Polymer artificial blood vessel or membrane material for rapid stabilization of endothelialization and preparation method and application thereof

Abstract

The application belongs to the field of biological materials, and particularly relates to a polymer artificial blood vessel or membrane material for realizing endothelialization quickly and stably, and a preparation method and application thereof. The polymer artificial blood vessel or membrane material comprises a polymer artificial blood vessel or membrane material and endothelial cells crosslinked on the surface of the polymer artificial blood vessel or membrane material; the polymer artificial blood vessel or membrane material and the endothelial cells are crosslinked and connected through alkyne groups and azido groups. The technical scheme of the application utilizes biological orthogonal reaction to mediate the quick and stable combination between the surface modified polymer artificial blood vessel or membrane material and the azido labeled endothelial cells, so that the artificial blood vessel forms a complete and stable endothelial layer before transplantation, and becomes an ideal artificial blood vessel graft with anticoagulation, antithrombosis and long-term patency after being implanted into the body.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a polymeric artificial blood vessel or membrane material that rapidly and stably achieves endothelialization, its preparation method, and its application. Background Technology

[0002] Vascular graft replacement is a primary surgical treatment for patients with advanced cardiovascular disease. Autologous blood vessels are the gold standard in vascular surgery; however, many patients lack sufficient autologous blood vessel donors due to pre-existing conditions, trauma, or size mismatch. Clinically, surgeries such as coronary artery bypass grafting, peripheral vascular replacement below the knee, and liver or kidney transplantation require the use of small-diameter (<6mm) vessels. However, current research on their application shows unsatisfactory efficacy, mainly manifested in transplant failure due to problems such as thrombosis and intimal hyperplasia after implantation. The main reason for this predicament is the lack of rapid functional endothelialization within the graft.

[0003] In natural arteries, endothelial cells (ECs) play a crucial role in inhibiting thrombus formation, suppressing excessive proliferation of smooth muscle cells (SMCs), and maintaining their contractile phenotype. Therefore, timely endothelialization of small-diameter artificial vascular grafts is an effective way to improve graft patency and regenerative capacity. Compared to promoting uncertain in-situ endothelialization of artificial vascular grafts in vivo, constructing artificial blood vessels pre-implanted with ECs in vitro has been widely proven to immediately improve blood compatibility and increase post-transplant patency. However, current in vitro endothelialization techniques mainly rely on natural cell adhesion. To achieve sufficient EC coverage, cell seeding of at least two days or even longer and a large number of healthy ECs are required. Almost all existing in vitro endothelialization techniques cannot meet the immediate preparation requirements of vascular surgery, and generally suffer from key problems such as long seeding time and poor cell retention, limiting their translational application in clinical practice. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a polymeric artificial blood vessel or membrane material that can rapidly and stably achieve endothelialization, 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:

[0006] A polymeric artificial blood vessel or membrane material that rapidly and stably achieves endothelialization includes the polymeric artificial blood vessel or membrane material and endothelial cells cross-linked on the surface of the polymeric artificial blood vessel or membrane material; the polymeric artificial blood vessel or membrane material and the endothelial cells are cross-linked through alkyne and azide groups.

[0007] The surface of the polymer artificial blood vessel or membrane material is modified with an alkynyl compound; the alkynyl compound is cross-linked on the surface of the polymer artificial blood vessel or membrane material through an EDC / NHS reaction;

[0008] The grafting density of alkynyl compounds on the surface of polymer artificial blood vessels or membrane materials is 0.045-0.72 nmol / mg.

[0009] Preferably, the grafting density of the alkynyl compound on the surface of the polymer artificial blood vessel or membrane material is 0.09-0.36 nmol / mg; more preferably, it is 0.18 nmol / mg.

[0010] The alkynyl compound is at least one of dibenzocyclooctyne (DBCO), 4-dibenzocyclooctyl alcohol (DIBO), or dicyclononyne (DIBAC), preferably dibenzocyclooctyne (DBCO).

[0011] The endothelial cell surface is modified with an azide compound; preferably, the endothelial cells are modified with an azide compound through the principle of glucose metabolism.

