Process for the preparation of endothelialized matrix artificial blood vessels

Abstract

The application provides a preparation method of an endothelium matrix vascular prosthesis. The method comprises the following steps: injecting a cell suspension into the inner wall of the vascular prosthesis to adhere, then supplying a culture medium under a perfusion circulation system to simulate an in-vivo vascular environment, so that an endothelium tissue is formed on the inner wall of the vascular prosthesis, and finally, the endothelium tissue vascular prosthesis is subjected to a decellularization treatment to obtain the endothelium matrix vascular prosthesis. The method can provide elasticity and mechanical properties for the vascular prosthesis by using a cell internal growth technology, and meanwhile, the adhesion and growth of cells are induced by using the topological structure of the vascular prosthesis. The method improves the biocompatibility of the vascular prosthesis while performing the endothelium matrix treatment on the vascular prosthesis, and finally, a biological vascular prosthesis with high elasticity, high mechanical strength and high biocompatibility is obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical textiles, and particularly relates to a preparation method of an endothelial matrix artificial blood vessel. BACKGROUND

[0002] The endothelial layer of blood vessels, as an internal barrier of blood vessels, plays a key role in many physiological and pathological processes. However, endothelial damage caused by artificial blood vessels and vascular stent implantation and the like can cause problems such as excessive proliferation of smooth muscle cells and activation of platelets, and ultimately cause intimal hyperplasia and thrombosis. Therefore, selectively promoting endothelial cell growth and promoting rapid endothelialization is one of the most effective strategies to maintain long-term patency of blood vessels. Rapidly forming an endothelial monolayer is the key to the success of manufacturing small-diameter vascular grafts, which is very important for treating dangerous and even fatal vascular diseases. However, it is a difficult problem to uniformly position endothelial cells on the inner lumen of a small-diameter tubular stent, which also increases the difficulty of preparing an endothelial artificial blood vessel.

[0003] In the prior art, the patent application file with the application number CN202310044325.9 and the publication date of March 17, 2023 and the name of "a functionalized degradable drug-loaded artificial bionic blood vessel and a preparation method thereof" uses electrospinning technology to prepare an artificial bionic blood vessel layer by layer, wherein the inner layer is a polyurethane urea-collagen layer, and the vascular endothelial growth factor is embedded; the middle layer is a polyurethane-collagen layer, which provides mechanical properties; and the outer layer is a polyurethane urea-collagen layer. In the above technical solution, the drug-loaded nanoparticles are prepared by mixing inorganic nanoparticles and drugs, and then electrospinning is performed after mixing polyurethane and collagen to obtain an artificial bionic blood vessel with a three-layer bionic structure, which improves the drug release property of the bionic blood vessel and improves the biocompatibility of the blood vessel. However, the drug release rate is uncontrollable, which can easily cause drug burst release, and can easily cause adverse symptoms such as massive hemorrhage and fever in the human body.

[0004] In addition, in the prior art, a patent document with the application number CN202110943333.8 and the publication date of November 16, 2021 and the name of "a blood vessel stent and a preparation method thereof" discloses that a heat-induced sensing type shape memory polymer is used as a blood vessel stent material, which can be deformed by the influence of human body temperature to support the two ends of the disconnected blood vessel and realize the smooth flow of blood; the addition of vascular endothelial growth factor in the blood vessel stent material can realize the recruitment of endothelial cells in the blood to accelerate the rate of endothelialization, so that the most superficial cells of the human body's vascular intima grow to the lumen surface of the artificial blood vessel to form a membrane-like tissue of endothelial cells; and the loading of heparin on the inner wall of the blood vessel stent can induce a reduction in the number of platelets to alleviate the symptoms of thrombosis. However, since the above process is to first prepare a blood vessel and then use the adhesion of cells in the body on the blood vessel to form endothelial tissue, the in-vivo endothelialization process is long, uncontrollable and complex in the drug loading preparation process, and it is difficult to achieve the expected goal of rapid endothelialization.

