Nanostructured low-immunogenic biological artificial blood vessel and preparation method therefor

Abstract

A nanostructured low-immunogenic biological artificial blood vessel, a preparation method therefor and a use thereof. The preparation method comprises: decellularizing a pre-treated animal blood vessel to obtain a decellularized blood vessel; treating the decellularized blood vessel in an enzyme solution to obtain an enzyme-treated blood vessel, wherein the enzyme solution comprises nuclease and / or a biological enzyme; and using a cross-linking agent to cross-link the enzyme-treated blood vessel to obtain a nanostructured low-immunogenic biological artificial blood vessel, which is used as a blood vessel transplantation material. The artificial blood vessel overcomes the defects of decreased mechanical properties in decellularized biological tissues, in-vivo calcification after long-term use, and the presence of immunogenicity, antigenic components such as vascular wall cells and cell nuclei are fully removed, and original collagen and other extracellular matrix structural proteins in the tissue are retained to a greater extent, avoiding the in-vivo calcification of the blood vessel upon long-term implantation, enhancing the durability of use of the blood vessel; and the method has a short preparation cycle, low cost and high long-term patency rate.

Description

A nanostructured low-immunogenic biological artificial blood vessel and its preparation method

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on July 28, 2023, with application number 202310942899.8 and invention name “A nanostructured low immunogenic bioartificial blood vessel and its preparation method”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the technical field of medical devices, and in particular relates to a nanostructured low-immunogenic biological artificial blood vessel and a preparation method thereof. Background Art

[0003] Nowadays, with the development of society, the World Health Organization has shown that the prevalence of cardiovascular diseases is getting higher and higher. It has become one of the killers endangering human health and the number one cause of death for humans. According to statistics, there are currently more than 11 million patients with coronary heart disease in my country, and more than 5 million patients who need revascularization. In 2022, the number of coronary artery bypass grafting surgeries in the country will be close to 100,000, a 10-fold increase from 10 years ago. In 2019, there were approximately 45.3 million patients with peripheral atherosclerosis in my country, and about 8% of patients required revascularization. The current surgical penetration rate, including interventional surgery, is only 0.3%, and the market space is huge. The China Hemodialysis Case Information Registration System (CNRDS) shows that the hemodialysis rate has increased year by year (13% to 14% / year). There are more than 130 million patients with chronic renal insufficiency in my country, and more than 3 million patients who need hemodialysis. There were approximately 200,000 registered dialysis patients in 2011, 630,000 in 2019, 690,000 in 2020, and 750,000 in 2021. It is estimated that there will be approximately 1.27 million cases in 2027 and more than 1.6 million cases in 2030. Among them, more than 80% of patients used conventional surgical arteriovenous fistulas, 12% used central venous catheters, and about 7.2% used artificial vascular access (data from the CNRDS data released at the 6th Pengcheng Summit Forum on Nephrology, Renal Intervention and Blood Purification Equipment in November 2022); there are approximately 650,000 long-term chronic hemodialysis patients in the United States each year, of which about 65% used conventional surgical arteriovenous fistulas, about 15% used central venous catheters, and about 20% used artificial vascular access. After conventional arteriovenous fistula surgery, patients must wait 3 to 6 months for the fistula to mature before it can be used as a dialysis access. In addition, about 1 / 3 to 1 / 2 of domestic patients cannot mature after 3 to 6 months of fistula surgery and cannot undergo dialysis. They have no choice but to use small-caliber artificial blood vessels. In addition, this access is prone to aneurysms and decay; central venous catheterization can only be used as a temporary dialysis access, and the annual infection rate of patients is as high as 200%, with many complications and a high mortality rate; traditional expanded polytetrafluoroethylene (ePTFE) artificial blood vessels are prone to infection and intraluminal thrombosis, with a one-year patency rate of about 10-30% and a service life of about 2 years, which seriously limits clinical effectiveness. Therefore, the development of small-caliber blood vessels suitable for hemodialysis vascular access in patients with coronary heart disease, lower limb artery replacement, and end-stage renal disease is of great clinical significance.

[0004] Vascular repair and replacement is one of the most effective options for treating vascular diseases. Currently, the sources of blood vessels generally include autologous blood vessels, polymer-based artificial blood vessels, and allogeneic blood vessels, and tissue-engineered blood vessels are in the research and development stage. Autologous internal mammary arteries or radial arteries have excellent medium- and long-term patency rates, with a 10-year patency rate of approximately 90% or more for coronary artery bypass grafting. Autologous great saphenous veins have a 5-year patency rate of approximately 60-70% and a 10-year patency rate of approximately 40-50%. Furthermore, except for coronary artery bypass grafting, they cannot be used for other clinical indications due to mismatched internal diameters. Furthermore, the source of autologous blood vessels is limited. 10-30% of patients cannot obtain the great saphenous vein due to varicose veins, obesity, diabetes, and other factors. Furthermore, obtaining the autologous great saphenous vein itself can cause secondary trauma, with complications such as wound infection, lower limb edema, and prolonged surgery and anesthesia time, which can affect the patient's postoperative recovery. Although artificial blood vessels made of polymer materials are easy to produce on a large scale commercially and their caliber and length are easy to control, polymer material vascular products have low long-term patency rates, are prone to infection and decay, and have a short service life, which seriously affects clinical effects.

[0005] Allogeneic blood vessels are categorized as homografts and xenografts. Homografts are restricted in their source and availability for ethical and regulatory reasons. Xenografts are generally animal-derived, harvested directly from mammals, and can be used as vascular grafts for lower limb artery replacement, coronary artery bypass grafting, dialysis, and other procedures.

[0006] In addition to blood vessels, another animal-derived medical device, the biocompatible heart valve (BHV), does not require lifelong anticoagulation therapy after implantation in the human body. However, calcification of the valve leaflets after implantation of the BHV in the body can lead to valve structural degeneration, causing valve stenosis and / or regurgitation. The body's harmful immune response to foreign antigens in the BHV can participate in the occurrence of calcification and structural degeneration, so its durability in the body is limited, affecting its service life.

[0007] Recently, Nature Medcine published a research paper titled "The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration." The study analyzed the hemodynamics, immune response factors, and the pathogenesis of leaflet failure in 1,668 patients up to 15 years after BHV implantation. The results showed that BHV implantation caused an increase in anti-α-Gαl and anti-Neu5Gc antibodies in the first six months, while the control group did not significantly increase the levels of these two antibodies (lower than patients with BHV implantation). α-Gαl is present in a large number of non-primate mammals, and anti-α-Gal antibodies exist in humans. The combination of α-Gal antigen and anti-α-Gal antibodies forms a xenotransplantation immune barrier. In order to ensure the safe and effective use of animal-derived medical devices, animal-derived materials should be selected and appropriate processes should be used to reduce or remove α-Gal antigens.

[0008] Currently available animal-derived medical devices, such as bioprosthetic valves and biogenic blood vessels, have been treated with chemical reagents to remove most of their immunogenicity, but some cellular antigens still remain. Therefore, animal-derived medical devices must be completely decellularized and cross-linked to enhance their mechanical strength before use. However, existing decellularization methods destroy the extracellular matrix components of the animal-derived blood vessel walls during the treatment process, and the removal of wall cells and antigens is incomplete. This can easily lead to calcification and decay during clinical use, affecting vascular remodeling in vivo and long-term patency.

