Preparation method of a biomimetic vascular graft and its application in liver preservation

Abstract

The application belongs to the technical field of biomedical materials, and provides a preparation method of a biomimetic vascular graft and application of the biomimetic vascular graft in liver preservation. The preparation method comprises the following steps: obtaining a decellularized blood vessel by removing connective tissue, freeze-thawing treatment and trypsin treatment on a normal blood vessel; incubating the decellularized blood vessel in a functional modification solution to obtain a heparinized blood vessel; and incubating the heparinized blood vessel in an IL-4 solution to obtain the biomimetic vascular graft. The application can remove cell components and retain extracellular matrix as much as possible. The preparation method of the biomimetic vascular graft is simple, has strong applicability and is easy to mass-produce. The application improves the blood compatibility and patency of the biomimetic vascular graft by functional modification of the decellularized blood vessel, realizes local release of IL-4 molecules, and the biomimetic vascular graft can be used as a vascular bridge to establish a 'living preservation chamber' in the abdominal cavity of a recipient, so that the donor liver can continuously perform physiological perfusion, thereby providing a new strategy for liver preservation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and in particular relates to a method for preparing a biomimetic vascular graft and its application in liver preservation. Background Technology

[0002] Liver transplantation remains the only effective treatment for end-stage liver disease. Despite advancements in surgical techniques and the development of immunosuppressive drugs, donor shortages and suboptimal preservation methods still limit patient benefits. Good organ preservation methods are crucial for successful organ transplantation. Static cold storage (SCS) is the standard preservation technique, with organ preservation solutions playing a vital role. However, with increasing clinical demands for organ preservation, the development of organ preservation solutions has stagnated; furthermore, SCS suffers from poor preservation efficacy and difficulty in assessing organ condition. Machine perfusion (MP) is a promising alternative due to its ability to better maintain graft viability and assess organ quality. However, this technique relies on complex and expensive perfusion equipment, is difficult to transport, and requires specialized personnel, limiting its clinical application. Therefore, new technological advancements are urgently needed in the field of liver preservation.

[0003] Decellularized vascularization refers to the removal of immunogenic cellular components from blood vessels of allogeneic or xenogeneic origin, while preserving the original components and structure of the extracellular matrix (ECM) to the greatest extent possible. The ECM is a peripheral component that helps regulate a range of cellular behaviors and is secreted by specific cells in a tissue or organ. Components that can be preserved in the ECM include various proteins such as fibronectin, collagen, elastin, and laminin, as well as glycosaminoglycans and some bioactive factors. However, the direct exposure of the main ECM components in decellularized vascular stents to the blood environment can significantly increase the risk of thrombosis. Therefore, improving the blood compatibility of decellularized vascular stents warrants further investigation.

[0004] Heparin has been successfully used in clinical practice, and its combination with biomaterials can avoid the side effects of systemic heparin administration. ECM, the main component of decellularized blood vessels, has abundant functional modification sites that can react with the amino and carboxyl groups in heparin molecules, binding and immobilizing onto the surface of decellularized blood vessels via ionic or covalent bonds.

[0005] Interleukin-4 (IL-4) is a direct inducer of M2 macrophages. Macrophages, as key innate immune cells, play a crucial role in regulating the immune microenvironment of donor livers. Research on reducing donor liver damage by regulating the polarization state of macrophages within the liver is progressing and shows broad application prospects. IL-4 can increase the number of anti-inflammatory M2 macrophages and decrease the number of pro-inflammatory and related cell damage M1 macrophages, thereby inducing immune tolerance and promoting tissue repair. However, systemic application of IL-4 is limited by difficulties in dose control and the potential for side effects. Therefore, new drug delivery strategies are still needed to achieve targeted drug delivery.

[0006] Therefore, this invention proposes a method for preparing a biomimetic vascular graft and its application in liver preservation. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing biomimetic vascular grafts and their application in liver preservation, aiming to solve the problems of shortcomings in existing organ preservation methods, poor blood compatibility of decellularized blood vessels, and limitations in the systemic application of IL-4.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for preparing a biomimetic vascular graft includes the following steps:

[0010] Preparation of decellularized blood vessels: Normal blood vessels are obtained by removing connective tissue, freezing and thawing, and treating with trypsin.