[0005] Therefore, it is necessary to design an improved preparation method of endothelialized artificial blood vessels to solve the above problems. SUMMARY

[0006] The purpose of the present application is to provide a preparation method of endothelialized artificial blood vessels.

[0007] To achieve the above-mentioned purpose of the application, the present application provides a preparation method of endothelialized artificial blood vessels, comprising the following steps:

[0008] S1, first preparing a blood vessel stent with a topological structure on the inner wall, then cutting the blood vessel stent to the required length and sterilizing to obtain a sterilized artificial blood vessel;

[0009] S2, transferring the cell suspension obtained by subculturing and digesting the cells in a sterile environment to a sterile syringe; then, connecting the artificial blood vessel prepared in step S1 between the connecting rubber tubes on the two end interfaces of the 3D perfusion circulating system; introducing the cell suspension in the sterile syringe into the artificial blood vessel, and the introduced cells deposit and adhere to the topological structure on the inner wall of the artificial blood vessel; after standing for 0-12 h, a preliminary cell-adhered artificial blood vessel is prepared;

[0010] S3, starting the 3D perfusion circulating system to make the preliminary cell-adhered artificial blood vessel obtained in step S3 rotate at a uniform speed, and then after decellularization, cleaning, sterilization and drying, an endothelialized artificial blood vessel is obtained.

[0011] Preferably, the topography is a groove with a density of 0.4-0.8 mm, a height of 0.2-0.4 mm, and a spiral distribution on the inner wall of the vascular stent; preferably, the endothelialized artificial blood vessel has a thickness of 1-2 mm and a diameter of 4-10 mm.

[0012] Preferably, in step S1, the artificial blood vessel has a length of 1-10 cm and a diameter of 4-8 mm.

[0013] Preferably, in step S2, the cell suspension has a density of 1×10 6 -5×10 5 cells / mL

[0014] Preferably, in step S2, the cell suspension is introduced into the artificial blood vessel at a rate of 0.1-0.5 mL / min.

[0015] Preferably, in step S2, the rotation speed of the uniform rotation is 6-15 r / min.

[0016] Preferably, in step S3, the decellularization treatment is performed as follows: the artificial blood vessel with intact endothelial tissue on the inner wall is placed in a CHAPS and Tris-HCl buffer, shaken on a shaker at a temperature of 25-30°C for 12-48 h to obtain a first treated artificial blood vessel; then, the first treated artificial blood vessel is washed with deionized water for 5-10 times, each time for 10-15 min, and then placed in a Tris-HCl buffer containing CHAPS, tributyl phosphate, sulfobetaine-14, and 3-(decyldimethylammonio)propane-1-sulfonate, shaken on a shaker at a temperature of 25-30°C for 12-48 h to obtain a second treated artificial blood vessel; then, the second treated artificial blood vessel is rinsed with a phosphate buffer for 3-5 times, each time for 5-10 h, and then placed in a Tris-HCl buffer containing MgCl2 and Benzonase nuclease, shaken on a shaker at a temperature of 25-30°C for 12-48 h, and then rinsed with the phosphate buffer again.

[0017] Preferably, in step S2, the cells are one of human umbilical vein endothelial cells, vascular endothelial cells, and human microvascular endothelial cells.

[0018] Preferably, in step S2, the artificial blood vessel is a small-diameter artificial blood vessel; preferably, the small-diameter artificial blood vessel is one of a polyurethane artificial blood vessel, a polyester artificial blood vessel, and a polycaprolactone artificial blood vessel.

[0019] Preferably, in step S3, the drying temperature is -50°C, and the drying time is 48-72 h.