[0009] The use of traditional cross-linkers is another significant factor in initiating calcification. Existing experiments have shown that 1) Calcification of BHVs after implantation is primarily a passive mechanism, with the reaction of xenogeneic cells, residual cell components, and cell membranes / associated phospholipid molecules with calcium ions being the primary initiator of calcification. Although crosslinkers utilize aldehyde groups to form Schiff reactions with amino groups in the collagen protein of the valve material, forming a five-membered ring of carbon atoms, stabilizing collagen, resisting tissue enzymatic degradation, and improving mechanical properties, they also cause cell death and retention in the bioprosthetic valve tissue, disrupting calcium pumps on the cell membrane and increasing the chances of calcium ions coming into contact with phosphates. 2) Residual crosslinkers in the product can directly bind to calcium ions. 3) They are unable to stabilize elastin and glycosaminoglycans, reducing valve durability and promoting calcification. Metalloproteinases and cathepsins can promote elastin degradation and the production of proinflammatory peptides, inducing calcification deposition. 4) Crosslinker treatment reduces the number of amino acid residues, exposing phosphate bonds in collagen and increasing the negative charge on the valve surface, which binds to positively charged calcium ions. 5) BHVs lack the anti-calcification barrier of endothelial cells found on human valve surfaces. Calcification occurs due to the adhesion of platelets and cellulose, as well as the penetration of calcium and phosphate. Studies have confirmed that removing elastin from tissue can significantly reduce the occurrence of calcification after implantation.

[0010] Vascular calcification has been clinically proven to pose a significant threat to patients with chronic kidney disease. Vascular calcification rates are very high in patients with chronic renal failure and are associated with poor prognosis. Four-year follow-up results from a Chinese dialysis calcification study showed that the incidence of vascular calcification increased from 77.4% in the first year to 90% in the fourth year.

[0011] Therefore, developing a biological blood vessel suitable for hemodialysis patients has important clinical value in improving the quality of life of patients, reducing the number of interventions, and prolonging the life of patients.

[0012] The present invention develops a nanostructured, low-immunogenic bio-artificial blood vessel and a preparation method thereof. A decellularization reagent with minimal impact on the extracellular matrix of the vascular wall is used to remove cells while preserving the vascular extracellular matrix and the vessel wall structure as much as possible without affecting the mechanical properties and compliance of the blood vessels. Enzymatic treatment is then used to remove the cell nuclei and antigen components remaining after decellularization, and a new cross-linking agent is used to increase the mechanical properties of the decellularized blood vessels. The decellularized blood vessels have excellent biocompatibility and an extremely low incidence of vascular calcification, and the long-term patency rate is improved.

[0013] Summary of the Invention

[0014] In view of this, the technical problem to be solved by the present invention is to provide a nanostructured low-immunogenicity bioartificial blood vessel and a preparation method thereof. The bioartificial blood vessel prepared by this method has good mechanical properties, extremely low immunogenicity and vascular calcification incidence, and significantly improves the long-term patency rate.

[0015] The present invention provides a method for preparing a nanostructured low-immunogenic biological artificial blood vessel, comprising the following steps:

[0016] S1) performing a decellularization treatment on the pretreated animal blood vessels to obtain decellularized blood vessels;

[0017] S2) treating the decellularized blood vessel in an enzyme solution to obtain an enzyme-treated blood vessel; the enzyme solution comprises nuclease and / or biological enzyme;

[0018] S3) cross-linking the blood vessel after the enzyme treatment with a cross-linking agent to obtain a nanostructured low-immunogenic bio-artificial blood vessel.

[0019] Preferably, the animal blood vessels are selected from arteries or veins of large animals; the large animals are selected from pigs, sheep, dogs, cattle or horses;

[0020] The animal blood vessel is selected from the group consisting of aorta, pulmonary artery, superior vena artery, inferior vena artery, common carotid artery, internal jugular vein, external jugular vein, femoral artery, femoral vein, iliac artery, iliac vein, superior mesenteric artery, inferior mesenteric artery, rectal artery, median sacral artery or lower limb artery;

[0021] The decellularization reagent used in the decellularization treatment in step S1) includes a detergent or a detergent and a chelating agent; the concentration of the detergent in the decellularization reagent is 0.01 to 500 mmol / L; the concentration of the chelating agent in the decellularization reagent is 0.01 to 500 mmol / L;

[0022] The temperature of the decellularization treatment is 10° C. to 38° C.; the time of the decellularization treatment is 2 to 72 hours; and the decellularization reagent is replaced every 1 to 24 hours during the decellularization treatment.

[0023] Preferably, when the decellularization reagent includes a detergent, the detergent is selected from one or more of a nonionic detergent, an anionic detergent, a cationic detergent, and a zwitterionic detergent;

[0024] When the decellularization reagent includes a detergent and a chelating agent, the detergent is selected from one or more of a nonionic detergent, an anionic detergent, a cationic detergent and a zwitterionic detergent;

[0025] The nonionic detergent is selected from one or more of polyethylene glycols, polyethylene glycol octylphenyl ether, polyols, polyoxyethylene fatty alcohol ethers and polyoxyethylene alkylphenol ethers;

[0026] The anionic detergent is selected from one or more of sodium lauryl sulfate, lithium lauryl sulfate, sodium lauryl sulfonate, sodium cholate and sodium deoxycholate;

[0027] The cationic detergent is selected from benzalkonium bromide and / or cetyltrimethylammonium bromide;

[0028] The zwitterionic detergent is selected from 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt and / or n-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate;

[0029] The chelating agent is an inorganic chelating agent and / or an organic chelating agent;

[0030] The inorganic chelating agent is selected from one or more of sodium tripolyphosphate, sodium hexametaphosphate and sodium pyrophosphate;

[0031] The organic chelating agent is selected from one or more of aminotriacetic acid, ethylenediaminetetraacetic acid, ethylene glycol ditetraacetic acid, ethylenediaminediacetic acid, cyclohexanediaminetetraacetic acid, S,S-ethylenediamine disuccinic acid, diethyltriacetic acid, diethylenetriaminepentaacetic acid and its salts, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid and dihydroxyethylglycine;

[0032] The decellularization reagent also includes sodium chloride, sodium hydroxide and water;

[0033] The concentration of sodium chloride in the decellularization reagent is 0.01 to 1.0 mmol / L;

[0034] The concentration of sodium hydroxide in the decellularization reagent is 0.1-2.0 mol / L.

[0035] Preferably, the concentration of the nuclease in the enzyme solution is 1 to 50,000 KU / L; and / or the concentration of the biological enzyme in the enzyme solution is 1 to 50,000 KU / L;

[0036] The enzyme solution further comprises 1 to 50 wt% of human serum or animal serum;

[0037] The enzyme solution also includes physiological saline or buffer;

[0038] The temperature of the enzyme treatment in step S2) is 36° C. to 37° C.; the time of the enzyme treatment is 2 to 72 hours; and the enzyme solution is replaced every 1 to 24 hours during the enzyme treatment.