[0011] Preparation of biomimetic vascular grafts: Decellularized blood vessels were incubated in a functionalized modification solution to obtain heparinized blood vessels, which were then incubated in an IL-4 solution to obtain biomimetic vascular grafts.

[0012] Furthermore, the specific process of freeze-thaw treatment is as follows: the blood vessel is placed in phosphate buffer and treated at -70℃ and -37℃ for 2 hours each, and this process is repeated multiple times; after the last treatment, it is treated at -70℃ and room temperature for 1 hour each.

[0013] Furthermore, the freeze-thaw process also includes a defatting process, specifically: the repeatedly frozen and thawed blood vessel is placed in alcohol, the alcohol is replaced at preset time points, and a syringe is used to fill the blood vessel with alcohol during the replacement process.

[0014] Furthermore, in the pancreatic enzyme treatment, the mass fraction of the pancreatic enzyme solution was 0.125%, and the treatment conditions were: treatment at 37°C and 15 rpm for 1.5 h.

[0015] Furthermore, the preparation process of the functionalized modification solution is as follows: heparin sodium, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added sequentially to 2-morpholine ethanesulfonic acid buffer, and the mixture is incubated under ice bath conditions.

[0016] Furthermore, the specific conditions for incubating the decellularized blood vessels in the functionalized modified solution were: incubation at 37°C for 24 hours.

[0017] Furthermore, the concentration of the IL-4 solution was 1 μg / mL, and the incubation conditions were: incubation at 37°C for 4 h.

[0018] A biomimetic vascular graft is prepared using the above-described method for preparing biomimetic vascular grafts.

[0019] The application of the above-mentioned biomimetic vascular graft in the preparation of vascular grafts for intraperitoneal liver preservation.

[0020] Furthermore, biomimetic vascular grafts have one or more of the following functions:

[0021] (1) Promotes the polarization of M1 macrophages in the liver towards M2 type;

[0022] (2) Reduces liver damage and decreases hepatocyte apoptosis;

[0023] (3) Improve blood vessel patency and blood compatibility.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. The biomimetic vascular graft prepared by the present invention is obtained by a combined decellularization method of repeated freeze-thaw and pancreatic enzyme treatment, which can remove immunogenic cellular components and retain the extracellular matrix as much as possible. The preparation method is simple, highly applicable, and easy to mass-produce.

[0026] 2. The biomimetic vascular graft of the present invention improves vascular patency and blood compatibility by functionalizing decellularized blood vessels, thereby achieving local sustained release of IL-4 molecules.

[0027] 3. The biomimetic vascular graft of the present invention can serve as a vascular bridge to establish a "living preservation chamber" in the recipient's abdominal cavity, enabling the donor liver to undergo continuous physiological perfusion, thus providing a new strategy for liver preservation. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the preparation of decellularized blood vessels.

[0029] Figure 2Characterization of decellularized blood vessels; where A represents HE staining and Masson staining results, and B represents DNA content monitoring results.

[0030] Figure 3 The distribution changes of ECM components in decellularized blood vessels.

[0031] Figure 4 The content of fixed heparin in heparinized cross-linking systems with different heparin concentrations.

[0032] Figure 5 This is the Fourier transform infrared spectrum.

[0033] Figure 6 Results of in vitro IL-4 release experiment for biomimetic vascular grafts.

[0034] Figure 7 The result is from toluidine blue staining.

[0035] Figure 8 Results of staining for live and dead cells.

[0036] Figure 9 Characterization of the decellularized, heparinized, and H / IL-4 groups; where A shows the HE and Masson staining results of vascular grafts in each group, B shows scanning electron microscope images of the inner surface of vascular grafts in each group, and C shows... Figure 9 Quantitative statistics of A in the middle, D is Figure 9 Quantitative statistics of B in China.

[0037] Figure 10 Image showing the results of iNOS and CD206 immunofluorescence staining in the transplanted liver.

[0038] Figure 11 The results of aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) in rats in the Control group, Decellularized group, Heparinized group, and H / IL-4 group are shown; where A is the AST result and B is the ALT result.

