treatment time is 30min.

[0014] Optionally, the matrix material is a polymer material commonly used in artificial blood vessels.

[0015] Specifically, the common polymer material for artificial blood vessels is at least one of polycaprolactone, polyglycolic acid, polylactic acid-co-glycolic acid, poly(3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly-L-lactide-caprolactone, and polydioxanone, or a mixture of any proportions thereof. Preferably, the common polymer material for artificial blood vessels is polycaprolactone.

[0016] Optionally, the organic solvent is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol is 5:1.

[0017] Optionally, the operation of modifying the active factor on the artificial blood vessel is: placing the aminolysis-crosslinked artificial blood vessel material horizontally and rotating it at a constant speed, delivering the active factor solution to the middle part of the artificial blood vessel and keeping the artificial blood vessel rotating at a constant speed; wherein,

[0018] The concentration of the active factor solution is 10-400 ng / mL, preferably 50-200 ng / mL, more preferably 100 ng / mL, the delivery speed is 1 mL / h, the delivery time is 30 min; and the horizontal rotation speed of the artificial blood vessel material is 10 rpm.

[0019] The third technical purpose of the present invention is to provide an artificial blood vessel with gradient-modified endothelialization-promoting active factors as described above for use in the field of tissue engineering.

[0020] Specifically, the artificial blood vessel can change its shape to be used as a vascular patch, a valved blood vessel, or a heart valve.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) The present invention performs aminolysis and uniform heparin coupling on the electrospun artificial blood vessels, and then performs gradient modification on the artificial blood vessels by uniformly rotating the artificial blood vessels in a horizontal state and infusing the active factor solution in the middle part, thereby obtaining artificial blood vessels with high active factor concentration in the middle and low concentration at both ends, which can guide the adjacent endothelial cells to migrate from the two ends to the middle, thereby achieving rapid endothelialization of the artificial blood vessels.

[0023] 2) The present invention loads VEGF onto artificial blood vessels by gradient modification, which not only has a simple aminolysis method but also does not affect the mechanical properties of the artificial blood vessels; and the endothelialization-promoting active factor with a concentration gradient can promote the migration of endothelial cells along the direction of high concentration active factors; the loading method is simple and can effectively maintain the activity of the active factors; the prepared artificial blood vessels with gradient-modified endothelialization-promoting active factors can both resist acute coagulation and accelerate the endothelialization rate of blood vessels. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0025] Figure 1 It is a statistical diagram of vascular axial mechanics after ammonolysis.

[0026] Figure 2 It is a statistical diagram of radial mechanical properties of blood vessels after ammonolysis.

[0027] Figure 3 It is a statistical chart of the endothelial coverage of implanted blood vessels.

[0028] Figure 4 It is the contact angle test diagram of comparative example 1 and embodiment 3.

[0029] Figure 5 These are CD31 immunofluorescence staining images of Comparative Example 1 and Example 3.

[0030] Figure 6 The present invention is a flow chart of the preparation process of the artificial blood vessel with gradient-modified endothelialization-promoting active factors. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0033] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0034] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0035] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0036] The invention discloses an artificial blood vessel with gradient-modified endothelialization-promoting active factors and a preparation method thereof.

[0037] The present invention first uses modified VEGF as the experimental active factor and performs the following specific operations:

[0038] 1. Preparation of solution:

[0039] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at a rate of 0.25 g / mL, the vial cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 h at room temperature in the dark.

[0040] 2. Electrospinning

[0041] Preparation of electrospun artificial blood vessels:

[0042] The stirred solution was loaded into a 10 mL glass syringe. The syringe was inverted to remove all bubbles and then a 21G needle was installed and fixed. The syringe was pushed forward using a syringe pump. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min. The electrospun artificial blood vessels were prepared according to the above parameters.

[0043] (III) Aminolysis of artificial vascular materials

[0044] The electrospun artificial blood vessels were soaked in 40% alcohol for 15 minutes, washed with PBS for 3 times, and then soaked in 0.1M-1M (0.1M, 0.25M, 0.5M, 0.75M, 1M) hexamethylenediamine solution for 30 minutes to obtain artificial blood vessels containing amino groups.

[0045] (IV) Aminolysis-crosslinking of artificial vascular materials

[0046] After washing with PBS five times, the artificial blood vessels were immersed in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. The aminolyzed artificial blood vessels were then placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) and reacted at 37°C for 4 h.