[0020] The beneficial effects of the present application are:

[0021] 1. The preparation method of the endothelialized artificial blood vessel provided by the present application, by injecting cell suspension into the inner wall of the artificial blood vessel for adhesion, then providing culture medium under the perfusion circulation system to simulate the in-vivo vascular environment, so that the inner wall of the blood vessel forms a layer of endothelial tissue, and finally decellularizing the endothelial tissue artificial blood vessel to obtain the endothelialized artificial blood vessel, which provides the vascular endothelialization matrix and further improves the biocompatibility of the artificial blood vessel to meet the needs of practical application. Through the above process, the cell internal growth technology can be used to provide elasticity and mechanical properties for the artificial blood vessel, at the same time, the adhesion and growth of cells are induced by the topological structure of the artificial blood vessel, which improves the biocompatibility of the artificial blood vessel during the endothelialization process of the artificial blood vessel, and finally a biological artificial blood vessel with high elasticity, high mechanical strength and high biocompatibility is obtained.

[0022] 2. The preparation method of the endothelialized artificial blood vessel provided by the present application, by growing endothelial tissue based on the spiral arranged grooves formed on the inner wall of the artificial blood vessel during the preparation process, the cell flow in the groove can be used to quickly spread the entire inner wall of the artificial blood vessel during the process of injecting cell suspension into the artificial blood vessel and rotating, at the same time, the spiral grooves can limit the cells, so that the cells are uniformly and stably present on the inner wall of the artificial blood vessel, ensuring that the surface morphology of the formed endothelial tissue is uniform; by placing the artificial blood vessel in the 3D perfusion circulation system, the in-vivo environment and the expansion of the blood vessel are simulated by the circulation system, and the cells are uniformly distributed on the inner wall of the blood vessel by rotating during the endothelialization process, so that the cells are uniformly and completely formed in the artificial blood vessel, in addition, the circulation system can also provide nutrients for the proliferation and growth of cells in the artificial blood vessel, and collect the waste to ensure the normal endothelialization process, finally obtain the artificial blood vessel with natural endothelial tissue, and endow the artificial blood vessel with anticoagulant property and biocompatibility.

[0023] 3、The preparation method of the endothelialized matrix artificial blood vessel provided by the application, by selecting appropriate cell types for endothelialization treatment of the artificial blood vessel, not only facilitates the adhesion and growth of cells in the artificial blood vessel, but also ensures the biocompatibility of the finally prepared endothelialized matrix artificial blood vessel, at the same time, the use of water-soluble, fat-soluble, nucleic acid and other decellularization solvents for decellularization treatment eliminates the immunogenicity of the endothelialized artificial blood vessel, and provides space and induction for the in vivo cell regrowth on the blood vessel, so that the human cells can proliferate and cover on the endothelialized matrix artificial blood vessel quickly. The implementation of the above process is mainly realized by means of a 3D perfusion circulating system, which has the advantages of simple process, strong controllability, and industrial production; secondly, the length of the blood vessel and the cell concentration and culture time in the process of growing endothelial tissue are regulated to adjust the endothelialization degree of the blood vessel, and the interface length and width of the perfusion circulating system are adjusted to regulate the caliber and length of the blood vessel, so as to ensure that the prepared endothelialized matrix artificial blood vessel can meet the needs of different application scenarios, and has high application value. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 SEM image of the artificial blood vessel before endothelialization matrix;

[0025] Figure 2 Endothelialized matrix artificial blood vessel prepared in Example 1 of the application;

[0026] Figure 3 3D optical microscope schematic diagram of the endothelialized matrix artificial blood vessels prepared in Examples 1 to 5;

[0027] Figure 4 Cell activity after co-culture of the endothelialized matrix blood vessel and cells of the application;

[0028] Figure 5 Cycle proliferation graph of the endothelialized matrix artificial blood vessels prepared in Examples 1 to 5;

[0029] Figure 6 Cell activity of the inner wall of the artificial blood vessel under different adhesion times. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the application clearer, the application will be described in detail below with reference to the drawings and specific examples.

[0031] Here, it also needs to be explained that, in order to avoid obscuring the application due to unnecessary details, only the structures and / or processing steps closely related to the scheme of the application are shown in the drawings, and other details not closely related to the application are omitted.