[0039] Figure 12 HE staining and TUNEL immunofluorescence staining results of donor livers from rats in the Control group, Decellularized group, Heparinized group, and H / IL-4 group after implantation; where A is the HE staining result and B is the TUNEL immunofluorescence staining result. Detailed Implementation

[0040] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the methods used in this invention are conventional methods in this technical field. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0041] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0042] Example 1: Preparation of biomimetic vascular grafts;

[0043] 1. Preparation of decellularized blood vessels (see preparation flowchart) Figure 1 );

[0044] (1) After removing the surrounding connective tissue from the normal blood vessels obtained from the abdominal aorta of rats, they were placed in an EP tube filled with ultrapure water and placed in a shaker at 37°C for 24 hours at 15 rpm.

[0045] (2) Replace the ultrapure water in the above EP tube with phosphate buffer (pH 7.4), and then treat it at -70℃ and -37℃ for 2 hours each, for a total of 3 times; after the last treatment, treat it at -70℃ and room temperature for 1 hour each. Then, place the repeatedly frozen and thawed blood vessel into an EP tube containing 70% ethanol (alcohol) for defatting treatment, and change the alcohol at 2, 4, 8 and 14 hours. During the solution change, a syringe can be used to fill the blood vessel with alcohol.

[0046] (3) Immerse the blood vessel completely in a 0.125% trypsin solution (dissolved in PBS, without EDTA, containing phenol red), place it on a shaker at 37°C, and centrifuge at 15 rpm for 1.5 h, changing the solution every 30 min. After washing repeatedly 5-8 times, store at -20°C to obtain decellularized blood vessels.

[0047] 2. Preparation of biomimetic vascular grafts;

[0048] (1) 200 mg of heparin sodium, 108 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 67 mg of N-hydroxysuccinimide (NHS) were added sequentially to 40 mL of 30 mmol / L 2-morpholinoethanesulfonic acid (MES) buffer. After mixing, the mixture was incubated at 0°C on an ice bath for 2 h. Then, decellularized blood vessels were added to this solution and incubated at 37°C for 24 h. The obtained blood vessels were then transferred to phosphate buffer and washed 5 times to obtain heparinized blood vessels.

[0049] (2) The heparinized blood vessel was completely immersed in an IL-4 solution with a concentration of 1 μg / mL and incubated at 37°C for 4 hours to obtain a biomimetic blood vessel graft (H / IL-4).

[0050] Example 2: Characterization of biomimetic vascular grafts;

[0051] HE staining results showed that, compared to natural blood vessels (Normal), the cell nuclei stained blue in decellularized blood vessels were completely removed, confirming that their cellular components had been effectively removed; Masson staining results showed that the cytoplasmic components stained red and the cell nuclei stained blue-black were completely removed, while the fibrous structure of the blood vessel wall was preserved. Figure 2 (A). DNA content monitoring results indicate that ( Figure 2 In the B-cell region, the DNA content of natural blood vessels was 919.93±104.20 ng / mg, while the DNA content of decellularized blood vessels was 24.94±3.72 ng / mg, both below the decellularization standard of 50 ng / mg. Furthermore, immunofluorescence staining showed (…). Figure 3 Compared to natural blood vessels, the distribution of α-SMA, Collagen I, Collagen IV, and Lamin in decellularized vascular ECM components showed no significant change. Changing the heparin sodium content (100, 150, 200, 250, 300 mg) in the heparin cross-linking system and reacting for 24 hours resulted in… Figure 4It was observed that satisfactory heparin fixation was achieved in all heparinized vessels after 24 hours. To ensure experimental stability and drug conservation, the heparin sodium content in the cross-linking system was determined to be 200 mg in subsequent experiments. Toluidine blue staining showed that only the cell nuclei of natural vessels were stained blue, while the extracellular matrix components remained unstained; decellularized vessels were unstained due to the removal of cellular components; heparinized vessels, however, appeared blue due to the formation of a complex between heparin molecules and toluidine blue, revealing the distribution of heparin. Zoom-in images further showed that the heparin bound along the extracellular matrix was distributed as blue bands. Figure 7 Fourier transform infrared spectroscopy shows ( Figure 5 Heparinized vessels at 1630 cm -1 The presence of a characteristic absorption peak for amide bonds at 1224 cm⁻¹ indicates the formation of covalent bonds; simultaneously, at 1224 cm⁻¹... -1 and 1030 cm -1 -SO3 is displayed at the location - The characteristic absorption peak of the group indicates that heparin is covalently fixed on the vascular stent. Toluidine blue staining and Fourier transform infrared spectroscopy jointly confirmed that heparin molecules are covalently bound to the decellularized vascular surface. In vitro IL-4 release assay results showed ( Figure 6 IL-4 molecules can be rapidly released within 1 day and can continue to be released for 14 days.