[0039] Preferably, after the enzyme treatment in step S2), the blood vessel is further treated with a solution containing a biological enzyme to obtain an enzyme-treated blood vessel;

[0040] The nuclease is selected from DNA enzyme and / or RNA enzyme; the biological enzyme is selected from one or more of pepsin, lipase, trypsin, cathepsin, papain, ficin and subtilisin; the concentration of the biological enzyme in the solution containing the biological enzyme is 1 to 50,000 KU / L; the solvent of the solution containing the biological enzyme is physiological saline, buffer solution or special solution for enzyme preparation;

[0041] The temperature for treating with the solution containing the biological enzyme is 36° C. to 37° C.; the treatment time is 0.25 to 48 hours; and the solution containing the biological enzyme is replaced every 1 to 24 hours during the treatment process.

[0042] Preferably, the cross-linking agent is selected from one or more of glutaraldehyde solution, oxidized starch solution, dialdehyde starch solution and Jeffamine buffer;

[0043] The concentration of the glutaraldehyde solution is 0.1 to 30 wt %;

[0044] The concentration of the oxidized starch solution is 0.01 to 10 wt %.

[0045] The concentration of the dialdehyde starch solution is 0.01 to 10 wt %;

[0046] The concentration of the Jeffamine buffer is 0.01-1.0 mol / L.

[0047] Preferably, after the cross-linking treatment in step S3), heparin is covalently bound using a chemical method to obtain a biological artificial blood vessel.

[0048] Preferably, the solution used for chemically covalently binding heparin is an MES buffer solution containing carbodiimide, N-hydroxysulfosuccinimide and heparin; the concentration of heparin in the solution used for chemically covalently binding heparin is 1-200 mg / ml.

[0049] The present invention also provides a nanostructured low-immunogenicity bio-artificial blood vessel prepared by the above-mentioned preparation method, wherein the bio-artificial blood vessel comprises a vascular collagen or a vascular collagen and elastin nanofiber structure.

[0050] Preferably, the vascular collagen includes type I collagen, type III collagen and / or type IV collagen;

[0051] When the bio-artificial blood vessel comprises vascular collagen, the content of the vascular collagen is 30% to 85% of the dry weight of the bio-artificial blood vessel;

[0052] When the biological artificial blood vessel comprises vascular collagen and elastin, the content of the vascular collagen is 20% to 55% of the dry weight of the biological artificial blood vessel, and the content of the elastin is 20% to 55% of the dry weight of the biological artificial blood vessel.

[0053] The present invention also provides an application of the above-mentioned nanostructured low-immunogenic bio-artificial blood vessel in the preparation of vascular graft materials for chronic renal failure hemodialysis vascular access, vascular graft materials for lower limb artery trauma, vascular graft materials for peripheral arterial disease bypass surgery, or vascular graft materials for coronary artery bypass surgery.

[0054] The present invention also provides an application of the above-mentioned nanostructured low-immunogenic bioartificial blood vessel in the preparation of a vascular graft material for vascular access in chronic dialysis patients; the bioartificial blood vessel is used as a replacement repair graft material after chronic dialysis autologous arteriovenous fistula or artificial blood vessel fistula has decayed, infected, or formed aneurysms.

[0055] The present invention provides a method for preparing a nanostructured low-immunogenicity bio-artificial blood vessel, comprising the following steps: S1) decellularizing a pretreated animal blood vessel to obtain a decellularized blood vessel; S2) treating the decellularized blood vessel in an enzyme solution to obtain an enzyme-treated blood vessel; the enzyme solution contains nuclease and / or biological enzyme; S3) cross-linking the enzyme-treated blood vessel using a cross-linking agent to obtain a nanostructured low-immunogenicity bio-artificial blood vessel and a method for preparing the same. Compared with the existing technology, the present invention adopts a method with less impact on the extracellular matrix of the blood vessel wall for decellularization. Cells are removed while retaining the extracellular matrix and wall structure of the blood vessel as much as possible, without affecting the mechanical properties and compliance of the blood vessel. Then, enzyme treatment is used to remove the cell nuclei and antigen components remaining after decellularization, significantly reducing the immunogenicity of the decellularized blood vessels. Finally, a cross-linking agent is used for treatment to improve the mechanical properties of the decellularized blood vessels, while blocking foreign protein antigen sites, further reducing immunogenicity, and increasing the durability of the blood vessels after implantation in the body, reducing the occurrence of luminal thrombosis and vascular calcification and decay. The bio-artificial blood vessels prepared by the present invention completely remove cells while reducing the impact on the damage to the extracellular matrix of the blood vessel wall and the mechanical properties. At the same time, it is beneficial to the adhesion and growth of endothelial cells after implantation in the body, improving the long-term patency rate. In addition, this method has a short preparation cycle and low cost, and can prepare bio-artificial blood vessels of different inner diameters and lengths for clinical use.

[0056] At the same time, the nanostructured low-immunogenicity bio-artificial blood vessels prepared by the present invention solve the defects of reduced mechanical properties of biological tissue after decellularization, calcification in the body after long-term use, and immunogenicity. They fully remove antigenic components such as tube wall cells and cell nuclei, while retaining the original extracellular matrix structural proteins such as collagen in the tissue to a greater extent. Foreign protein antigens are blocked by cross-linking agents to avoid calcification of blood vessels after long-term implantation in the body, thereby increasing the durability of blood vessel use. At the same time, this method has a short preparation cycle, low cost, and high long-term patency rate, making it suitable for commercial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figures 1a and 1b are diagrams showing the histological test results of biological blood vessels after treatment with 5.2% ficin for 1 / 2 hour in Example 1 of the present invention;

[0058] Figures 2a, 2b, 2c, and 2d are diagrams showing the histological test results of biological blood vessels treated with 5.2% ficin for 3 hours in Example 1 of the present invention;

[0059] FIG3a is a diagram showing the histological test results of the biological vascular decellularization method A in Example 1 of the present invention;

[0060] FIG3 b is a diagram showing the histological test results of the biological vascular decellularization method B in Example 1 of the present invention;

[0061] FIG4a is a diagram showing the collagen staining results of biological vascular tissue after decellularization by method A in Example 1 of the present invention;

[0062] FIG4 b is a diagram showing the elastin staining results of biological vascular tissue after decellularization by method A in Example 1 of the present invention;

[0063] FIG5a is a diagram showing the collagen staining results of biological vascular tissue after decellularization according to method B in Example 1 of the present invention;

[0064] FIG5 b is a diagram showing the elastin staining results of biological vascular tissue after decellularization according to method B in Example 1 of the present invention;

[0065] FIG6 is a photograph of an untreated fresh blood vessel in Example 1 of the present invention;

[0066] FIG7 is a photograph of the bioartificial blood vessel obtained in Example 1 of the present invention;

[0067] Figures 8a, 8b, 8c, and 8d are SEM photographs of the cross-section of the bioartificial blood vessel obtained in Example 1 of the present invention at different magnifications;

[0068] Figures 9a, 9b, 9c and 9d are SEM photographs of the intima of the bio-artificial blood vessel obtained in Example 1 of the present invention at different magnifications;

[0069] Figures 10a, 10b, 10c, and 10d are SEM photographs of the adventitia of the bio-artificial blood vessel obtained in Example 1 of the present invention at different magnifications;

[0070] FIG11 is a DAPI-stained photograph of untreated fresh blood vessels in Example 1 of the present invention;