[0047] 5. VEGF gradient modification of artificial blood vessels

[0048] The artificial vascular material after aminolysis and cross-linking was placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 10-400 ng / mL (10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL) of VEGF solution was delivered to the middle part of the artificial blood vessel at a rate of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 minutes to allow VEGF to diffuse from the middle to both ends, thereby obtaining an artificial blood vessel with a gradually decreasing VEGF concentration from the middle to both ends.

[0049] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0050] Example 1

[0051] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0052] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.1 M hexamethylenediamine for 30 min.

[0053] After the aminolysis of the artificial blood vessel, it was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 30 minutes. Then, the aminolysis of the artificial blood vessel was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 hours at 37°C. After the reaction was completed, the aminolysis-cross-linked artificial blood vessel was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 100 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 minutes. After 30 minutes, the delivery of the solution was stopped, and the blood vessel was rotated at a constant speed of 10 rpm for 4 hours. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0054] Example 2

[0055] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0056] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.25 M hexamethylenediamine for 30 min.

[0057] After the aminolysis of the artificial blood vessel, it was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the aminolysis of the artificial blood vessel was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 h at 37 ° C. After the reaction was completed, the aminolysis-cross-linked artificial blood vessel was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 100 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of the solution was stopped, and the blood vessel was allowed to rotate at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0058] Example 3

[0059] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0060] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.5 M hexamethylenediamine for 30 min.

[0061] After the aminolysis of the artificial blood vessel, it was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the aminolysis of the artificial blood vessel was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 h at 37 ° C. After the reaction was completed, the aminolysis-cross-linked artificial blood vessel was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 100 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of the solution was stopped, and the blood vessel was allowed to rotate at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0062] Example 4

[0063] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0064] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.75 M hexamethylenediamine for 30 min.

[0065] After the aminolysis of the artificial blood vessel, it was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the aminolysis of the artificial blood vessel was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 h at 37 ° C. After the reaction was completed, the aminolysis-cross-linked artificial blood vessel was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 100 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of the solution was stopped, and the blood vessel was allowed to rotate at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0066] Example 5

[0067] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0068] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 1 M hexamethylenediamine for 30 min.

[0069] After the aminolysis of the artificial blood vessel, it was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the aminolysis of the artificial blood vessel was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 h at 37 ° C. After the reaction was completed, the aminolysis-cross-linked artificial blood vessel was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 100 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of the solution was stopped, and the blood vessel was allowed to rotate at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0070] The specific parameters of Examples 1-5 are shown in Summary Table 1

[0071] Table 1 Summary of specific parameters of Examples 1-5

[0072] Example 6

[0073] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0074] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.5 M hexamethylenediamine for 30 min.

[0075] After the aminolysis, the artificial blood vessels were washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the artificial blood vessels were placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) at 37 ° C for 4 h. After the reaction was completed, the artificial blood vessels after aminolysis and cross-linking were passed through the stainless steel rod, and then placed horizontally on the rotating shaft and rotated at a constant speed of 10 rpm. 10 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of solution was stopped, and the blood vessel was rotated at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0076] Example 7

[0077] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0078] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.5 M hexamethylenediamine for 30 min.

[0079] After the aminolysis of the artificial blood vessel, it was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the aminolysis of the artificial blood vessel was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 h at 37 ° C. After the reaction was completed, the aminolysis-cross-linked artificial blood vessel was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 50 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of the solution was stopped, and the blood vessel was rotated at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0080] Example 8

[0081] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0082] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.5 M hexamethylenediamine for 30 min.

[0083] After the aminolysis of the artificial blood vessel, it was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the aminolysis of the artificial blood vessel was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 h at 37 ° C. After the reaction was completed, the aminolysis-cross-linked artificial blood vessel was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 100 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of the solution was stopped, and the blood vessel was allowed to rotate at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0084] Example 9

[0085] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0086] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.5 M hexamethylenediamine for 30 min.

[0087] After the aminolysis of the artificial blood vessel, it was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the aminolysis of the artificial blood vessel was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 h at 37 ° C. After the reaction was completed, the aminolysis-cross-linked artificial blood vessel was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 200 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of the solution was stopped, and the blood vessel was rotated at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0088] Example 10

[0089] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0090] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.5 M hexamethylenediamine for 30 min.