[0012] The endothelial cells are endothelial cells iPSC-ECs differentiated from induced pluripotent stem cells, endothelial cells ESC-ECs differentiated from embryonic stem cells, endothelial progenitor cells or circulating monocytes differentiated from endothelial cells, human umbilical vein endothelial cells HUVECs, or endothelial cells isolated from natural vein donors, etc.; the azido group compound is tetraacetyl-N-azidoacetylmannosamine.

[0013] The polymer artificial blood vessel or membrane is made of synthetic or natural polymer materials; preferably, the synthetic polymer material is a mixture of at least one or more of polycaprolactone, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly(3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly-L-lactide-caprolactone, and polydioxane-hexanone in any proportion; the natural polymer material is one or more of gelatin, collagen, hyaluronic acid, and fibrin glue.

[0014] The present invention also includes a method for preparing a polymeric artificial blood vessel or membrane material that rapidly and stably achieves endothelialization, characterized by comprising the following steps: 1) preparing a polymeric artificial blood vessel or membrane material with a surface modified with an alkyne compound; 2) preparing endothelial cells with a surface modified with an azide compound; inoculating the endothelial cells with azide compound onto the polymeric artificial blood vessel or membrane material with an alkyne compound and incubating them to obtain a polymeric artificial blood vessel or membrane material that rapidly and stably achieves endothelialization;

[0015] Preferably, endothelial cells with azide-based compounds on their surface are used at a concentration of 3 × 10⁻⁶. 5 pcs / cm 2For polymeric artificial blood vessels or membrane materials with surface-modified alkyne compounds, the inoculation density is appropriate; the incubation time is 60-150 min; preferably 120 min.

[0016] The present invention also includes the application of the polymeric artificial blood vessel or membrane material that achieves rapid and stable endothelialization.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] Traditional static endothelial cell implantation is time-consuming and has weak erosion resistance. The technical solution of this application utilizes bioorthogonal reaction to mediate the rapid and stable binding between surface-modified polymer artificial blood vessel or membrane material and azide-labeled endothelial cells, so that the artificial blood vessel forms a complete and stable endothelial layer before transplantation, and becomes an ideal artificial blood vessel graft with anticoagulation, antithrombosis and long-term patency after implantation.

[0019] Bioorthogonal chemistry exhibits excellent biocompatibility, specificity, and speed under physiological conditions, representing an advanced method for rapidly forming covalent bonds applicable to living cells. This application combines cell modification techniques from metabolic glycoengineering to non-destructively anchor bioorthogonal azide groups to the cell surface, providing a foundation for bioorthogonal reactions. The combination of bioorthogonal chemistry and living-cell metabolic glycoengineering serves as a chemical "bridge" to facilitate rapid endothelial cell implantation for artificial blood vessels. This endothelial cell implantation process accelerates the in vitro preparation of endothelialized artificial blood vessels, resolving the challenges in the development and transformation of small-diameter artificial blood vessels. Attached Figure Description

[0020] Figure 1 Scanning electron microscope images of the electrospun PCL artificial blood vessel and membrane material constructed in Example 3.

[0021] Figure 2 Comparative Example 2, Examples 1-5: iPSC-ECs cell adhesion at different cell seeding densities.

[0022] Figure 3 The image shows the evaluation results of mouse carotid artery implantation 4 weeks after Comparative Example 4.

[0023] Figure 4 The image shows the evaluation results of mouse carotid artery implantation 4 weeks after Example 8. Detailed Implementation

[0024] 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.

[0025] Example:

[0026] 1. Preparation of artificial blood vessel materials with surface-modified polymers containing alkyne compounds;

[0027] 1) PCL was dissolved in a mixed organic solution of chloroform and methanol to prepare 10% and 25% PCL (MW = 80,000) electrospinning solutions, respectively. Electrospinning filament layers were prepared using the 10% PCL solution, and then immersed in 1 mol / L NaOH solution for alkaline hydrolysis for 6 h. The reaction was terminated in 10 mmol / L glacial acetic acid solution for 30 min to expose carboxyl groups available for subsequent modification.