[0052] Example 3: Biocompatibility of biomimetic vascular grafts;

[0053] The biocompatibility of the biomimetic vascular grafts was evaluated using a live / dead cell staining assay. Four groups were set up: Control group (blank control); Decellularized group (decellularized vascular graft group); Heparinized group (heparinized vascular graft group); and H / IL-4 group (biomimetic vascular graft group). Human umbilical vein endothelial cells were used for in vitro cytotoxicity testing. Results showed ( Figure 8 On day 1 of culture, all groups showed green fluorescence signals from live cells, with no obvious red fluorescence signals from dead cells. On day 3, the number of cells in each group increased, and the green fluorescence signal intensified. On day 7, the cells in each group completely filled the field of view, with dense green fluorescence and no obvious red fluorescence signals from dead cells. The results indicate that the biomimetic vascular graft has low toxicity to human umbilical vein endothelial cells and good biocompatibility.

[0054] Example 4: The dual function of biomimetic vascular grafts;

[0055] To investigate the in vivo effects of biomimetic vascular grafts, healthy SD rats were selected, and allogeneic liver transplantation models were constructed using different vascular materials as vascular bridges. The specific construction process is as follows:

[0056] 1. Preparation of experimental animals: SPF-grade male SD rats, weighing approximately 200g, were purchased from Speford Biotechnology Co., Ltd. The rats were housed in a clean environment with a 12-hour circadian rhythm and free access to food and water. They were fasted for 8 hours prior to surgery, but water was allowed. All surgeries were performed under clean surgical conditions.

[0057] 2. Donor Liver Acquisition: Induce anesthesia with 3%-4% isoflurane. Maintain anesthesia with 1.5%-2% isoflurane at an airflow rate of 1.5 L / min. Remove hair from the rat's abdomen and disinfect with povidone-iodine. Drape the area to expose the surgical site. Administer 5 mL of saline subcutaneously and penicillin intramuscularly preoperatively. Make a midline abdominal incision to fully expose the xiphoid process and the bladder. Elevate the rat's back to fully expose the liver; suspend the xiphoid process cephalad using 4-0 sutures and pull it towards the head; use abdominal hooks to fully expose the surgical field on both sides of the abdominal cavity. Gently move the intestines to one side and cover with moist gauze.

[0058] Using microsurgical scissors, cut the falciform ligament, separate the left phrenic vein and the superior and inferior vena cava, ligate and tranverse the left inferior phrenic vein. Then, cut downwards the left triangular ligament, carefully separate the ligaments on the posterior wall of the superior and inferior vena cava, and embed a suture in the posterior space for later use. Turn the liver upwards and cover it with moist gauze. Expose the right adrenal vein and ligate it away from the liver. Expose the hepatic hilum, ligate and tranverse the hepatogastric and hepatoesophageal ligaments. After exposing the portal vein, ligate and tranverse the hepatic artery, then bluntly dissect the connective tissue around the portal vein to expose it, embedding two sutures. Adjust the isoflurane concentration to 0.5%-1%, and clamp the inferior vena cava and portal vein above the right renal vein with vascular clamps. Pull upwards the sutures embedded in the superior and inferior vena cava to provide sufficient space for the vascular clamps to occlude the superior and inferior vena cava near the diaphragm. The superior and inferior vena cava are transected away from the liver, the portal vein is transected at its left and right branches, and the inferior vena cava can be transected away from the liver. The liver is rinsed with 50 mL of normal saline until it gradually turns a light yellowish-brown color. The removed liver is placed in 4°C normal saline and then on an ice pack.