[0032] It is also important to note that the term "comprising" or "including" or any other variation thereof is intended to cover a non-exclusive inclusion, such that processes, methods, articles, or apparatuses that comprise a list of elements are not necessarily limited to those elements, but can include other elements not expressly listed or inherent to such processes, methods, articles, or apparatuses.

[0033] Referring to Figure 1 As shown in the drawings, the present application provides a preparation method of endothelial matrix artificial blood vessel, comprising the following steps:

[0034] S1, preparation of artificial blood vessel: first, a vascular stent with a topological structure on the inner wall is prepared, and after being cut to the required length, it is subjected to sterilization treatment to obtain a sterilized artificial blood vessel;

[0035] S2, preparation of cell-adherent artificial blood vessel: in a sterile environment, the cell suspension obtained after subculture digestion of cells is transferred to a sterile syringe; then, the artificial blood vessel prepared in step S1 is connected between the connecting rubber tubes on the two end interfaces of a 3D perfusion circulating system, one of which is connected with a culture medium bottle containing nutrients, and the other is connected with a recovery bottle for transporting waste; the cell suspension is introduced into the artificial blood vessel through the sterile syringe, and the introduced cells are deposited and adhered to the topological structure on the inner wall of the artificial blood vessel; after standing for 0-12 h, a preliminary cell-adherent artificial blood vessel is prepared;

[0036] S3, formation of endothelial tissue on the inner wall of the artificial blood vessel: the 3D perfusion circulating system is started, and the preliminary cell-adherent artificial blood vessel obtained in step S3 is rotated at a uniform speed, so that the cells are uniformly distributed and grown inside the blood vessel, and finally an artificial blood vessel with complete inner wall growth and endothelial tissue is obtained; then, the artificial blood vessel is subjected to decellularization, washing, sterilization and drying in sequence, and an endothelial matrix artificial blood vessel is prepared.

[0037] Preferably, in step S1, the artificial blood vessel is a small-diameter artificial blood vessel, specifically one of polyurethane artificial blood vessel, polyester artificial blood vessel, and polycaprolactone artificial blood vessel.

[0038] Preferably, in step S1, the topological structure is a groove with a density of 0.4-0.8 mm and a height of 0.2-0.4 mm spirally distributed on the inner wall of the vascular stent, and the preparation method of the vascular stent with the groove is as follows: the artificial blood vessel is an inner wall radial groove structure artificial blood vessel prepared by wet spinning combined with winding.

[0039] Preferably, in step S1, the artificial blood vessel is 1-10 cm long, 4-8 mm in diameter, and 1 mm in wall thickness.

[0040] Preferably, in step S2, the density of the cell suspension is 1x106 -5 x 10 5 cells / mL.

[0041] Preferably, in step S2, the pushing speed of the cell suspension into the artificial blood vessel is 0.1-0.5 mL / min.

[0042] Preferably, in step S2, the rotating speed of the uniform rotation is 6-15 r / min. When the rotating speed is within the above range, the cells are uniformly distributed on the inner wall of the artificial blood vessel, and the thickness of the endothelial tissue obtained by the growth of the cells on the inner wall of the artificial blood vessel is uniform.

[0043] Preferably, in step S2, the cells are one of human umbilical vein endothelial cells, vascular endothelial cells, and human microvascular endothelial cells.

[0044] Preferably, in step S3, the decellularization treatment is performed as follows: the artificial blood vessel with the endothelial tissue grown on the inner wall is placed in a biological buffer (Tris-HCl buffer) containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid inner salt (CHAPS) and tributyl phosphate, and shaken on a shaker at a temperature of 25-30℃ for 24 h to obtain the artificial blood vessel after the first treatment; then, the artificial blood vessel after the first treatment is washed with deionized water for 5-10 times, each time for 10-15 min, and then placed in a Tris-HCl buffer containing CHAPS, tributyl phosphate, sulfobetaine-14, and 3-(decyldimethylammonio)propane-1-sulfonic acid inner salt, and shaken on a shaker at a temperature of 25-30℃ for 24 h to obtain the artificial blood vessel after the second treatment; then, the artificial blood vessel after the second treatment is rinsed with a phosphate buffer for 3-5 times, each time for 5-10 h, and then placed in a Tris-HCl buffer containing MgCl2 and Benzonase nuclease, and shaken on a shaker at a temperature of 25-30℃ for 24 h, and then the above rinsing step with the phosphate buffer is repeated.