[0071] FIG12 is a DAPI-stained photograph of the bio-artificial blood vessel obtained in Example 1 of the present invention;

[0072] FIG13 shows the radial tensile strength test results of bio-artificial blood vessels according to Examples 3, 4, 5, and 6 of the present invention;

[0073] FIG14 shows the bursting pressure test results of bio-artificial blood vessels according to Examples 3, 4, 5, and 6 of the present invention;

[0074] FIG15 is a picture of the e-PTFE control group during the animal experiment in Example 7 of the present invention;

[0075] FIG16 is a picture of a bio-artificial blood vessel during an animal experiment in Example 7 of the present invention;

[0076] FIG17 is an ultrasound image of a bio-artificial blood vessel after animal experiment in Example 7 of the present invention;

[0077] FIG18 is an ultrasound image of blood vessels at the arterial anastomosis after surgery in an animal experiment in Example 7 of the present invention;

[0078] FIG19 is a picture of angiography of an arteriovenous fistula to check patency of a vascular graft after surgery in an animal experiment in Example 7 of the present invention;

[0079] FIG20 is a picture of the right common carotid artery replacement during the animal experiment in Example 7 of the present invention;

[0080] FIG21 is an angiographic image of the right common carotid artery after surgery in the animal experiment in Example 7 of the present invention;

[0081] FIG22 is a picture of the right common carotid artery vascular replacement angiography during the follow-up of the animal experiment after surgery in Example 7 of the present invention, showing the patency of the biotype blood vessels and the autologous blood vessels near the anastomosis. DETAILED DESCRIPTION

[0082] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0083] The present invention provides a method for preparing a nanostructured low-immunogenicity bio-artificial blood vessel, comprising the following steps: S1) performing a decellularization treatment on a pretreated animal blood vessel to obtain a decellularized blood vessel; S2) performing an enzyme treatment on the decellularized blood vessel in an enzyme solution to obtain an enzyme-treated blood vessel; the enzyme solution contains a nuclease or a biological enzyme; S3) performing a cross-linking treatment on the enzyme-treated blood vessel using a cross-linking agent to obtain a nanostructured low-immunogenicity bio-artificial blood vessel.

[0084] Among them, the source of the animal blood vessel raw materials of the present invention complies with the national standards for animal breeding of animal-derived medical devices.

[0085] In the present invention, the animal blood vessels are preferably taken from the arteries or veins of large animals such as pigs, sheep, dogs, cattle, and horses; the types of animal blood vessels include but are not limited to the aorta, pulmonary artery, superior vena artery, inferior vena artery, common carotid artery, internal jugular vein, external jugular vein, femoral artery, femoral vein, iliac artery, iliac vein, mesenteric (superior and inferior) artery, rectal artery (superior and inferior) and median sacral artery, lower limb artery, etc.; the pretreated animal blood vessels are pretreated by methods well known to those skilled in the art, including disinfecting the obtained animal blood vessels and removing the fat and connective tissue around the blood vessels, but without damaging the vascular adventitia and vascular wall.

[0086] The pretreated animal blood vessels are subjected to a decellularization treatment to obtain decellularized blood vessels; the decellularization agent used in the decellularization treatment preferably includes a detergent or a detergent and a chelating agent; when the decellularization agent includes a detergent, the detergent is preferably one or more of a nonionic detergent, an anionic detergent, and a cationic detergent and a zwitterionic detergent; the molar ratio of the one or more of the nonionic detergent, anionic detergent, and cationic detergent to the zwitterionic detergent is preferably (1-3):(5-15), more preferably (1.5-2.5):(6-12), and even more preferably (1.5-2.5) :(6~10), most preferably (1.8~2.5):(8~10); when the decellularization reagent includes a detergent and a chelating agent, the detergent is preferably one or more of a nonionic detergent, an anionic detergent, a cationic detergent and a zwitterionic detergent; the nonionic detergent is preferably one or more of polyethylene glycols, polyethylene glycol octylphenyl ether, polyols, polyoxyethylene fatty alcohol ether and polyoxyethylene alkylphenol ether; the anionic detergent is preferably one or more of sodium lauryl sulfate, lithium lauryl sulfate, sodium lauryl sulfonate, sodium cholate and sodium deoxycholate; the cationic detergent is preferably one or more of Benzalkonium bromide and / or hexadecyltrimethylammonium bromide are selected; the zwitterionic detergent is preferably 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt and / or n-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate; the concentration of the detergent in the decellularization reagent is preferably 0.01 to 500 mmol / L, more preferably 1 to 100 mmol / L, more preferably 1 to 50 mmol / L, and most preferably 1 to 20 mmol / L; in the embodiment provided by the present invention, the concentration of the detergent in the decellularization reagent is specifically 9.8 mmol / L, 8 mmol / L, 10mmol / L or 6mmol / L; the chelating agent is preferably an inorganic chelating agent and / or an organic chelating agent; the inorganic chelating agent is preferably one or more of sodium tripolyphosphate, sodium hexametaphosphate and sodium pyrophosphate; the organic chelating agent is preferably one or more of aminotriacetic acid, ethylenediaminetetraacetic acid, ethylene glycol ditetraacetic acid, ethylenediaminediacetic acid, cyclohexanediaminetetraacetic acid, S,S-ethylenediamine disuccinic acid, diethyltriacetic acid, diethylenetriaminepentaacetic acid and its salts, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid and dihydroxyethylglycine; the concentration of the chelating agent in the decellularization reagent is preferably 0.01~500mmol / L, more preferably 1~300mmol / L, more preferably 1~100mmol / L, more preferably 1~50mmol / L, most preferably 10~30mmol / L; In the embodiment provided by the present invention, the concentration of the chelating agent in the decellularization reagent is specifically 25mmol / L, 30mmol / L or 20mmol / L; In the present invention, the decellularization reagent further includes sodium chloride, sodium hydroxide and water; The concentration of sodium chloride in the decellularization reagent is preferably 0.01~1mmol / L, more preferably 0.01-0.5mmol / L, more preferably 0.08-0.2mmol / L, more preferably 0.1-0.15mmol / L, most preferably 0.1-0.12mmol / L; in the embodiment provided by the present invention, the concentration of sodium chloride in the decellularization reagent is specifically 0.12mmol / L, 0.15mmol / L or 0.1mmol / L; the concentration of sodium hydroxide in the decellularization reagent is preferably 0.1-2mol / L, more preferably 0.8-1.5mol / L, More preferably, it is 0.8 to 1.3 mol / L, and most preferably, it is 1 to 1.3 mol / L. In the embodiment provided by the present invention, the concentration of sodium hydroxide in the decellularization reagent is specifically 1 mol / L, 1.2 mol / L or 0.8 mol / L; the temperature of the decellularization treatment is preferably 10°C to 38°C, more preferably 20°C to 38°C, more preferably 25°C to 38°C, and most preferably 36°C to 38°C; the time of the decellularization treatment is preferably 2 to 72 h, more preferably 8 to 48 h, more preferably 10 to 45 h, and most preferably 36°C to 38°C. The preferred time is 15 to 40 hours, more preferably 20 to 40 hours, more preferably 25 to 35 hours, and most preferably 30 hours. During the decellularization process, the decellularization reagent is preferably replaced every 1 to 24 hours, more preferably every 2 to 12 hours, more preferably every 2 to 8 hours, and most preferably every 5 to 8 hours. In the embodiments provided by the present invention, the decellularization reagent is specifically replaced every 2 to 8 hours. The number of times the decellularization reagent is replaced is preferably 1 to 6 times, more preferably 2 to 6 times, and most preferably 3 to 6 times.