[0091] After the aminolysis of the artificial blood vessel, it was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the aminolysis of the artificial blood vessel was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 h at 37 ° C. After the reaction was completed, the aminolysis-cross-linked artificial blood vessel was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 400 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of the solution was stopped, and the blood vessel was allowed to rotate at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0092] The specific parameters of Examples 6-10 are shown in Summary Table 2

[0093] Table 2 Summary of specific parameters of Examples 6-10

[0094] Comparative Example 1

[0095] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0096] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in PBS for 30 min.

[0097] After washing with PBS for 5 times, the artificial blood vessels were soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. The artificial blood vessels after aminolysis were then placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) for 4 h at 37 ° C. After the reaction was completed, the artificial blood vessels after aminolysis and cross-linking were passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. 100 ng / mL VEGF solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of the solution was stopped, and the blood vessel was allowed to rotate at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS for 6 times, and finally dried under nitrogen for use.

[0098] Comparative Example 2

[0099] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added at an amount of 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer and stirred continuously for 12 hours at room temperature and away from light. The above solution was used for electrospinning to prepare electrospun PCL artificial blood vessels. The specific operation and parameters are as follows: the stirred solution was loaded into a 10 mL glass syringe, the syringe was inverted to remove bubbles, and a 21G needle was installed and fixed, and a syringe pump was used for propulsion. The flow rate of the solution was set to 8 mL / h, the high voltage DC voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 4 min.

[0100] The electrospun artificial blood vessels were immersed in 40% alcohol for 15 min, washed three times with PBS, and then placed in 0.5 M hexamethylenediamine for 30 min.

[0101] After the aminolysis, the artificial blood vessel was washed with PBS for 5 times and then soaked in MES with a pH of 5.6 and a concentration of 0.05 mol / L for 0.5 h. Then, the artificial blood vessel after aminolysis was placed in a mixed solution containing EDC, NHS and heparin (0.2% EDC + 0.12% NHS + 0.12% heparin) at 37 ° C for 4 h. After the reaction was completed, the artificial blood vessel after aminolysis and cross-linking was passed through a stainless steel rod, and then placed horizontally on a rotating shaft and rotated at a constant speed of 10 rpm. PBS solution was delivered to the middle part of the artificial blood vessel at a speed of 1 mL / h and the blood vessel was kept rotating at a constant speed for 30 min. After 30 min, the delivery of solution was stopped, and the blood vessel was allowed to rotate at a constant speed of 10 rpm for 4 h. After that, the blood vessel was removed, washed with PBS 6 times, and finally dried under nitrogen for use.

[0102] The specific parameters of Comparative Examples 1-2 are shown in Summary Table 3

[0103] Table 3 Summary of specific parameters of comparative examples 1-2

[0104] The beneficial effects of the present invention are fully illustrated below through the display and analysis of relevant results.

[0105] The axial tension, radial tension, suture strength, bursting pressure, contact angle and N content of the electrospun PCL artificial blood vessels after aminolysis of Examples 1-10 and Comparative Examples 1-2 were tested, and the specific method was as follows:

[0106] 1. Characterization of axial tension

[0107] The axial tension of the electrospun PCL artificial blood vessel after aminolysis was tested using an Instron-3345 tensile testing machine. The testing method was as follows: the upper and lower ends of a 2.5 cm long blood vessel were fixed to the upper and lower clamps of the tensile testing machine to ensure that the distance between the upper and lower clamps was controlled at 1 cm. Then, various parameters of the sample were input, the stretching speed was adjusted to 20 mm / min, the tension in the software was adjusted to zero, and the tensile testing machine was started until the sample was completely broken. The Young's modulus, stress and maximum elongation at break of the sample were obtained based on the stress-strain curve.

[0108] 2. Radial tension characterization

[0109] The radial tension of the electrospun PCL artificial blood vessel after aminolysis was tested using an Instron-3345 tensile testing machine. The testing method was as follows: a sample with a length of about 5 mm was fixed to the tensile testing machine through a steel ring, various parameters of the sample were input, the stretching speed was adjusted to 10 mm / min, the tension of the software was adjusted to zero, and the tensile testing machine was started until the sample was completely broken. The Young's modulus, stress and maximum elongation at break of the sample were obtained based on the stress-strain curve.