[0045] Preferably, in step S3, the drying temperature is -50℃, and the drying time is 48-72 h.

[0046] Preferably, in step S3, the thickness of the endothelial matrix artificial blood vessel is 1-2 mm, and the diameter is 4-10 mm.

[0047] The preparation method of the endothelial matrix artificial blood vessel of the present application will be further described below in combination with specific examples:

[0048] Example 1

[0049] In this example, an endothelial matrix artificial blood vessel is prepared, and the specific preparation method comprises the following steps:

[0050] S1, Preparation of artificial blood vessel: first, a polyurethane blood vessel stent with an inner wall having a density of 0.4 mm and a height of 0.2 ditch and a diameter of 5 mm was prepared, cut to 8 cm and then sterilized to obtain a sterile artificial blood vessel;

[0051] S2, Preparation of cell-adhesive artificial blood vessel: in a sterile environment, human umbilical vein endothelial cells were subcultured and digested, 5 mL of medium was added for blowing and resuspension to obtain a cell suspension with a density of 1 x 10 6 cells / mL, the cell suspension was transferred to a sterile syringe; then, the artificial blood vessel prepared in step S1 was connected between the connecting rubber tubes on the two end interfaces of the 3D perfusion circulating system, one of which was connected to the medium bottle containing nutrients, and the other was connected to the recovery bottle for transporting waste; the cell suspension was introduced into the artificial blood vessel at a pushing speed of 0.1 mL / min, the introduced cells were deposited and adhered to the topological structure of the inner wall of the artificial blood vessel, and after standing for 4 h, a preliminary cell-adhesive artificial blood vessel was prepared;

[0052] S3, Formation of endothelial tissue in the inner wall of artificial blood vessel: the 3D perfusion circulating system was started, and the preliminary cell-adhesive artificial blood vessel obtained in step S3 was rotated at a speed of 6 r / min to make the cells uniformly distributed and grown inside the blood vessel, and finally an artificial blood vessel with complete inner wall growth and endothelial tissue was obtained; then, the artificial blood vessel was subjected to decellularization, washing, sterilization and drying in sequence to obtain an endothelialized artificial blood vessel. The sterilization was performed in an ethylene oxide drying cabinet; the decellularization process specifically included the following steps: the complete endothelial tissue artificial blood vessel was placed in a CHAPS solution with a concentration of 2% and a Tris-HCl buffer solution with a concentration of 2 mmol / L and pH = 7.8, and was shaken on a shaker at room temperature for 24 h; then it was rinsed in deionized water for 6 times, each time for more than 10 min; it was placed in a Tris-HCl buffer solution containing 2% CHAPS, 2 mmol / L tributyl phosphate and 1% ASB-14 and 2% SB3-10, and was continuously shaken at room temperature for 24 h; then it was taken out and rinsed in phosphate buffer solution (PBS) for 4 times, each time for 6 h; then it was placed in a Tris-HCl buffer solution containing 1 mmol / L MgCl2 and 100 units / mL Benzonase nuclease with pH = 8, and was continuously shaken at room temperature for 24 h; finally, it was rinsed with PBS for 4 times, each time for 6 h, to obtain an endothelialized artificial blood vessel.

[0053] The SEM image of the endothelialized artificial blood vessel prepared in this example is shown in Figure 2 , and the comparison Figure 1The SEM image of the artificial blood vessel before endothelial matrix shows that the grooves of the inner wall of the artificial blood vessel are completely covered after endothelial treatment, and a complete and uniform endothelial tissue layer is formed.