[0028] 2) Based on the fine fiber layer in 1), it is further subjected to bidirectional spinning with 10% and 25% PCL solutions, and then unidirectional spinning with 25% PCL for subsequent modification and use.

[0029] 3) Next, the carboxyl groups on the inner surface of the electrospun PCL artificial blood vessel were fully activated into NHS groups in a 0.1M EDC / NHS reaction solution prepared with MES buffer. Subsequently, it was cross-linked with dibenzocyclooctene groups with amino-terminal ends (abbreviated as DBCO). Based on different DBCO modification concentrations, electrospun PCL artificial blood vessels with actual DBCO modification densities of 0.045±0.004 nmol / mg (Example 1), 0.09±0.015 nmol / mg (Example 2), 0.18±0.033 nmol / mg (Example 3), 0.36±0.071 nmol / mg (Example 4), and 0.72±0.123 nmol / mg (Example 5) were finally prepared. At the same time, the corresponding modified PCL membrane materials were prepared for the evaluation of cell seeding conditions, the artificial blood vessel with an inner diameter of 2.0 mm was used for the stability evaluation in the perfusion bioreactor, and the artificial blood vessel with an inner diameter of 0.7 mm was used for the in vivo evaluation of mouse carotid artery transplantation.

[0030] Comparative Example 1: PCL was dissolved in a mixed organic solution of chloroform and methanol to prepare electrospinning solutions of 10% fine filaments and 25% coarse filaments (MW = 80,000). The inner layer of electrospun fine filaments was prepared using the 10% PCL solution, followed by bidirectional spinning with 10% and 25% PCL solutions, and then unidirectional spinning with 25% PCL to prepare an artificial blood vessel with a composite structure of inner fine filaments and outer coarse filaments. No modification was performed during the process. The corresponding untreated PCL membrane material was used to evaluate cell seeding conditions.

[0031] The results showed that the vascular materials obtained in Examples 1-5 and Comparative Example 1 all had good fibrous morphology. For example, as... Figure 1As shown, the polymer artificial blood vessels with a diameter of 0.7 mm (for mouse carotid artery transplantation) and 2 mm (for in vitro erosion resistance experiments) prepared in Example 3 exhibit a composite structure of inner thin filaments and outer coarse filaments.

[0032] 2. Endothelial cells with surface-modified azide compounds were seeded into polymer artificial blood vessels with surface-modified alkynyl compounds and incubated to obtain polymer artificial blood vessels that rapidly and stably achieve endothelialization.

[0033] 2.1 Analysis of the adhesion and growth capacity of iPSCs-ECs planted on PCL fiber membranes with different DBCO modification densities.

[0034] Polymer membrane materials from Examples 1-5 and Comparative Example 1 (hereinafter referred to as membrane materials) were perforated and placed in 48-well plates in ECM basal medium. After iPSC-ECs were treated with 20 μM Ac4ManNAz for 48 h of sugar metabolism, their surfaces were modified with azide compounds. Cells were collected and cultured at 3 × 10⁻⁶ cells / well. 5 pcs / cm 2 The density was seeded onto the membrane materials of Examples 1-5 and Comparative Example 1.

[0035] Simultaneously, a comparative example 2 was set up: after perforating the membrane material of comparative example 1, it was placed in a 48-well plate in ECM basal medium, and cells were seeded using untreated iPSC-ECs at a concentration of 3 × 10⁻⁶. 5 pcs / cm 2 Cells were seeded at a density equal to that of Comparative Example 1 polymer material. After incubation for 2 hours, unbound cells were washed away, and DAPI staining was used to visualize the results. Figure 2 The adhesion patterns were observed and statistically analyzed. Results showed that the cell adhesion amounts of the membrane materials in Example 1, Comparative Example 1, and Comparative Example 2 were extremely low, with no significant difference among the three. In Examples 2 and 3, the number of adhered cells increased with increasing surface azide group modification concentration, and both groups were significantly higher than the number of cells adhered to the membrane materials in Examples 1, Comparative Example 1, and Comparative Example 2. The number of cells adhered to the membrane materials in Examples 4 and 5 was comparable to that in Example 3, without further improvement. These results indicate that azide-modified iPSC-ECs can rapidly undergo a bioorthogonal reaction with DBCO on the membrane material, thereby achieving rapid and stable cell seeding. Furthermore, the DBCO modification density on the surface of the material in Example 3 allows cells to achieve saturated adhesion efficiency.