[0087] According to the present invention, after decellularization, the treated blood vessels are preferably washed in physiological saline or buffer to obtain decellularized blood vessels; the physiological saline is preferably 0.9% (V / V) physiological saline; the buffer is preferably PBS buffer; the washing temperature is preferably 10°C to 38°C, more preferably 20°C to 38°C, more preferably 25°C to 38°C, and most preferably 36°C to 38°C; the washing time is preferably 10 to 60 minutes, more preferably 20 to 50 minutes, more preferably 35 to 45 minutes, and most preferably 40 minutes.

[0088] The decellularized blood vessel is subjected to enzymatic digestion in an enzyme solution; the enzyme solution contains nucleases and / or biological enzymes; the nucleases are preferably DNA enzymes and / or RNA enzymes; in the embodiments provided herein, the nucleases are specifically DNase II enzymes, DNase I enzymes, or Benzonase nucleases; the biological enzymes are preferably one or more of pepsin, lipase, trypsin, cathepsin, papain, ficin, and subtilisin; the concentration of the nuclease in the enzyme solution is preferably 1 to 50,000 KU / L, more preferably 1 to 4,000 KU / L, and even more preferably 5 to 1,000 KU / L. Most preferably, the concentration of the biological enzyme in the enzyme solution is preferably 5-100KU / L; and / or, the concentration of the biological enzyme in the enzyme solution is preferably 1-50000KU / L, more preferably 100-50000KU / L, and even more preferably 500-10000KU / L; in the embodiment provided by the present invention, the concentration of the nuclease and / or biological enzyme in the enzyme solution is specifically 3KU / L, 9KU / L, 15KU / L, 20KU / L, 50KU / L, 5000KU / L, 1000KU / L or 20000KU / L; the enzyme solution preferably further comprises 1-1 0wt% serum, physiological saline or buffer; the serum is preferably human serum or animal serum; the serum concentration is preferably 1-30wt%, more preferably 4-20wt%, more preferably 4-10wt%, more preferably 4-6wt%, and most preferably 5wt%; the physiological saline is preferably 0.9% (V / V) physiological saline; the buffer is preferably PBS buffer; the enzyme treatment temperature is preferably 36°C to 37°C; the enzyme treatment time is preferably 2-72h, more preferably 12-72h, more preferably 24-72h, and more preferably 3 6 to 60 hours, more preferably 40 to 50 hours, most preferably 48 hours; during the enzyme treatment, the enzyme solution is preferably replaced every 1 to 24 hours, more preferably every 5 to 10 hours, and more preferably every 6 to 8 hours; the number of times the enzyme solution is replaced is preferably 1 to 8 times, more preferably 3 to 7 times, more preferably 4 to 6 times, and most preferably 5 times; if a nuclease enzyme solution is used for treatment, it is preferably treated with a solution containing a biological enzyme after the enzyme treatment to obtain an enzyme-treated blood vessel; the biological enzyme is preferably pepsin, fat One or more of enzymes, trypsin, cathepsin, papain, ficin and subtilisin; the concentration of the enzyme in the solution containing the enzyme is preferably 1 to 50,000 KU / L, more preferably 1 to 10,000 KU / L, more preferably 1 to 5,000 KU / L, and most preferably 100 to 1,000 KU / L; or in terms of mass percentage, the concentration of the enzyme in the solution containing the enzyme is preferably 1 to 10 wt %, more preferably 3 to 8 wt %, more preferably 5 to 6 wt %, and most preferably 5.2 to 5.5wt%; in the embodiments provided by the present invention, the concentration of the enzyme in the solution containing the enzyme is specifically 3KU / L, 1000KU / L or 100KU / L; the solvent of the solution containing the enzyme is physiological saline, a buffer solution or a special solution for enzyme preparation; the physiological saline solution is preferably 0.9% (V / V) physiological saline; the buffer solution is preferably PBS buffer; the temperature for treatment with the solution containing the enzyme is preferably 36°C to 37°C; the treatment time is preferably 0.25 to 48 hours, more preferably 6 to 48 hours, more preferably 12 to 48 hours, more preferably 20 to 36 hours, and most preferably 24 to 36 hours; during the treatment process, the solution containing the enzyme is preferably replaced every 1 to 24 hours, more preferably every 5 to 15 hours, more preferably every 5 to 10 hours, and most preferably every 6 to 8 hours; during the treatment process, the solution containing the enzyme is preferably replaced 1 to 5 times, more preferably 2 to 3 times.

[0089] In the present invention, after enzyme treatment or treatment with a solution containing a biological enzyme, it is preferably washed with physiological saline or a buffer solution to obtain an enzyme-treated blood vessel; the physiological saline is preferably 0.9% (V / V) physiological saline; the buffer solution is preferably PBS buffer; the washing temperature is preferably 10°C to 38°C, more preferably 20°C to 38°C, more preferably 25°C to 38°C, and most preferably 36°C to 38°C; the washing time is preferably 10 to 60 minutes, more preferably 20 to 50 minutes, more preferably 35 to 45 minutes, and most preferably 40 minutes.

[0090] The blood vessel after enzyme treatment is cross-linked using a cross-linking agent; the cross-linking agent is preferably one or more of glutaraldehyde solution, oxidized starch solution, dialdehyde starch solution and Jeffamine buffer; the concentration of the glutaraldehyde solution is preferably 0.1-30wt%, more preferably 1-10wt%, more preferably 3-8wt%, more preferably 4-6wt%, and most preferably 5wt%; the pH value of the glutaraldehyde solution is preferably 7-11, more preferably 8-10, and more preferably 9; the concentration of the oxidized starch solution is preferably 0.01-10wt%, more preferably 1-5wt%, more preferably 2-4wt%, and most preferably 3wt%; the pH value of the dialdehyde starch solution is preferably 7-11, more preferably 8-10, and more preferably 9; the concentration of the dialdehyde starch solution is preferably 0.01-15wt%. t%, more preferably 1-10wt%, more preferably 2-8wt%, most preferably 6wt%; the pH value of the dialdehyde starch solution is preferably 7-11, more preferably 8-10, and more preferably 9; the concentration of the Jeffamine buffer is preferably 0.01-1mol / L, more preferably 0.03-0.08mmol / L, more preferably 0.04-0.06mmol / L, and most preferably 0.05mmol / L; the pH value of the Jeffamine buffer is preferably 8-13, more preferably 9-12, and more preferably 10-11; the temperature of the cross-linking treatment is preferably 20°C-30°C, more preferably 23°C-27°C, and more preferably 25°C; the time of the cross-linking treatment is preferably 12-36h, more preferably 18-30h, and more preferably 20-24h.