[0054] Examples 2 to 3

[0055] Examples 2 to 3 are different from Example 1 only in that in step S2, the fiber density of the artificial blood vessel is different from that of Example 1, and the other steps are basically the same as those of Example 1, which will not be repeated here. The fiber density of the artificial blood vessel in Examples 1 to 3 and the performance of the endothelial matrix artificial blood vessel prepared under the corresponding conditions are shown in Table 1. As can be seen from the table, the cell activity of the artificial blood vessel with a fiber density of 0.4 mm is the best. The fiber density determines the distribution of the groove structure, and the fiber density of 0.4 mm is the most suitable for cell adhesion and proliferation. Figure 3 As can be seen from the table, the inner wall with different densities presents different size groove structures, which has a certain influence on cell adhesion and proliferation. As can be seen from the table, Figure 4 As can be seen from the table, the cell density of the endothelial matrix artificial blood vessel increases with time, gradually covering the inner surface of the artificial blood vessel. The cell activity of the endothelial matrix artificial blood vessel prepared under different fiber densities is shown in Table 1 and Figure 5 As can be seen from the table, Figure 5 As can be seen from the table, the cell activity of the artificial blood vessel with a fiber density of 0.4 mm is 62.2%, which has the highest cell activity. Compared with the adhesion rate of 15.3% of the pure PU group, the endothelial matrix artificial blood vessel prepared in this application shows better biocompatibility. At the same time, compared with the TCP hole plate control group, the endothelial matrix artificial blood vessel prepared in this application also shows higher cell activity.

[0056] Table 1 Fiber density of artificial blood vessel in Examples 1 to 3 and performance of endothelial matrix artificial blood vessel prepared under corresponding conditions

[0057] Item Fiber density (mm) Endothelial basal matrix human vascular cell activity (%) Example 1 0.4 62.2 Example 2 0.5 57.2 Example 3 0.6 55.6 Example 4 0.7 45.4 Example 5 0.8 39.1

[0058] Examples 6 to 7

[0059] Examples 6 to 7 are different from Example 1 only in that in step S2, the adhesion time of the cell suspension introduced into the artificial blood vessel is different from that of Example 1, and the other steps are basically the same as those of Example 1, which will not be repeated here. The performance of the endothelial matrix artificial blood vessel of Example 1 and Examples 6 to 7 and the corresponding conditions is shown in Table 2. As can be seen from the table, the longer the cell adhesion time in the early stage, the greater the cell density on the inner wall of the artificial blood vessel. As shown in Figure 6 As shown in the table, the cell density on the preliminary adhesion artificial blood vessel obtained when the cell adhesion time in the early stage is 12h is 100%. The cells adhered in the early stage are sufficient to cover the groove structure of the inner wall of the blood vessel, providing a basis for the subsequent cell proliferation to form endothelial tissue.

[0060] Table 2 Performance of endothelialized artificial blood vessels prepared by pushing and corresponding conditions of Example 1 and Examples 6-7

[0061] Item Adhesion time (h) Cell density (%) Example 1 4 70 Example 6 0 20 Example 7 12 100

[0062] In summary, the preparation method of the endothelialized artificial blood vessel provided by the present application, by injecting cell suspension into the inner wall of the artificial blood vessel for adhesion, then providing culture medium under the perfusion type circulation system to simulate the in-vivo blood vessel environment, so that a layer of endothelial tissue is formed on the inner wall of the blood vessel, and finally the endothelial tissue artificial blood vessel is subjected to decellularization treatment to obtain the endothelialized artificial blood vessel, which provides the blood vessel endothelialization matrix and further improves the biocompatibility of the artificial blood vessel to meet the needs of practical application. Through the above process, the cell internal growth technology can be used to provide the artificial blood vessel with elasticity and mechanical properties, at the same time, the adhesion and growth of cells are induced by the topological structure of the artificial blood vessel, and the biocompatibility of the artificial blood vessel is improved while the artificial blood vessel is subjected to endothelialization treatment, finally the biological artificial blood vessel with high elasticity, high mechanical strength and high biocompatibility is obtained, and a preparation method of the endothelialized artificial blood vessel with simple process, strong controllability and industrial production is provided.

