Artificial blood vessels or membrane materials anchored to endothelial cells based on hydrophobic interactions, their preparation methods and applications

Abstract

This invention belongs to the field of biomaterials, specifically relating to an artificial blood vessel or membrane material anchored to endothelial cells based on hydrophobic interactions, its preparation method, and its application. The artificial blood vessel or membrane material anchored to endothelial cells based on hydrophobic interactions includes the artificial blood vessel or membrane material and PEGylated phospholipids modified on the surface of the artificial blood vessel or membrane material. This application utilizes the ability of phospholipid polyethylene glycol to embed into the cell membrane through hydrophobic interactions to anchor HUVECs, thereby achieving efficient and stable HUVEC implantation. This allows the artificial blood vessel / membrane to form a complete and stable endothelial layer before transplantation, and after implantation, it becomes an ideal artificial blood vessel graft with anticoagulant, antithrombotic, and long-term patency properties.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to an artificial blood vessel or membrane material for anchoring endothelial cells based on hydrophobic interactions, its preparation method, and its application. Background Technology

[0002] Vascular graft replacement is a primary surgical treatment for patients with advanced cardiovascular disease. Small-diameter artificial blood vessels (inner diameter <6mm) are in urgent need in clinical treatments such as coronary artery bypass grafting, peripheral vascular replacement below the knee, and liver / kidney transplantation. However, when used for small-diameter vascular grafts, the lack of bioactivity on the material surface easily induces thrombosis, intimal hyperplasia, and long-term vascular occlusion after implantation, resulting in consistently poor long-term patency rates and failing to meet the actual needs of clinical treatment.

[0003] Constructing a complete and functionally stable endothelial layer is a core approach to improving the blood compatibility of artificial blood vessels and reducing postoperative complications. In vitro pre-seeding of endothelial cells (ECs) allows artificial blood vessels to form an endothelial layer with anticoagulant and antiproliferative functions before implantation, representing a mainstream research direction for rapid endothelialization. However, conventional natural cell seeding methods have significant limitations: cells adhere to the material only through non-specific interactions, resulting in low binding efficiency, long incubation periods, and insufficient adhesion strength. After implantation, cells are easily detached due to physiological blood flow, making it difficult to form a continuous and complete endothelial layer, which greatly limits the in vivo application efficacy of artificial blood vessels. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an artificial blood vessel or membrane material for anchoring endothelial cells based on hydrophobic interactions, as well as its preparation method and application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An artificial blood vessel or membrane material for anchoring endothelial cells based on hydrophobic interactions includes the artificial blood vessel or membrane material and PEGylated phospholipids modified on the surface of the artificial blood vessel or membrane material. The phospholipids of the PEGylated phospholipids are embedded in the cell membrane through hydrophobic interactions, thereby anchoring endothelial cells and achieving rapid in vitro endothelialization.

[0006] The PEGylated phospholipid has a saturated grafting density of 0.356 ± 0.042 nmol / mg in artificial blood vessels or membrane materials.

[0007] The PEGylated phospholipids include one or more of DSPE-PEG, DOPE-PEG, DMPE-PEG, DPPE-PEG, DLPE-PEG, POPE-PEG, DOPC-PEG, or DSPC-PEG; preferably, the PEGylated phospholipids are DSPE-PEG2000.

[0008] The membrane material is obtained by spinning an inner spinning solution composed of a mixture of low molecular weight PCL and high molecular weight PCL. The artificial blood vessel comprises an inner layer, an outer layer, and an intermediate transition layer; the inner layer is obtained by spinning an inner layer spinning solution composed of a mixture of low molecular weight PCL and high molecular weight PCL; the outer layer is obtained by spinning an outer layer spinning solution of high molecular weight PCL; the transition layer is obtained by cross-spinning the inner layer spinning solution and the outer layer spinning solution; preferably, the Mw of the low molecular weight PCL is 2000; and the Mw of the high molecular weight PCL is 80000.

[0009] The present invention also includes a method for preparing the artificial blood vessel or membrane material based on hydrophobic interaction anchoring endothelial cells, comprising the following steps: modifying an amino-PEGylated phospholipid into an artificial blood vessel or membrane material containing carboxyl groups through chemical crosslinking; preferably, modifying it into the inner layer of an artificial blood vessel containing carboxyl groups; preferably, the amino-PEGylated phospholipid is DSPE-PEG2000-NH2.