[0110] 3. Characterization of suture strength

[0111] The flat end (0°) suture strength of the artificial blood vessels prepared in Examples 1-6 and Comparative Examples 1-2 was tested according to ISO7198:2016 A.5.7 suture strength test standard. The test method is as follows: cut the blood vessel into a small segment with a length of about 1 cm, pass a 6-0 medical suture through the sample tube wall at a distance of about 2 mm from the horizontal end of the blood vessel, tie a knot on the suture to form a closed loop, clamp the knotted suture with the air pump clamp of the tensile testing machine, clamp the other end of the blood vessel with the air pump clamp of the tensile testing machine, balance the tensile testing machine, and adjust to zero; stretch at a tensile speed of 8.00 mm / min until the 6-0 suture penetrates the end of the sample.

[0112] 4. Burst pressure characterization

[0113] The artificial blood vessels prepared in Examples 1-6 and Comparative Examples 1-2 were tested in accordance with 8.3.3.3 Pressurized Burst Strength - Balloon Method in YY0500-2004 / ISO7198. The specific operation method is as follows: evenly apply vaseline lubricant on the balloon, carefully place the balloon into the artificial blood vessel sample, directly connect one end of the blood vessel to the catheter of the sensor, and seal the other end, and introduce CO2 gas at a rate of kilopascals per second until the blood vessel bursts. The change process of the pressure value is recorded by the paperless pressure recorder on the other side of the sensor, and the maximum pressure value is retrieved, and the bursting pressure of the blood vessel is calculated based on the conversion standard of kPa and mmHg.

[0114] 5. Contact angle characterization

[0115] The vascular material was spread flat on a glass slide with double-sided tape, and the micrometer was rotated to make water droplets fall on the surface of the material. A photo was taken every 2 seconds, and the process lasted for 2 minutes. The contact angle was tested using Image J software.

[0116] 6. Characterization of N element content

[0117] X-ray photoelectron spectroscopy (XPS) was used to analyze the surface elements of artificial blood vessels. The samples were cut into pieces with an area of ​​0.50 cm×0.50 cm, and then XPS detection was performed to analyze the contents of the three elements: carbon, nitrogen, and oxygen (C, N, O).

[0118] 7. Data Analysis

[0119] The data were analyzed using GraphPad Prism 9.0.0 software, and all data were expressed as mean ± standard error.

[0120] Result analysis:

[0121] Example 3 and Examples 6-10 and Comparative Example 2 adopted the same aminolysis conditions, and the following analysis focuses on Examples 1-5 and Comparative Example 1.

[0122] The axial tensile force data (Table 4) show that the maximum stress, Young's modulus and elongation at break all decrease from Example 1 to Example 5. In particular, the axial mechanical properties begin to decrease significantly from Example 4 ( Figure 1 ).

[0123] The radial tensile force data (Table 4) show that the maximum stress, Young's modulus and elongation at break all decrease from Example 1 to Example 5. Consistent with the axial mechanics, the mechanics begin to decrease significantly from Example 4 ( Figure 2 ).

[0124] The data of suture strength (Table 4) show that the suture strength decreases from Example 1 to Example 5, and the magnitude of the suture strength decreases significantly from Example 4.

[0125] The bursting pressure data (Table 4) also showed a similar pattern to the tension and suture strength. In particular, the bursting pressures in Examples 4 and 5 were both lower than the minimum requirement of 1600 mmHg for clinical vascular transplantation, indicating that the blood vessels prepared in Examples 4 and 5 did not have the potential for clinical application.

[0126] Table 4. Results of vascular mechanics, contact angle and N element content (parallel experiments, n = 3, mean ± standard error)

[0127] The contact angle results show that the contact angle of Comparative Example 1 is 122.11±1.85°. Compared with Comparative Example 1, the contact angles of Examples 1-5 are all reduced, indicating that the hydrophilicity of the blood vessels is increased ( Figure 4 ). The quantitative results of N element show that compared with comparative example 1, the content of N gradually increases from example 1 to example 5, indicating that amino groups are indeed introduced into the blood vessels, indicating that the aminolysis method is feasible and effective, and also confirms the change of contact angle.