[0091] For the blood vessels after cross-linking treatment, it is preferred to use a chemical method to covalently bind heparin to perform lumen surface modification treatment. The solution used for the chemical method to covalently bind heparin is an MES buffer solution containing carbodiimide, N-hydroxysulfosuccinimide and heparin; the concentration of carbodiimide in the solution is preferably 1-100 mg / ml, more preferably 10-80 mg / ml; more preferably 20-70 mg, most preferably 30-50 mg / ml; the concentration of N-hydroxysulfosuccinimide in the solution is preferably 1-100 mg / ml, more preferably 1-80 mg / ml, more preferably 5-60 mg / ml, most preferably 10-50 mg / ml; the concentration of heparin in the solution is preferably 1-100 mg / ml, more preferably 1-80 mg / ml, more preferably 5-60 mg / ml, most preferably 10-50 mg / ml. The concentration is preferably 1-200 mg / ml, more preferably 10-150 mg / ml, more preferably 20-100 mg / ml, and most preferably 30-80 mg / ml; the pH value of the solution used for covalently binding heparin by the chemical method is preferably 5-13, more preferably 6-12, and more preferably 7-11; the temperature for covalently binding heparin is preferably 20°C-37°C, more preferably 23-30°C, and more preferably 25°C; the time for covalently binding heparin is preferably 0.5-24 h, more preferably 0.5-12 h, and more preferably 0.5-5 h.

[0092] After covalently binding heparin, it is preferably washed with physiological saline or buffer to obtain a nanostructured low-immunogenic bio-artificial blood vessel with heparin anticoagulant activity on the inner wall of the lumen and a preparation method thereof; the physiological saline is preferably 0.9% (V / V) physiological saline; the buffer is preferably PBS buffer; the washing temperature is preferably 10°C to 38°C, more preferably 20°C to 38°C, more preferably 25°C to 38°C, and most preferably 36°C to 38°C; the washing time is preferably 10 to 60 minutes, more preferably 20 to 50 minutes, more preferably 35 to 45 minutes, and most preferably 40 minutes.

[0093] In the present invention, unless otherwise specified, all the reagents mentioned above are sterilized by filtration.

[0094] The present invention adopts a method with less impact on the extracellular matrix of the blood vessel wall for decellularization. This method removes cells while retaining the extracellular matrix and wall structure of the blood vessel as much as possible, without affecting the mechanical properties and compliance of the blood vessel. Then, enzyme treatment is used to remove the cell nuclei and antigen components remaining after decellularization, significantly reducing the immunogenicity of the decellularized blood vessels. Finally, a cross-linking agent is used to improve the mechanical properties of the decellularized blood vessels, while blocking foreign protein antigen sites, further reducing immunogenicity, and increasing the durability of the blood vessels after implantation in the body, reducing the occurrence of luminal thrombosis and vascular calcification and decay. The bio-artificial blood vessels prepared by the present invention reduce the damage to the extracellular matrix of the blood vessel wall and the impact on mechanical properties while completely removing cells. At the same time, it is beneficial to the adhesion and growth of endothelial cells after implantation in the body, improving the long-term patency rate. In addition, this method has a short preparation cycle and low cost, and can prepare bio-artificial blood vessels of different inner diameters and lengths for clinical use, making it suitable for commercial production.

[0095] The present invention also provides a nanostructured, low-immunogenic bioartificial blood vessel prepared by the above method; the bioartificial blood vessel comprises a nanofiber structure of vascular collagen or vascular collagen and elastin; and also comprises a small amount of fibronectin, laminin, proteoglycan and glycoprotein; the mechanical properties of the bioartificial blood vessel exceed those of the human body's own great saphenous vein, and are similar to or better than those of the human body's small-caliber arteries.

[0096] The bio-artificial blood vessel provided by the present invention can be implanted in the body for a long time.

[0097] According to the present invention, specifically, the vascular collagen includes type I collagen, type III collagen and (or) type IV collagen.

[0098] According to the present invention, when the biological artificial blood vessel is mainly composed of vascular collagen, the content of the vascular collagen is preferably 30% to 85% of the dry weight of the biological artificial blood vessel.

[0099] When the biological artificial blood vessel is mainly composed of vascular collagen and elastin, the content of the vascular collagen is 20% to 55% of the dry weight of the biological artificial blood vessel, and the content of the elastin is 20% to 55% of the dry weight of the biological artificial blood vessel.

[0100] The present invention also provides an application of the above-mentioned nanostructured low-immunogenic bio-artificial blood vessel in the preparation of clinical indications such as establishment of hemodialysis vascular access for chronic renal failure, replacement of blood vessels for lower limb artery trauma, bypass grafting of peripheral arterial disease, coronary artery bypass grafting, and replacement or bypass grafting of blood vessels with matching caliber in other parts of the human body.

[0101] The present invention also provides the use of the above-mentioned nanostructured low-immunogenic bio-artificial blood vessels in establishing vascular access for chronic dialysis patients: as an alternative repair graft material after the decay, infection, and aneurysm formation of autologous arteriovenous fistulas or artificial blood vessel fistulas in chronic dialysis; the product can be implanted in the body to quickly perform hemodialysis and can be used as early as 1 to 10 days after surgery, avoiding the need for traditional polymer material blood vessels to wait for 3 to 4 weeks after surgery.

[0102] The bio-artificial blood vessels provided by the present invention can rapidly endothelialize after implantation in the body, reducing lumen thrombosis. Their short-, medium-, and long-term patency rates in the body are higher than those of small-caliber blood vessels made of traditional polymer materials such as expanded polytetrafluoroethylene (ePTFE) and polyurethane. At the same time, the bio-artificial blood vessels have extremely low immunogenicity, can resist infection, and quickly heal with surrounding tissues of the body, reducing or avoiding leakage of plasma or blood from the blood vessel walls.

[0103] To further illustrate the present invention, a method for preparing a nanostructured low-immunogenic biological artificial blood vessel provided by the present invention is described in detail below with reference to examples.

[0104] The reagents used in the following examples are all commercially available.

[0105] The present invention obtains a heterogeneous acellular matrix conduit, i.e., a nanostructured low-immunogenic bioartificial blood vessel and its preparation method by decellularizing, enzymatically treating, and cross-linking directly obtained blood vessels. The specific scheme is divided into the following steps:

[0106] (1) Blood vessels obtained directly from a slaughterhouse are sterilized and the fat and connective tissue surrounding the blood vessels are removed using medical scissors to obtain pre-treated blood vessels.

[0107] (2) Using chemical reagent decellularization methods, the cell components in the blood vessels are removed to obtain xenogeneic decellularized matrix conduits.

[0108] (3) Treating the acellular matrix conduit with nuclease to remove residual nuclear antigen components in the tissue; or treating the acellular matrix conduit with biological enzymes to remove vascular wall elastin.

[0109] (4) The enzyme-treated decellularized matrix blood vessels are treated with a solution containing aldehydes, oxidized starch, and dialdehyde starch crosslinkers to increase the mechanical properties of the blood vessels and block the xenogeneic extracellular matrix protein antigen sites while maintaining vascular compliance, thereby obtaining the final nanostructured low-immunogenicity bio-artificial blood vessels.

[0110] Example 1

[0111] (1) Obtain bovine arteries from a slaughterhouse. The bovine arteries were healthy, weighing 800 kg and passing quarantine. The inner diameter of the blood vessels was 3 to 10 mm. The fat and connective tissue surrounding the blood vessels were carefully removed using medical scissors without damaging the adventitia to obtain the pre-treated blood vessels.