[0010] Specifically, the steps include: immersing the artificial blood vessel or membrane material in an activation solution for activation; and adding the activated artificial blood vessel or membrane material to an aminoPEGylated phospholipid solution for reaction. Preferably, the concentration of aminoPEGylated phospholipid is 100-600 μmol / L; more preferably 400 μmol / L; the solvent for the aminoPEGylated phospholipid solution is PBS buffer. The activation solution is a mixture of EDC and NHS in a 1:1 molar ratio; the solvent of the activation solution is MES buffer.

[0011] The present invention also includes an application of the aforementioned artificial blood vessel or membrane material based on hydrophobic interactions to anchor endothelial cells.

[0012] Preferably, the method is applied to in vitro implantation and specifically includes the following steps: injecting an endothelial cell suspension into the artificial blood vessel or membrane material that anchors endothelial cells based on hydrophobic interactions, and incubating it in a cell culture incubator; preferably, the incubation time is 30-120 min; more preferably, it is 90 min.

[0013] Endothelial cell seeding density in artificial blood vessels or membrane materials: (1-5) × 10 6 pcs / cm 2 Preferably 3×10 6 pcs / cm 2 .

[0014] Compared with the prior art, the beneficial effects of the present invention are: This application utilizes PEGylated phospholipids to embed into the cell membrane through hydrophobic interactions to anchor HUVECs, thereby achieving efficient and stable HUVEC implantation. This allows the artificial blood vessel to form a complete and stable endothelial layer before transplantation, and after implantation, it becomes an ideal artificial blood vessel graft with anticoagulation, antithrombosis, and long-term patency. Attached Figure Description

[0015] Figure 1 The images shown are scanning electron microscope images of the electrospun PCL artificial blood vessel material constructed in Example 10. A is an overall image of the 2.0 mm diameter blood vessel, B is a magnified view of a part, C is an inner surface image, and D is an outer surface image. Figure 2 The diagram shows the cell adhesion of HUVECs in Comparative Examples 1-5 and Examples 1-5. A represents (1-5) × 10⁻⁶ cells. 6 pcs / cm 2 Representative CLSM images of adherent cells 2 h after gradient density seeding of HUVECs, B is the quantitative statistic of adherent cell density; Figure 3 Figure showing the growth capability analysis of HUVECs on electrospun fiber membranes; Figure 4 The images show the cell adhesion of HUVECs in Comparative Examples 6-9 and Examples 6-9. A is a CLSM image of cell adhesion at different incubation times after cell seeding, and B is a quantitative statistical graph of adherent cell density. Figure 5 The diagram shows the effect of DSPE-PEG2000-NH2 modification on the resistance of HUVECs to blood flow erosion on electrospun fiber tubes. A is a representative fluorescence image that changes over time, and B is a diagram showing the retention of HUVECs cells on the graft. Figure 6 The images shown are evaluation images of Comparative Example 10 after implantation into the abdominal aorta of rats 4 weeks later. A is an immunofluorescence image, and B is a scanning electron microscope image. Figure 7 The images shown are evaluation images of rat abdominal aorta implanted in Example 10 4 weeks later. A is an immunofluorescence image, and B is a scanning electron microscope image. Figure 8 CD31 for Comparative Example 10 and Example 10 + Endothelial cell coverage. Detailed Implementation

[0016] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments.

[0017] Example 1: A method for preparing a membrane material that embeds into the cell membrane based on hydrophobic interactions and thereby anchors endothelial cells, comprising the following steps: dissolving PCL (Mw = 2000) and PCL (Mw = 80000) in a 1:1 mass ratio in a mixed organic solvent of chloroform and methanol, preparing a 25% (w / v) PCL electrospinning solution as the inner layer spinning solution, and performing electrospinning to obtain the membrane material.