[0128] The VEGF loading capacity, endothelial cell proliferation capacity, NO release level, and endothelial cell migration capacity of the artificial blood vessels prepared in Examples 1-10 and Comparative Examples 1-2 were analyzed by the following specific methods:

[0129] 1. VEGF loading

[0130] The VEGF Elisa kit was used to test the VEGF loading in the artificial blood vessel. The specific operation was as follows: the middle part (5 mm in length) was selected as the measurement sample in both the embodiment and the comparative example, and then the test was performed according to the instructions of the kit. At the end of the reaction, the absorbance value of the reaction solution at 450 nm was detected according to the instructions. The larger the absorbance value, the more VEGF loading.

[0131] 2. Endothelial cell proliferation

[0132] The vascular materials in the middle part of the embodiment and the comparative example were spread flat in a 48-well plate and then pressed with a film pressing ring. 3 HUVECs were added to the wells in the amount of 20 μg / mL. After culturing for 2 days, the absorbance of the supernatant at 450 nm was detected using CCK-8. The larger the absorbance, the better the cell proliferation.

[0133] 3. NO Release

[0134] The vascular materials in the middle part of the embodiment and the comparative example were spread flat in a 48-well plate and then pressed with a film pressing ring. 3 HUVECs were added to the wells in the amount of 10 μg / mL. After culturing for 2 days, the NO content in the supernatant was determined using a Griess Reaction kit. The final result was displayed as absorbance at 540 nm. The larger the absorbance value, the higher the NO content.

[0135] 4. Migration of endothelial cells

[0136] The blood vessels of the embodiment and the comparative example were cut in half from the middle, and the cut parts were marked. Then, the blood vessel materials were spread flat in a 6-well plate, and then pressed with a film pressing ring with a slot. A water blocking plate was placed through the slot at a position 1 / 3 of the distance from the unmarked end of the blood vessel material, and then 1×10 5 After HUVECs were placed in the incubator for 12 hours, the water blocking plate was removed and 1.5 ml of new culture medium was added. After culturing for 12 hours, the samples were fixed, stained and the migration efficiency of the cells was measured (migration efficiency = migration distance / distance from the indentation to the marked part*%).

[0137] 5. Data Analysis

[0138] The data were analyzed using GraphPad Prism 9.0.0 software, and all data were expressed as mean ± standard error.

[0139] Result analysis:

[0140] The loading amount of VEGF in Table 5 shows that compared with Comparative Example 1, the loading amount of VEGF gradually increases from Example 1 to Example 5, indicating that under the condition of constant VEGF concentration, the loading amount of VEGF changes with aminolysis. Compared with Comparative Example 2, the loading amount of VEGF also increases from Example 6 to Example 10, but no increase occurs after Example 8, indicating that under the condition of consistent aminolysis conditions, the loading of VEGF will have a saturation effect.

[0141] The proliferation data in Table 5 show that compared with Comparative Example 1, the values ​​of Examples 1 to 5 gradually increase, and are significant starting from Example 2. Compared with Comparative Example 2, the cell proliferation data from Examples 6 to 10 increase significantly, but do not change after Example 8.

[0142] The data of NO release in Table 5 show that compared with Comparative Example 1, the NO release from Example 1 to Example 5 gradually increases, and a significant change begins to appear from Example 2. Compared with Comparative Example 2, the NO release from Example 6 to Example 10 increases significantly, but there is no increase after Example 8.

[0143] The data of cell migration in Table 5 show similar patterns as cell proliferation and NO release, that is, compared with Comparative Example 1, the data from Example 1 to Example 5 show a gradual increase, and significant changes begin to appear from Example 2. Compared with Comparative Example 2, the migration rate increases significantly from Example 6 to Example 10, but there is no increase after Example 8.

[0144] Table 5. Microscopic and mechanical characteristics of blood vessels (parallel experiments, n = 3, mean ± standard error)

[0145] Animal Experimental Research

[0146] Based on the relevant data in Table 4 and Table 5, it can be seen that: Examples 3 and 8 are the same; Examples 4 and 5 have a burst pressure less than 1600 mmHg and exclude transplantation; the detection data of Examples 8, 9, and 10 are almost consistent with that of Example 3; therefore, based on the 3R principle of animal experiments, Comparative Example 1, Comparative Example 2, Examples 1-3, and Examples 6-7 were selected for animal experiments.