[0059] 3. Donor Liver Preparation: Remove excess adipose tissue around the portal vein and inferior vena cava. Pass the portal vein through a pre-fabricated portal vein cannula, fold the vein wall onto the cannula using straight forceps, and fix it with sutures. The inferior vena cava is prepared in the same manner as the portal vein. Connect the prepared donor liver to the vascular graft (bionic grafts, decellularized vessels, or heparinized vessels may be used) through the portal vein end. Insert two 8-0 sutures from the outside into both sides of the inferior vena cava, and tie the left suture. The sutures should not be too long to prevent entanglement. Store at 4°C after completion.

[0060] 4. Recipient Surgery: The recipient liver resection method is the same as the donor liver acquisition method, the difference being that when preserving blood vessels, each blood vessel is severed near the liver end, and the residual ends of each blood vessel are trimmed. The donor liver is removed and placed in the original anatomical position of the recipient rat's liver, aligning the venous ends. First, the sutures pre-reserved in the donor liver's superior and inferior vena cava are passed through the inner wall of the right side of the recipient's superior and inferior vena cava. Then, the tail suture is clamped with a vascular clamp and suspended on the right side of the recipient rat, bringing the anastomosis close. Next, using a microneedle, the suture needle pre-reserved on the left side of the recipient's superior and inferior vena cava is passed through the inner wall of the left side of the recipient's superior and inferior vena cava and tied. The suture needle is inserted into the posterior wall of the donor liver's inferior vena cava and continuous suturing is performed (from left to right relative to the surgeon). When reaching the left traction point, the needle exits from the anterior wall of the recipient, and the posterior wall suture is tightened appropriately. The needle is then inserted next to the anterior wall exit point and continuous suturing is performed (from right to left relative to the surgeon). When reaching the right end, physiological saline is injected into the inferior vena cava to expel air. The suture is tightened and tied with the remaining suture on the right side, and the tail suture is cut off. The portal vein end was anastomosed. The extended arm of the inferior vena cava cannula was held with microsurgical curved forceps and inserted into the recipient's inferior vena cava. Then, a suture was passed posteriorly to secure the recipient's inferior vena cava to the cannula. Releasing the clamp between the portal vein and the inferior vena cava revealed a rapid reddening of the liver. After vascular connection was completed, the intestines were restored, the abdominal cavity was flushed, fluid was aspirated, and the abdomen was closed layer by layer. Dezocine was administered subcutaneously for analgesia.

[0061] 5. Postoperative care: After surgery, place the patient on a 25℃ warming pad to rewarm until fully awake, and house them individually. Provide free access to 5% glucose solution for drinking water and food.

[0062] In this experiment, three allogeneic liver transplantation models were constructed in SD rats, using biomimetic vascular grafts, decellularized blood vessels, and heparinized blood vessels as vascular bridges, respectively. The models using decellularized blood vessels and heparinized blood vessels as vascular bridges served as the control group, while the model using biomimetic vascular grafts as vascular bridges served as the experimental group.

[0063] Hematoxylin and eosin (H&E) staining and Masson staining were used to determine the area of ​​vascular occlusion. Figure 9 Figures A and C show that the vascular occlusion area in the H / IL-4 group and the Heparinized group was significantly lower than that in the Decellularized group. Furthermore, the red blood cell count results under scanning electron microscopy (SEM) (…) Figure 9(B, D) shows that the number of erythrocytes adhering to the vascular wall in the H / IL-4 group and the Heparinized group was significantly lower than that in the Decellularized group. These results indicate that the vascular patency in the Heparinized group and the H / IL-4 group was superior to that in the Decellularized group, suggesting that heparinization modification helps improve the blood compatibility of biomimetic vascular grafts. Further immunofluorescence staining of the transplanted liver with iNOS (M1 marker) and CD206 (M2 marker) showed... Figure 10 The proportion of iNOS-positive M1 macrophages was significantly reduced in the H / IL-4 group (green fluorescence was significantly weakened), while the proportion of CD206-positive M2 macrophages was significantly increased (red fluorescence was significantly enhanced), indicating that the H / IL-4 group can promote the polarization of M1 macrophages in the liver to M2 type, while no such change was observed in other groups.