[0018] The modification of DSPE-PEG2000-NH2 membrane material via EDC / NHS coupling reaction includes the following steps: First, an activation solution (0.1 mol / L EDC + 0.1 mol / L NHS) is prepared using 0.5 M MES buffer. The dried electrospun PCL membrane material is then immersed in the activation solution and reacted for 2 h to obtain the activated PCL membrane material. The low molecular weight PCL (Mw = 2000) has a significantly higher carboxyl group density at the chain ends than the high molecular weight PCL alone (Mw = 80000), providing more reaction sites and thus effectively improving the EDC / NHS activation efficiency and the efficiency of subsequent amidation reactions.

[0019] To determine the optimal modification concentration of DSPE-PEG2000-NH2, a saturation concentration determination was performed beforehand: A series of concentration solutions (0, 10, 20, 50, 100, 200, 400, and 600 μmol / L) were prepared using DSPE-PEG2000-NH2 powder. Activated PCL membrane materials of the same specifications were immersed in each concentration solution and incubated for 2 h. After removing the PCL membrane materials, an equal amount of fluorescein (which specifically reacts with free amino groups to generate fluorescent products) was added to each solution. The fluorescence intensity was measured, and the remaining amino group concentration in the solution was calculated based on a standard curve. The difference between the initial amino group concentration and the remaining amino group concentration represents the amount of amino group consumed in the reaction. When the amount of amino group consumed tends to be constant (i.e., the amount of DSPE-PEG2000-NH2 bound no longer increases significantly with increasing concentration), it indicates that the modification has reached saturation. The experimentally determined saturation concentration was 400 μmol / L.

[0020] DSPE-PEG2000-NH2 powder was dissolved in PBS buffer (pH = 8.0) to prepare a 400 μmol / L crosslinking solution. The activated PCL membrane material was immersed in this crosslinking solution and reacted at room temperature for 2 h to allow the amino groups of DSPE-PEG2000-NH2 to undergo amidation with the activated carboxyl groups on the PCL surface, forming a covalent modification. The saturated modification density of DSPE-PEG2000 was 0.356 ± 0.042 nmol / mg.

[0021] PCL membrane material modified with DSPE-PEG2000 was placed in 48-well plates. Human umbilical vein endothelial cells (HUVECs) were seeded onto the PCL material under ECM basal culture conditions. The final seeding density of endothelial cells on the electrospun PCL artificial blood vessel material was set at 1×10⁻⁶. 6 pcs / cm 2 After incubation for 2 hours, wash away unbound cells.

[0022] Example 2: The only difference from Example 1 is that the final density of endothelial cells seeded on the electrospun PCL membrane material was set to 2 × 10⁻⁶. 6 pcs / cm 2 .

[0023] Example 3: The only difference from Example 1 is that the final density of endothelial cells seeded on the electrospun PCL membrane material was set to 3 × 10⁻⁶. 6 pcs / cm 2 .

[0024] Example 4: The only difference from Example 1 is that the final density of endothelial cells seeded on the electrospun PCL membrane material was set to 4 × 10⁻⁶. 6 pcs / cm 2 .

[0025] Example 5: The only difference from Example 1 is that the final density of endothelial cells seeded on the electrospun PCL membrane material was set to 5 × 10⁻⁶. 6 pcs / cm 2 .

[0026] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that the PCL membrane material prepared in Comparative Example 1 was not subsequently modified with DSPE-PEG2000-NH2. The final density of endothelial cells actually seeded on the electrospun PCL membrane material was set to 1×10⁻⁶. 6 pcs / cm 2 .

[0027] Comparative Example 2: The only difference from Comparative Example 1 is that the final density of endothelial cells seeded on the electrospun PCL membrane material was set to 2 × 10⁻⁶. 6 pcs / cm 2 .

[0028] Comparative Example 3: The only difference from Comparative Example 1 is that the final density of endothelial cells seeded on the electrospun PCL membrane material was set to 3 × 10⁻⁶. 6 pcs / cm 2 .

[0029] Comparative Example 4: The only difference from Comparative Example 1 is that the final density of endothelial cells seeded on the electrospun PCL membrane material was set to 4 × 10⁻⁶. 6 pcs / cm2 .

[0030] Comparative Example 5: The only difference from Comparative Example 1 is that the final density of endothelial cells seeded on the electrospun PCL membrane material was set to 5 × 10⁻⁶. 6 pcs / cm 2 .