[0147] Rat abdominal aorta transplantation

[0148] After anesthetizing 250 g SD rats, the artificial blood vessel materials were cut into 1.1 cm pieces and implanted into the abdominal aorta of rats using 9-0 sutures in an end-to-end interrupted suture manner. One month after transplantation, the patency of the blood vessels was detected using Vevo 2100 Doppler ultrasound. After that, the samples were taken and the removed vascular tissues were cut longitudinally. A sample was placed in the tissue embedding agent OCT and quickly frozen with liquid nitrogen for frozen section analysis. Immunofluorescence staining was performed using CD31 antibody and the endothelial coverage was calculated. The endothelial coverage = CD31-positive length of the luminal surface of the longitudinal section of the vessel / total length of the luminal surface of the longitudinal section of the vessel × 100%.

[0149] Result analysis:

[0150] One month after implantation in rats, the endothelial coverage of Comparative Example 1 was 30.40±2.77% ( Figure 5 ), while the endothelial coverage of the examples all increased, especially the endothelial coverage of Example 3 reached 93.43±2.36% ( Figure 5 ), significantly higher than the comparative example and other embodiments ( Figure 3 ).

[0151] Table 6. Animal test results (parallel experiments, n = 3, mean ± standard error)

[0152] Based on the above in vivo / in vitro results, the gradient-modified artificial blood vessel prepared by the present invention has excellent performance, which can resist acute coagulation and accelerate the endothelialization rate of blood vessels.

[0153] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An artificial blood vessel with gradient modified endothelialization active factors, characterized in that: The artificial blood vessel has uniform heparin modification, and the active factor is modified in a gradient manner. The modification is characterized in that the concentration in the middle of the tubular graft is high, the concentration at both ends is low, and there is a gradient decrease from the middle to the two ends; that is, the concentration of the active factor of the artificial blood vessel gradually decreases from the middle to the two ends.

2. The artificial blood vessel with gradient-modified endothelial-promoting active factors according to claim 1, characterized in that: The active factors include vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), C-type natriuretic peptide (CNP) or basic fibroblast growth factor (b-Fibroblast Growth Factor, b-FGF).

3. A method for preparing an artificial blood vessel with gradient-modified endothelial-promoting active factors as claimed in claim 1, characterized in that: The steps include: The matrix material is dissolved in an organic solvent; a matrix solution is formed after the polymer material is completely dissolved; the matrix solution is then prepared into a common artificial blood vessel by electrospinning, and then the active factors are modified on the artificial blood vessel by covalent cross-linking (aminolysis-cross-linking reaction) to obtain an artificial blood vessel with gradient-modified active factors.

4. The method for preparing an artificial blood vessel with gradient-modified endothelial-promoting active factors according to claim 3, characterized in that: The aminolysis treatment is carried out using hexamethylenediamine, the concentration of the hexamethylenediamine is 0.1M-1M, and the treatment time is 30min.

5. The method for preparing an artificial blood vessel with gradient-modified endothelial-promoting active factors according to claim 3, characterized in that: The matrix material is a common polymer material used in artificial blood vessels.

6. The method for preparing an artificial blood vessel with gradient-modified endothelial-promoting active factors according to claim 5, characterized in that: The common polymer material for artificial blood vessels is at least one of polycaprolactone, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly (3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly-L-lactide-caprolactone, and polydioxanone, or a mixture of any proportions of several of them.

7. The method for preparing an artificial blood vessel with gradient-modified endothelial-promoting active factors according to claim 3, characterized in that: The organic solvent is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol is 5:

1.

8. The method for preparing an artificial blood vessel with gradient-modified endothelial-promoting active factors according to claim 3, characterized in that: The operation of modifying the active factor on the artificial blood vessel is as follows: placing the aminolysis-crosslinked artificial blood vessel material horizontally and rotating it at a constant speed, delivering the active factor solution to the middle part of the artificial blood vessel and keeping the artificial blood vessel rotating at a constant speed; wherein, The concentration of the active factor solution is 10-400 ng / mL, the delivery speed is 1 mL / h, and the delivery time is 30 min; and the horizontal rotation speed of the artificial blood vessel material is 10 rpm.

9. Use of the artificial blood vessel with gradient-modified endothelialization-promoting active factors as claimed in claim 1 or the artificial blood vessel with gradient-modified endothelialization-promoting active factors prepared by the method as claimed in claim 3 in the field of tissue engineering.

10. The use according to claim 9, characterized in that: The artificial blood vessel can change its shape to be used as a blood vessel patch, a valved blood vessel, or a heart valve.

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