[0036] The membrane materials from Examples 1-5 and Comparative Example 1 were perforated and placed in 48-well plates. After iPSC-ECs were treated with 20 μM Ac4 ManNAz for 48 hours to induce glucose metabolism, their surfaces were modified with an azide compound. Cells were collected and seeded at 8000 cells / well, with the ECM medium changed daily. Comparative Example 2 was also set up: iPSC-ECs without glucose metabolism treatment were used for cell seeding, with other conditions the same as Comparative Example 1. CCK8 assays were performed after 1, 3, and 5 days of culture. The results showed that cells on the membrane materials of Examples 1-3, Comparative Example 1, and Comparative Example 2 continued to proliferate. The proliferation rates of Examples 1, Comparative Example 1, and Comparative Example 2 were comparable, while the proliferation rates of Examples 2-4 were significantly better than that of Example 1. Optimal cell proliferation was observed in Example 3. Cells on the membrane material of Example 5 showed virtually no proliferation, and the proliferation level on day 5 was lower than that of Comparative Example 1, indicating that excessively high DBCO modification density can cause cytotoxicity.

[0037] The adhesion and growth capabilities of endothelial cells with different azide group modification densities on PCL fiber membranes are shown in Table 1.

[0038] Table 1

[0039]

[0040] 2.2 The resistance of endothelial cells to blood flow erosion on PCL artificial blood vessels with different DBCO-modified densities.

[0041] After iPSC-ECs were treated with 20 μM Ac4ManNAz for 48 hours, their surface was modified with azide groups. Cells were then collected for DiR labeling and analyzed at 3 × 10⁻⁶. 5 pcs / cm 2 The artificial blood vessel materials with a diameter of 2.0 mm prepared in Examples 1-5 and Comparative Example 1 were seeded at the appropriate seeding density and incubated for 2 hours. The cells were rotated 90° every 15 minutes to ensure uniform seeding. Comparative Example 2 was also set up: cells were seeded using DiR-labeled iPSC-ECs without glucose metabolism treatment, along with the unmodified polymer material obtained in Comparative Example 1. The flow rate of the culture medium in the in vitro flow culture device was set to 10 cm / s to simulate the blood flow velocity of the human coronary artery. The artificial blood vessels seeded with endothelial cells were perfused at this flow rate for 72 hours. 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. It was found that the cell retention rate of the artificial blood vessel materials in Examples 1, 4, and 5 was comparable to that of the artificial blood vessels in Comparative Examples 1 and 2; the cell retention rates of Examples 2 and 3 were significantly higher than those of Comparative Examples 1, 2, 1, 4, and 5, respectively; among them, the artificial blood vessel in Example 3 had the highest cell retention rate.

[0042] The results of the analysis of the resistance to blood flow erosion of endothelial cells implanted on artificial blood vessels with different DBCO modification densities are shown in Table 2.

[0043] Table 2

[0044] Comparative Example 1 10.34±2.89 Comparative Example 2 10.54±3.65 Example 1 12.89±3.14 Example 2 50.56±6.43 Example 3 73.30±8.36 Example 4 14.21±7.23 Example 5 13.81±2.69