[0031] Performance testing I. Analysis of the effects of different planting densities on the adhesion and growth capacity of endothelial cells on electrospun fiber membranes with optimal DSPE-PEG2000-NH2 modification density; The membrane materials of Examples 1-5 and Comparative Examples 1-5 were stained with DAPI and then observed for cell adhesion under a fluorescence microscope. Figure 2 The results were analyzed and quantitatively measured. The results showed that, since the comparative examples relied solely on natural cell adhesion and lacked the hydrophobic interaction process mediated by DSPE-PEG2000-NH2, the cell adhesion on the membrane materials of each comparative example was significantly lower than that of the corresponding examples. The number of adhered cells in Examples 1-3 gradually increased due to the hydrophobic interaction mediated by DSPE-PEG2000-NH2, and all three groups were significantly higher than their corresponding comparative examples. The number of cells adhered on the membrane materials of Examples 4 and 5 was comparable to that of Example 3, without further improvement. These results indicate that HUVECs can rapidly bind to DSPE-PEG2000-NH2 on the membrane material surface through hydrophobic interactions, achieving efficient and stable cell seeding. Moreover, the DSPE-PEG2000-NH2 modification density corresponding to Example 3 was sufficient to saturate HUVECs with adhesion.

[0032] II. Cytotoxicity analysis of electrospun fiber membranes with DSPE-PEG2000-NH2 modification density; The membrane materials of Example 3 and Comparative Example 3 were perforated and placed in a 48-well plate with an 8×10⁻⁶ diameter. 3 HUVECs were seeded per well and cultured in ECM medium, with the medium changed every 24 hours. Cell viability was assessed using a CCK8 assay after 1, 3, and 5 days of culture. Figure 3 The results showed that cells from both Example 3 and Comparative Example 3 continued to proliferate at each time point, and there was no statistically significant difference in cell viability between the two groups, indicating that the modification of DSPE-PEG2000-NH2 did not produce cytotoxicity to HUVECs.

[0033] III. Analysis of the effects of different incubation times on the adhesion and growth of endothelial cells on electrospun fiber membranes with optimal DSPE-PEG2000-NH2 modification density; Based on the above experiments, the membrane material of Example 3 was selected as having the optimal HUVECs seeding density. Furthermore, the effect of different incubation times on endothelial cell seeding was investigated. Specific examples and comparative cases are as follows: Example 6: Cell seeding was performed using the DSPE-PEG2000-NH2 modified membrane material prepared in Example 3. Endothelial cells were collected at 3 × 10⁻⁶ m² / h². 6 pcs / cm 2 The inoculation density was applied to the membrane material and incubated for 30 minutes for planting.

[0034] Example 7: Same as Example 6, but the cell seeding (incubation) time is 60 min.

[0035] Example 8: Same as Example 6, but the cell seeding (incubation) time is 90 min.

[0036] Example 9: Same as Example 6, but the cell seeding (incubation) time is 120 min.

[0037] Comparative Example 6: Cells were seeded using the membrane material without DSPE-PEG2000-NH2 modification prepared in Comparative Example 3, and endothelial cells were collected at 3 × 10⁻⁶. 6 pcs / cm 2 The inoculation density was applied to the membrane material and incubated for 30 minutes for planting.

[0038] Comparative Example 7: Same as Comparative Example 6, but the cell seeding (incubation) time was 60 min.

[0039] Comparative Example 8: Same as Comparative Example 6, but the cell seeding (incubation) time was 90 min.

[0040] Comparative Example 9: Same as Comparative Example 6, but the cell seeding (incubation) time was 120 min.

[0041] After washing away unbound cells in Examples 6-9 and Comparative Examples 6-9, DAPI staining was performed and the results were quantified. Figure 4 The results showed that cell adhesion on the comparative membrane material relied solely on natural adhesion, lacking the hydrophobic interactions mediated by DSPE-PEG2000-NH2, and its adhesion amount was significantly lower than that of the examples. With prolonged incubation time, the number of adhered cells in Examples 6-8 increased sequentially, and was significantly higher than that in the comparative example; the number of adhered cells in Example 9 was comparable to that in Example 8, without further improvement. These results indicate that DSPE-PEG2000-NH2 can promote rapid adhesion of HUVECs, reaching saturation adhesion after 90 min of incubation.