[0112] The pretreated vessels were treated with 5.2% ficin (Sigma) at 37°C for 1 / 2 hour, 3 hours, 9 hours, 12 hours, and 24 hours. Samples were retained for testing at each time point, and the medium was changed every 8 hours for treatments exceeding 8 hours. After treatment, the vessels were transferred to 0.9% (v / v) saline and washed at 37°C for 40 minutes to obtain the enzymatically treated vessels. Tissue section staining, DNA quantification, and elastin quantification of the vessel wall were performed. The results showed that after 1 / 2 hour of enzymatic treatment, the vessel wall still contained a large number of cells (Figures 1a and 1b) and elastin (Table 1). After 3 hours of enzymatic treatment, the vessel wall contained little or only trace amounts of elastin (Table 1), but a large number of cell nuclei (Figures 2a and 2b) and DNA residues (Figures 2c and 2d) were still present in the vessel wall. Clinical use can trigger a strong immune response, so decellularization is required before enzymatic treatment.

[0113] Table-1 Comparison of quantitative and radial mechanical properties of elastin and collagen in bovine arteries treated with ficin at different time points

[0114] (2) There are two methods for decellularization:

[0115] A. Place the pretreated vessels in a decellularization reagent containing 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt (8 mmol / L), sodium lauryl sulfate (1.8 mmol / L), NaCl (0.12 mmol / L), NaOH (1 mol / L), and sterile deionized water at 37°C for 12 hours, changing the medium every 2 hours for a total of 6 times. After treatment, transfer the vessels to 0.9% (v / v) saline and rinse at 37°C for 40 minutes to obtain the decellularized vessels.

[0116] B. Place the pretreated vessels in a decellularization reagent containing 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt (8 mmol / L), EDTA (8 mmol / L), NaCl (0.12 mmol / L), NaOH (1 mol / L), and sterile deionized water at 37°C for 12 hours, changing the medium every 2 hours for a total of 6 times. After treatment, transfer the vessels to 0.9% (v / v) saline and rinse at 37°C for 40 minutes to obtain the decellularized vessels.

[0117] The blood vessels treated with two different decellularization methods were subjected to radial tensile and bursting pressure tests and compared. According to the test method in "YY / T 0500-2021 Cardiovascular Implants, Vascular Prostheses, Tubular Vascular Grafts and Vascular Patches Standard", the radial tensile strength of the decellularized vascular tissue of method A was 2.92±0.42N / mm (n=8), and the bursting pressure was 2642.4±116.7mmHg; the radial tensile strength of the decellularized vascular tissue of method B was 2.55±2.50N / mm (n=8), and the bursting pressure was 2014.2±121mmHg. The mechanical properties of the decellularized blood vessels of method A are better than those of method B.

[0118] (3) Enzyme treatment: The blood vessels after decellularization in the previous step were placed in 0.9% (V / V) saline containing DNase I (9 KU / L) and treated at 37°C for 48 hours, with the solution changed every 8 hours, for a total of 5 times. After treatment, the blood vessels were transferred to 0.9% (V / V) saline and washed at 37°C for 40 minutes to obtain blood vessels treated with DNase I. Histological and DNA quantitative detection was performed, and the tube walls were free of cell nuclei and DNA residues (Figure 3a shows decellularization followed by enzyme treatment in method A, and Figure 3b shows decellularization followed by enzyme treatment in method B). The tube walls mainly contained collagen and elastin. Figures 4 and 5 show the staining results of bio-type vascular tissue after decellularization using methods A and B, respectively. Figure 4a shows the collagen staining results of bio-type vascular tissue after decellularization using method A, and Figure 4b shows the elastin staining results of bio-type vascular tissue after decellularization using method A. Figure 5a shows the collagen staining results of bio-type vascular tissue after decellularization using method B, and Figure 5b shows the elastin staining results of bio-type vascular tissue after decellularization using method B. Decellularization method A preserves the mechanical properties of the vessel wall tissue and completely removes vessel wall cells and DNA after DNase treatment. Therefore, method A is preferred for subsequent experiments.

[0119] The decellularized blood vessels from Method A were treated with DNase I and then treated with 5.2% ficin (Sigma). The blood vessels were incubated in 0.9% (v / v) saline at 37°C for 1 / 2 hour, 3 hours, 9 hours, 12 hours, and 24 hours. Samples were retained for testing at each time point. The medium was changed every 8 hours for periods exceeding 8 hours. After treatment, the grafts were transferred to 0.9% (v / v) saline and rinsed at 37°C for 40 minutes to obtain the enzyme-treated vascular grafts.

[0120] The results of DNA content, fibrin, and elastin quantification and radial tensile testing of vascular tissues treated with ficin for different times are shown in Table 2. The preferred ficin treatment time was 3 hours for subsequent experiments.

[0121] Table-2 Quantitative analysis and radial tensile test results of collagen and elastin in vascular tissues treated with ficin at different times

[0122] (4) Cross-linking treatment: Finally, the blood vessels treated with DNase I for 48 h and ficin for 3 h in step (3) were placed in a 6% (wt) dialdehyde starch solution at 25°C for 30 h, and then transferred to 0.9% (v / v) saline for washing for 40 min to obtain a biological artificial blood vessel.

[0123] (5) Covalent binding of heparin: 1200 mg of EDC and 1000 mg of Sulfo-NHS were dissolved in 20 ml of MES buffer, and 1000 mg of heparin was dissolved in 20 ml of MES buffer. The two were mixed in equal volumes and the pH was adjusted to 7 to obtain a heparin grafting reagent. The bioartificial blood vessel obtained in the previous step was then placed in the reagent and shaken in the dark for 3 h to obtain a heparin-coated bioartificial blood vessel. The blood vessel was then implanted in the body for venous fistula and vascular replacement surgery.

[0124] (6) The untreated fresh blood vessels (Figure 6) and the bio-artificial blood vessels treated in step (5) (Figure 7) were subjected to radial tensile and burst pressure tests. The tensile strength of the untreated fresh blood vessels was 2.15±0.22N / mm (n=8), and the tensile strength of the treated bio-artificial blood vessels was 2.05±0.13N / mm (n=8). P>0.05, indicating no significant difference. The burst pressure of the untreated fresh blood vessels was 2252.5±220.8mmHg (n=8), and the burst pressure of the bio-artificial blood vessels treated in step (5) was 2388.8±433.3mmHg (n=8). P>0.05, indicating no significant difference. This indicates that decellularization has no effect on the mechanical properties of the blood vessels.

[0125] (7) The bio-artificial blood vessels and their preparation method after treatment in step (5) were dehydrated and sprayed with gold, and then photographed with a scanning electron microscope (SEM). The SEM photographs of the cross-section, endothelium, and adventitia at different magnifications of the blood vessels are shown in Figures 8, 9, and 10, respectively. The average diameter of the cross-section fibers was 74.04±11.37 nm (n=10, Figure 8c), the average diameter of the endothelium fibers was 41.02±5.36 nm (n=10, Figure 9c), and the average diameter of the adventitia fibers was 84.89±12.96 nm (n=10, Figure 10d). The comparison between the diameters of the cross-section fibers and the endothelium fibers was P<0.01, and the comparison between the diameters of the endothelium fibers and the adventitia fibers was P<0.01, indicating significant differences. The comparison between the diameters of the cross-section fibers and the adventitia fibers was P>0.05, indicating no significant differences.