[0045] 2.3 Vascular transplantation experiment: After iPSC-ECs were treated with 20 μM Ac4ManNAz glucose metabolism for 48 hours, their surface was modified with azide groups. Cells were then collected at 3 × 10⁻⁶ cells / year. 5 pcs / cm 2 The cells were seeded at the specified density into 0.7 mm diameter artificial blood vessel materials prepared in Examples 1-5 and Comparative Example 1, and incubated for 2 hours, rotating 90° every 15 minutes. Comparative Example 2 was also set up: DiR-labeled iPSC-ECs without glucose metabolism treatment were used for cell seeding, with other conditions the same as Comparative Example 1. After seeding, the cells were transplanted into the carotid arteries of nude mice. Four weeks after implantation, the patency rates were 0% for Comparative Examples 1 and 2, 0% for Example 1, 60% for Examples 2 and 4, 100% for Example 3, and 20% for Example 5. To evaluate the effect of implanted endothelial cells on the endothelialization rate of the artificial blood vessel, we analyzed the CD31 of the grafts. + Statistical analysis of endothelial cell coverage was performed. The results showed that the endothelial coverage of Example 1, Comparative Example 2, and Example 1 was 0%; the endothelial coverage of Example 2 was 51.23±8.63%; the highest endothelial coverage of Example 3 was 91.39±6.15%; the endothelial coverage of Example 4 was 55.53±9.87%; and the endothelial coverage of Example 5 was 4.82±5.42% (Table 3). These staining results indicate that the endothelial cells implanted under the conditions of Example 3 were most effective in resisting blood flow erosion and promoting the degree of reendothelialization of the vascular graft in the host. Table 3 shows the quantitative statistics of PCL vascular graft patency rate and CD31+ endothelial coverage rate 4 weeks after carotid artery implantation in nude mice.

[0046] Table 3

[0047]

[0048] Based on the above experiments, we screened the membrane and tube materials of Example 3 to find the optimal DBCO modification concentration. Following this, we investigated the effects of different incubation times on in vitro iPSCs-ECs. Specific examples and comparisons are as follows:

[0049] Example 6: Cell seeding was performed using the DBCO-modified electrospun PCL membrane and tube materials prepared in Example 3. After iPSC-ECs were treated with 20 μM Ac4ManNAz for 48 h of sugar metabolism, their surfaces were modified with azide. Cells were collected at 3 × 10⁻⁶ cells / cm². 5 pcs / cm 2 For membrane materials, inoculation density is 60 min for planting; for tube materials, it is 3 × 10⁶. 5 / cm 2 The azide-modified iPSC-ECs were planted at a specific density and incubated for 60 minutes, rotating 90° every 15 minutes to achieve uniform planting of the tube material.

[0050] Example 7: Same as Example 6, but the cell seeding time is 90 min.

[0051] Example 8: Same as Example 6, but the cell seeding time is 120 min.

[0052] Example 9: Same as Example 6, but the cell seeding time is 150 min.

[0053] Comparative Example 3: Cell seeding was performed using the unmodified membrane and tube materials prepared in Comparative Example 1. The seeding density and method of the azide-modified iPSC-ECs were the same as in Example 6, but the seeding time was 150 min. Comparative Example 3 shows seeding relying on natural cell adhesion. Natural adhesion is directly proportional to seeding time. For the unmodified membrane and tube materials, cell adhesion was highest between 60 and 150 min. Therefore, setting only one comparative example with a 150 min incubation time is sufficient to demonstrate the advantages of cell seeding based on orthogonal biological reactions.

[0054] Comparative Example 4: Cell seeding was performed using the DBCO-free membrane and tube materials prepared in Comparative Example 1. Azide-free iPSCs-ECs were used for seeding, with the same seeding density and method as in Example 6, but the seeding time was 150 min. Comparative Example 4 represents seeding relying on natural cell adhesion. Natural adhesion is directly proportional to seeding time. For the DBCO-free membrane and tube materials, cell adhesion was highest between 60 and 150 min. Therefore, a single comparative example with a 150-min incubation time is sufficient to demonstrate the advantages of cell seeding based on orthogonal biological reactions.

[0055] 2.4 Evaluation of the adhesion of endothelial cells to electrospun fiber membranes with optimal DBCO modification density at different planting times.

[0056] Cell adhesion in Comparative Examples 3 and 4 relied solely on natural cell adhesion, without any bioorthogonal click reaction accelerating the cell adhesion process. The number of adherent cells in Examples 6-8 gradually increased and was significantly greater than in Comparative Examples 3 and 4; the number of adherent cells in Example 9 was comparable to that in Example 8, without further significant increase. These results indicate that bioorthogonalization can promote cell adhesion, reaching saturation adhesion density after 120 min of incubation (Table 4). Table 4 shows the evaluation results of endothelial cell adhesion at the optimal seeding density on electrospun fiber membranes with the optimal DBCO modification concentration at different incubation times.