[0042] IV. Effect of DSPE-PEG2000-NH2 modification on the resistance of HUVECs to blood flow erosion on electrospun fiber tubes; Based on the above experiments, Example 8 (incubation for 90 min) was selected as the optimal cell seeding condition. To achieve quantitative fluorescence analysis of the residency ratio, HUVECs were labeled with the near-infrared fluorescent probe DiR and seeded at 3 × 10⁻⁶ cells / min. 6 / cm 2 Cells were seeded at a density equal to that of the DSPE-PEG2000-NH2 modified artificial blood vessel of Example 10 and the corresponding unmodified DSPE-PEG2000-NH2 artificial blood vessel of Example 10. The cells were incubated for 90 min, rotating 90° around the tube axis every 15 min to ensure uniform adhesion of cells to the inner wall of the lumen. The seeded artificial blood vessel material was then connected to an in vitro flow culture system. The flow rate of the culture medium in the in vitro flow culture device was set to 10 cm / s (simulating the human coronary artery blood flow velocity of 9.9 ± 3.5 cm / s). The artificial blood vessels seeded with endothelial cells were perfused at this flow rate for 72 h. Finally, the fluorescence intensity at 0 hours and after flushing was observed and quantitatively compared under in vivo fluorescence imaging. The ratio of fluorescence intensity after flushing to that before flushing was calculated as the cell retention rate. Figure 5 The results showed that the cell retention rate of the embodiment was significantly higher than that of the control group after 72 h of perfusion. These results indicate that DSPE-PEG2000-NH2-mediated cell adhesion can improve the cell's resistance to erosion.

[0043] The preparation method of the DSPE-PEG2000-NH2 modified artificial blood vessel in Example 10 includes the following steps: 1. Preparation of artificial blood vessels: 1) PCL (Mw = 2000) and PCL (Mw = 80000) were dissolved in a mixed organic solvent of chloroform and methanol at a mass ratio of 1:1 to prepare a 25% (w / v) PCL electrospinning solution as the inner layer spinning solution for the preparation of fine fibers. PCL (Mw = 80000) was dissolved in a mixed organic solvent of chloroform and methanol to prepare a 25% (w / v) PCL electrospinning solution as the outer layer spinning solution for the preparation of coarse fibers.

[0044] 2) Electrospun blood vessels were prepared based on the solution prepared in step 1): the inner layer was a fine fiber layer; the middle layer was a transition layer of coarse and fine fibers prepared by spinning two different spinning solutions; and the outer layer was a coarse fiber layer. The prepared PCL blood vessel material was used for subsequent modification and application. Figure 1 As shown.

[0045] 2. Modification of DSPE-PEG2000-NH2: First, prepare an activation solution (0.1 mol / L EDC + 0.1 mol / L NHS) using 0.5 M MES buffer. Immerse the dried electrospun PCL artificial blood vessel in the activation solution and react for 2 h to obtain the activated PCL artificial blood vessel. Dissolve the DSPE-PEG2000-NH2 powder in PBS buffer (pH = 8.0) to prepare a 400 μmol / L crosslinking solution. Immerse the activated PCL artificial blood vessel in the crosslinking solution and react at room temperature for 2 h to allow the amino groups of DSPE-PEG2000-NH2 to undergo an amidation reaction with the activated carboxyl groups on the PCL surface, forming a covalent modification.

[0046] Comparative Example 10: The only difference between Comparative Example 10 and Example 10 is that the artificial blood vessel is unmodified DSPE-PEG2000-NH2.

[0047] V. Vascular transplantation experiment; The artificial blood vessels of Example 10 (DSPE-PEG2000-NH2-mediated endothelial cell seeding) and Comparative Example 10 (unmodified PCL blank control) were transplanted into the abdominal aorta of rats, and the materials were harvested 4 weeks after implantation.

[0048] Macroscopic morphological observation: Comparative example 10 vascular grafts ( Figure 6 CD31 immunofluorescence staining showed that the luminal surface was almost entirely devoid of positive endothelial cells, and scanning electron microscopy (SEM) revealed exposed PCL fibers; Example 10: Vascular graft ( Figure 7 CD31 immunofluorescence staining showed that the lumen surface was covered with continuous and complete endothelial cells, and SEM observation showed that the cells were arranged in a typical cobblestone pattern with good morphology.