[0063] The above examples are only used to illustrate the technical solutions of the present application and not to limit it, although the present application 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 application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing an endothelial matrix-modified artificial blood vessel, characterized in that, Includes the following steps: S1. First, prepare a vascular stent with a topological structure on the inner wall, then cut the vascular stent to the required length and sterilize it to obtain a sterile artificial blood vessel. S2. Under sterile conditions, the cell suspension obtained after cell passage and digestion is transferred to a sterile syringe; then, the artificial blood vessel prepared in step S1 is connected between the connecting tubing at both ends of the 3D perfusion circulation system; then, the cell suspension in the sterile syringe is introduced into the artificial blood vessel, and the introduced cells are deposited and adhered to the topology of the inner wall of the artificial blood vessel. After standing for 0-12 hours, a preliminary cell-adhered artificial blood vessel is obtained. S3. Activate the 3D perfusion circulation system to make the artificial blood vessel with preliminary cell adhesion obtained in step S2 rotate at a uniform speed. After decellularization, washing, sterilization and freeze-drying, endothelial matrix-modified artificial blood vessel is obtained. The topological structure consists of grooves with a density of 0.4-0.8 mm and a height of 0.2-0.4 mm, spirally distributed on the inner wall of the vascular stent; the endothelial matrix artificial blood vessel has a thickness of 1-2 mm and a diameter of 4-10 mm. The cell suspension is introduced into the artificial blood vessel at a rate of 0.1-0.5 mL / min; The rotational speed of the uniform rotation is 6-15 r / min.

2. The method for preparing endothelial matrix-modified artificial blood vessels according to claim 1, characterized in that, In step S1, the artificial blood vessel is 1-10 cm long and 4-8 mm in diameter.

3. The method for preparing endothelial matrix-modified artificial blood vessels according to claim 1, characterized in that, In step S2, the density of the cell suspension is 1×10⁻⁶. 6 -5×10 5 cells / mL.

4. The method for preparing endothelial matrix-modified artificial blood vessels according to claim 1, characterized in that, In step S3, the decellularization process is performed as follows: Artificial blood vessels with intact endothelial tissue growing on their inner walls are placed in a CHAPS and Tris-HCl buffer solution and shaken on a shaker at 25-30°C for 12-48 hours to obtain the first-treated artificial blood vessel. Next, the first-treated artificial blood vessel is washed 5-10 times with deionized water, each wash lasting 10-15 minutes. It is then placed in a Tris-HCl buffer solution containing CHAPS, tributyl phosphate, amidosulfobetaine-14, and 3-(decyldimethylammonium)propane-1-sulfonic acid inner salt, and shaken on a shaker at 25-30°C for 12-48 hours to obtain the second-treated artificial blood vessel. Then, the second-treated artificial blood vessel is rinsed 3-5 times with phosphate buffer, each rinse lasting 5-10 hours. After rinsing, it is placed in a Tris-HCl buffer solution containing MgCl2 and Benzonase nuclease and shaken on a shaker at 25-30°C for 12-48 hours. The rinsing process is repeated using the phosphate buffer solution.

5. The method for preparing endothelial matrix-modified artificial blood vessels according to claim 1, characterized in that, In step S2, the cells are vascular endothelial cells.

6. The method for preparing endothelial matrix-modified artificial blood vessels according to claim 1, characterized in that, In step S2, the artificial blood vessel is a small-diameter artificial blood vessel; the small-diameter artificial blood vessel is one of polyurethane artificial blood vessels, polyester artificial blood vessels, and polycaprolactone artificial blood vessels.

7. The method for preparing endothelial matrix-modified artificial blood vessels according to claim 1, characterized in that, In step S3, the drying temperature is -50 ℃ and the drying time is 48-72 h.

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