[0057] Table 4

[0058]

[0059] 2.5 Evaluation of the effect of different implantation times on the resistance of endothelial cells to blood flow erosion on electrospun fiber tubes with optimal peptide modification density.

[0060] To achieve quantitative fluorescence analysis of cell retention rates, cells from Examples 6-9 and Comparative Examples 3 and 4 were labeled with the near-infrared fluorescent probe DiR. The seeded tubes were connected to an in vitro flow culture system, with the culture medium flow rate set to 10 cm / s to simulate the blood flow velocity of the human coronary artery. Endothelial cell-inoculated artificial blood vessels were perfused at this flow rate for 72 h, and cell retention efficiency was monitored at each time point using an IVIS imaging system. The results showed that the cell retention rates in Comparative Examples 3 and 4 were lower than those in Examples 1-8. The cell retention rates in Examples 6-8 increased with increasing adhesion time; the cell retention rate in Example 9 was comparable to that in Example 8, without further significant increase (Table 5). These results indicate that cell adhesion based on bioorthogonal structures can improve the cell's resistance to erosion, reaching saturation at 120 min of incubation. Table 5 shows the quantitative analysis of endothelial cell retention on PCL electrospun fiber tubes with optimal cell seeding efficiency after 72 h of blood flow erosion.

[0061] Table 5

[0062]

[0063] 2.6 Vascular Transplantation Experiment: Endothelial cells were implanted into electrospun fiber tubes with optimal DBCO modification density for different time periods, and then transplanted into the carotid arteries of nude mice. Four weeks after implantation, the patency rates of Comparative Examples 3 and 4 were 20%; the patency rates of Examples 6 were all 60%; the patency rates of Examples 7 were all 80%; and the patency rates of Examples 8-9 were all 100%. To examine the effect of implanted endothelial cells on the endothelialization rate of the artificial blood vessel, we analyzed the CD31 of the grafts. +Statistical analysis of endothelial cell coverage was performed, and the results showed that the endothelial coverage of Comparative Examples 3 and 4 was 0%; the endothelial coverage of Examples 6, 7, 8 and 9 were 55.73±6.34%, 63.82±10.33%, 89.74±7.51%, and 88.13±6.62%, respectively (Table 6).

[0064] Comparative Example 4: Clogged vascular grafts, such as Figure 3 As shown, H&E staining images reveal complete occlusion of the lumen, CD31 immunofluorescence staining images show almost no positive endothelial cells covering the lumen, and scanning electron microscopy images of the luminal surface show irregular thrombus deposition; the vascular graft of Example 8 is as follows. Figure 4 As shown, H&E staining images revealed patency of the lumen, CD31 immunofluorescence staining images showed continuous coverage by CD31-positive endothelial cells, and scanning electron microscopy images of the luminal surface showed that the covering endothelial cells were arranged in a cobblestone pattern with good morphology. These results indicate that endothelial cell seeding based on bioorthogonal reactions reaches saturation after 120 min of incubation, effectively improving the retention capacity of hiPSC-ECs and promoting the degree of reendothelialization of the vascular graft in the host. Table 6 shows the quantitative statistical analysis results of the patency rate and CD31+ endothelial coverage rate of PCL vascular grafts with optimal DBCO modification density after endothelial cell seeding at different times, 4 weeks after implantation in the carotid artery of nude mice.

[0065] Table 6

[0066]

[0067]

[0068] In summary, the technical solution of this application utilizes bioorthogonal reaction to mediate a rapid and stable binding between surface-modified polymer artificial blood vessels or membrane materials and membrane-modified endothelial cells, enabling the artificial blood vessel to form a complete and stable endothelial layer before transplantation. After implantation, it becomes an ideal artificial blood vessel graft with anticoagulation, antithrombosis, and long-term patency.