[0049] Quantitative statistical analysis: To further verify the promoting effect of implanted endothelial cells on the endothelialization rate of artificial blood vessels, CD31 on the luminal surface of the graft was analyzed. + Quantitative analysis of endothelial cell coverage was performed. Figure 8 The results showed that the CD31 positivity coverage rate in Comparative Example 10 was 18.74% ± 2.48%, while that in Example 10 was 88.43 ± 6.98%, with a statistically significant difference between the two groups. These results indicate that DSPE-PEG2000-NH2-mediated endothelial cell implantation can significantly improve the in vivo reendothelialization of vascular grafts, providing an effective strategy for the rapid endothelialization of artificial blood vessels.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An artificial blood vessel or membrane material for anchoring endothelial cells based on hydrophobic interactions, characterized in that, It includes artificial blood vessels or membrane materials and PEGylated phospholipids modified on the surface of the artificial blood vessels or membrane materials; the phospholipids of the PEGylated phospholipids are embedded in the cell membrane through hydrophobic interactions, thereby anchoring endothelial cells and achieving rapid in vitro endothelialization.

2. The artificial blood vessel or membrane material based on hydrophobic interactions to anchor endothelial cells according to claim 1, characterized in that, The saturated grafting density of the PEGylated phospholipid in artificial blood vessels or membranes is 0.356 ± 0.042 nmol / mg.

3. The artificial blood vessel or membrane material for anchoring endothelial cells based on hydrophobic interactions according to claim 1, characterized in that, The PEGylated phospholipids include one or more of DSPE-PEG, DOPE-PEG, DMPE-PEG, DPPE-PEG, DLPE-PEG, POPE-PEG, DOPC-PEG, or DSPC-PEG.

4. The artificial blood vessel or membrane material for anchoring endothelial cells based on hydrophobic interactions according to claim 1, characterized in that, The membrane material is obtained by spinning an inner spinning solution composed of a mixture of low molecular weight PCL and high molecular weight PCL. The artificial blood vessel comprises an inner layer, an outer layer, and an intermediate transition layer; the inner layer is obtained by spinning an inner layer spinning solution composed of a mixture of low molecular weight PCL and high molecular weight PCL; the outer layer is obtained by spinning an outer layer spinning solution of high molecular weight PCL; and the transition layer is obtained by spun together the inner layer spinning solution and the outer layer spinning solution. The low molecular weight PCL has an Mw of 2000; the high molecular weight PCL has an Mw of 80000.

5. A method for preparing an artificial blood vessel or membrane material based on hydrophobic interactions to anchor endothelial cells as described in any one of claims 1-4, characterized in that, The process includes the following steps: modifying amino-PEGylated phospholipids into artificial blood vessels or membrane materials containing carboxyl groups through chemical cross-linking.

6. The preparation method according to claim 5, characterized in that, Specifically, the steps include: immersing the artificial blood vessel or membrane material in an activation solution for activation; and adding the activated artificial blood vessel or membrane material to an aminoPEGylated phospholipid solution for reaction. The concentration of aminoPEGylated phospholipids is 100-600 μmol / L; the solvent for the aminoPEGylated phospholipid solution is PBS buffer. The activation solution is a mixture of EDC and NHS in a molar ratio of 1:1; the solvent of the activation solution is MES buffer.

7. The application of the artificial blood vessel or membrane material based on hydrophobic interactions to anchor endothelial cells according to any one of claims 1-4, characterized in that, This technology is used for in vitro seeding of endothelial cells, wherein the endothelial cells include one or more of the following: stem / progenitor cell-induced differentiated endothelial cells, human umbilical vein endothelial cells, or human arterial endothelial cells.

8. The application according to claim 7, characterized in that, Specifically, the steps include: injecting an endothelial cell suspension into the artificial blood vessel or membrane material that anchors endothelial cells based on hydrophobic interactions, and incubating it in a cell culture incubator.

9. The application according to claim 8, characterized in that, The incubation temperature was 37℃; the incubation time was 30-120 min; the density of endothelial cells seeded in artificial blood vessels or membrane materials was (1-5) × 10⁻⁶. 6 pcs / cm 2 .

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