[0064] Example 5: The protective effect of biomimetic vascular grafts on donor liver;

[0065] This embodiment further examined the liver function levels of SD rats in each group after implantation. The results showed that the liver function levels of the rats increased after the bridging biomimetic vascular grafts were implanted. Figure 11 The results showed that the aspartate aminotransferase (AST) level in the control group (using normal rats as a reference) was 42.99±37.61 (U / L), the AST level in the decellularized group was 343.44±20.49 (U / L), the AST level in the heparinized group was 264.93±58.84 (U / L), and the AST level in the H / IL-4 group was 126.44±40.94 (U / L). Figure 11 The results showed that in the alanine aminotransferase (ALT) assay, the ALT levels in the blank group, decellularized group, heparinized group, and H / IL-4 group were 13.38±8.60 (U / L), 380.46±99.20 (U / L), 365.89±113.19 (U / L), and 147.83±24.83 (U / L), respectively. It is evident that compared with the decellularized and heparinized groups, the H / IL-4 group showed significantly lower levels of both AST and ALT, indicating a reduction in liver inflammation. HE staining (…) Figure 12 (A) showed that the vacuolar degeneration and inflammatory cell infiltration of hepatocytes in the H / IL-4 group were significantly less than those in the Heparinized and Decellularized groups, and were close to those in the control group. TUNEL immunofluorescence staining ( Figure 12 Figure B shows that the number of TUNEL-positive cells in the H / IL-4 group was lower than that in the Heparinized and Decellularized groups, indicating a decreased level of apoptosis in the H / IL-4 group. These results collectively suggest that biomimetic vascular graft treatment can alleviate liver damage and reduce hepatocyte apoptosis.

[0066] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention.

Claims

1. A method for preparing a biomimetic vascular graft, characterized in that, Includes the following steps: Preparation of decellularized blood vessels: Normal blood vessels are obtained by removing connective tissue, freezing and thawing, and treating with trypsin. Preparation of biomimetic vascular grafts: Decellularized blood vessels were incubated in a functionalized modification solution to obtain heparinized blood vessels, which were then incubated in an IL-4 solution to obtain biomimetic vascular grafts.

2. The method for preparing the biomimetic vascular graft according to claim 1, characterized in that, The specific process of the freeze-thaw treatment is as follows: the blood vessel is placed in phosphate buffer and treated at -70℃ and -37℃ for 2 hours each, and this process is repeated multiple times; after the last treatment, it is treated at -70℃ and room temperature for 1 hour each.

3. The method for preparing the biomimetic vascular graft according to claim 1, characterized in that, The freeze-thaw process also includes a degreasing process, which involves placing the repeatedly frozen and thawed blood vessel into alcohol, changing the alcohol at preset time points, and using a syringe to fill the blood vessel with alcohol during the liquid change.

4. The method for preparing the biomimetic vascular graft according to claim 1, characterized in that, In the pancreatic enzyme treatment, the mass fraction of the pancreatic enzyme solution was 0.125%, and the treatment conditions were: treatment at 37°C and 15 rpm for 1.5 h.

5. The method for preparing the biomimetic vascular graft according to claim 1, characterized in that, The preparation process of the functionalized modification solution is as follows: heparin sodium, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added sequentially to 2-morpholine ethanesulfonic acid buffer, and the mixture is incubated under ice bath conditions.

6. The method for preparing the biomimetic vascular graft according to claim 1 or 5, characterized in that, The specific conditions for incubating the decellularized blood vessels in the functionalized modified solution were: incubation at 37°C for 24 hours.

7. The method for preparing the biomimetic vascular graft according to claim 1, characterized in that, The concentration of the IL-4 solution was 1 μg / mL, and the incubation conditions were: incubation at 37°C for 4 hours.

8. A biomimetic vascular graft, characterized in that, It was prepared using the preparation method of the biomimetic vascular graft according to any one of claims 1-7.

9. The use of the biomimetic vascular graft according to claim 8 in the preparation of vascular grafts for intraperitoneal liver preservation.

10. The application according to claim 9, characterized in that, The biomimetic vascular graft has one or more of the following functions: (1) Promotes the polarization of M1 macrophages in the liver towards M2 type; (2) Reduces liver damage and decreases hepatocyte apoptosis; (3) Improve blood vessel patency and blood compatibility.

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