[0069] Bioorthogonal chemistry exhibits excellent biocompatibility, specificity, and speed under physiological conditions, representing an advanced method for rapidly forming covalent bonds applicable to living cells. This application combines cell modification techniques from metabolic glycoengineering to non-destructively anchor bioorthogonal azide groups to the cell surface, providing a foundation for bioorthogonal reactions. The combination of bioorthogonal chemistry and living-cell metabolic glycoengineering serves as a chemical "bridge" to facilitate rapid endothelial cell implantation for artificial blood vessels. This endothelial cell implantation process accelerates the in vitro preparation of endothelialized artificial blood vessels, resolving the challenges in the development and transformation of small-diameter artificial blood vessels.

[0070] 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. A polymeric artificial blood vessel or membrane material for rapid and stable endothelialization, characterized in that, It includes polymeric artificial blood vessels or membrane materials and endothelial cells cross-linked on the surface of the polymeric artificial blood vessels or membrane materials; the polymeric artificial blood vessels or membrane materials and the endothelial cells are cross-linked and connected by click reaction through alkynyl groups and azide groups; The surface of the polymer artificial blood vessel or membrane material is modified with an alkynyl compound; the alkynyl compound is cross-linked on the surface of the polymer artificial blood vessel or membrane material via an EDC / NHS reaction; the grafting density of the alkynyl compound on the surface of the polymer artificial blood vessel or membrane material is 0.09-0.36 nmol / mg; The endothelial cells are surface-modified with an azide compound; the endothelial cells with the azide compound surface-modified are used at a rate of 3 × 10⁻⁶. 5 pcs / cm 2 Inoculation density is applied to polymer artificial blood vessels or membrane materials whose surfaces are modified with alkynyl compounds.

2. The polymeric artificial blood vessel or membrane material for rapid and stable endothelialization according to claim 1, characterized in that, The grafting density of alkynyl compounds on the surface of polymer artificial blood vessels or membrane materials is 0.18 nmol / mg.

3. The polymeric artificial blood vessel or membrane material for rapid and stable endothelialization according to claim 1, characterized in that, The alkynyl compound is at least one of dibenzocyclooctyne (DBCO), 4-dibenzocyclooctyl alcohol (DIBO), or dicyclononyne (DIBAC).

4. The polymeric artificial blood vessel or membrane material for rapid and stable endothelialization according to claim 1, characterized in that, Endothelial cells were modified with an azide compound based on the principle of glucose metabolism. The azide compound was tetraacetyl-N-azidoacetylmannosamine Ac4ManNAz.

5. The polymeric artificial blood vessel or membrane material for rapid and stable endothelialization according to claim 1, characterized in that, The endothelial cells mentioned are endothelial cells iPSC-ECs differentiated from induced pluripotent stem cells, endothelial cells ESC-ECs differentiated from embryonic stem cells, endothelial progenitor cells or circulating monocytes differentiated from endothelial cells, human umbilical vein endothelial cells HUVECs, or endothelial cells isolated from natural vein donors.

6. The polymeric artificial blood vessel or membrane material for rapid and stable endothelialization according to claim 1, characterized in that, The polymer artificial blood vessel or membrane is made of synthetic or natural polymer materials; the synthetic polymer material is a mixture of at least one or more of polycaprolactone, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly(3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly-L-lactide-caprolactone, and polydioxane in any proportion; the natural polymer material is one or more of gelatin, collagen, hyaluronic acid, and fibrin glue.

7. A method for preparing a polymeric artificial blood vessel or membrane material for rapid and stable endothelialization as described in any one of claims 1-6, characterized in that, The process includes the following steps: 1) preparing polymer artificial blood vessels or membrane materials with surface-modified alkyne compounds; 2) preparing endothelial cells with surface-modified azide compounds; and incubating the endothelial cells with surface-modified azide compounds into polymer artificial blood vessels or membrane materials with surface-modified alkyne compounds to obtain polymer artificial blood vessels or membrane materials that achieve rapid and stable endothelialization.

8. The preparation method according to claim 7, characterized in that, The incubation time in step 2) is 60-150 minutes.

9. The use of a polymeric artificial blood vessel or membrane material according to any one of claims 1-6 for rapidly and stably achieving endothelialization in the preparation of artificial blood vessels or membrane materials.

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