[0126] Example 2

[0127] (1) The blood vessels prepared in Example 1 were sliced ​​before and after decellularization and then stained with DAPI (Figures 11 and 12). Blue fluorescence represents cell nuclei. Many cell nuclei can be observed in the stained photos of the blood vessel slices before decellularization, but no cell nuclei can be detected in the staining of the blood vessel slices after decellularization, indicating that the vessel wall cells have been completely removed.

[0128] The staining process of the bio-artificial blood vessels before and after decellularization is as follows: 1) prepare 4 μm tissue sections; 2) air-dry at room temperature for 15 minutes; 3) fix with fixative for 10 minutes and wash with PBS for 5 minutes; 4) stain with DAPI for 5 minutes; 5) wash with PBS for 10 minutes; 6) seal with anti-fluorescence quenching sealing medium.

[0129] Unless otherwise specified, the following enzymatically treated blood vessels were obtained by first decellularizing them using method A and then continuing the enzymatic treatment.

[0130] (3) Suture tension: The suture tension of the fresh bovine blood vessels in Example 1 was 206±7 g (n=8). The suture tension of the vascular graft without elastin in Example 1 (i.e., the blood vessels treated with DNase I and ficin in step (3) and cross-linked with dialdehyde starch) was 201±11 g (n=8). P>0.05, indicating no significant difference.

[0131] Example 3

[0132] (1) Obtain the superior rectal artery of a healthy bovine weighing 800 kg from a slaughterhouse. The vessel diameter is 4-8 mm. Use medical scissors to carefully remove the fat and connective tissue surrounding the vessel without damaging the adventitia.

[0133] (2) Decellularization: The pretreated blood vessels were placed in a decellularization reagent containing 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt (10 mmol / L), sodium lauryl sulfate (2.5 mmol / L), NaCl (0.12 mmol / L), NaOH (1 mol / L), and sterile deionized water. The cells were treated at 20°C for 30 h, with the solution changed every 8 h for a total of three times. After treatment, the cells were transferred to 0.9% (v / v) physiological saline and washed at 37°C for 40 min to obtain the decellularized blood vessels.

[0134] (3) Enzyme treatment: The blood vessels treated in the previous step were placed in 0.9% (V / V) saline containing Benzonase nuclease (15KU / L) and treated at 37°C for 48 hours, with the solution changed every 8 hours, for a total of 5 times. After the treatment, they were transferred to 0.9% (V / V) saline and washed at 37°C for 40 minutes; the blood vessels treated with the above Benzonase nuclease were treated with ficin (3KU / L) in 0.9% (V / V) saline, and treated at 37°C for 24 hours, with the solution changed every 8 hours, for a total of 3 times. After the treatment, they were transferred to 0.9% (V / V) saline and washed at 37°C for 40 minutes to obtain enzyme-treated blood vessels.

[0135] (4) Cross-linking treatment: Finally, the enzyme-treated blood vessels were placed in a solution containing 6% (wt) dialdehyde starch at 25°C for 24 h, and then transferred to 0.9% (V / V) saline for washing for 40 min to obtain biological artificial blood vessels.

[0136] (5) Covalent binding of heparin: 800 mg of EDC and 400 mg of Sulfo-NHS were dissolved in 20 ml of MES buffer. Similarly, 800 mg of heparin was dissolved in 20 ml of MES buffer. The two were mixed in equal volumes and the pH was adjusted to 7 to obtain a heparin grafting reagent. The bioartificial blood vessel obtained in the previous step was then placed in the reagent and shaken in the dark for 3 h to obtain a heparin-coated bioartificial blood vessel for heart bypass surgery.

[0137] (6) The blood vessels treated in step (5) were subjected to radial tensile and bursting pressure tests according to the method in Example 2. The test results showed that the radial tensile strength was 2.11±0.25 N / mm (n=8) (as shown in Figure 13) and the bursting pressure strength was 2364±203 mmHg (n=8) (as shown in Figure 14); the DNA content was 26.42±2.21 ng / ml (n=8) when tested according to the method for determining the residual DNA content of animal-derived biological materials.

[0138] (7) The collagen and elastin content of the bio-artificial blood vessels obtained in step (5) were quantitatively tested. The collagen content accounted for 80.32±9.06% (n=8) of the blood vessel dry weight, and the elastin content accounted for 0% (n=8) of the blood vessel dry weight.

[0139] Example 4

[0140] (1) The median sacral artery of a healthy bovine weighing 900 kg and passing quarantine was obtained from a slaughterhouse. The vessel diameter was 4-8 mm. The fat and connective tissue surrounding the vessel were carefully removed using medical scissors without damaging the adventitia.

[0141] (2) Decellularization: The pretreated blood vessels were placed in a decellularization reagent containing 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid (6 mmol / L), EDTA (8.0 mmol / L), NaCl (0.1 mmol / L), NaOH (0.8 mol / L), and sterile deionized water. The cells were treated at 20°C for 30 h, with the solution changed every 8 h for a total of three times. After treatment, the cells were transferred to 0.9% (v / v) saline and washed at 37°C for 40 min to obtain the decellularized blood vessels.

[0142] (3) Enzyme treatment: The treated blood vessels were placed in 0.9% (V / V) saline containing ficin (1000 KU / L) at 37°C for 48 h, with the solution changed every 8 h for a total of 5 times. After treatment, the vessels were transferred to 0.9% (V / V) saline and washed at 37°C for 40 min to obtain the enzyme-treated blood vessels.

[0143] (4) Cross-linking treatment: Finally, the blood vessels after enzyme treatment were placed in a solution containing 5% (wt) dialdehyde starch at 25°C for 24 hours, and then transferred to 0.9% (V / V) physiological saline for washing for 40 minutes to obtain a biological artificial blood vessel.

[0144] (5) Covalent binding of heparin: 1500 mg of EDC and 800 mg of Sulfo-NHS were dissolved in 20 ml of MES buffer. Similarly, 1000 mg of heparin was dissolved in 20 ml of MES buffer. The two were mixed in equal volumes and the pH was adjusted to 7 to obtain a heparin grafting reagent. The bioartificial blood vessel obtained in the previous step was then placed in the reagent and shaken in the dark for 3 h to obtain a heparin-coated bioartificial blood vessel.

[0145] (6) The blood vessels treated in step (5) were subjected to radial tensile and bursting pressure tests according to the method in Example 2. The test results showed that the radial tensile strength was 2.09±0.18N / mm (n=8) (as shown in Figure 13) and the bursting pressure strength was 2627±185mmHg (n=8) (as shown in Figure 14); the DNA content was 42.57±2ng / ml (n=8) when tested according to the method for determining the residual DNA content of animal-derived biological materials.

[0146] (7) The collagen and elastin content of the vascular wall of the bio-artificial blood vessel obtained in step (5) were quantitatively tested. The collagen content accounted for 82.32±4.65% (n=8) of the dry weight of the blood vessel, and the elastin content accounted for 0% (n=8) of the dry weight of the blood vessel.

[0147] Example 5

[0148] (1) Obtain the femoral vein of a healthy pig weighing 100 kg from a slaughterhouse. The diameter of the blood vessel should be 4-8 mm. Use medical scissors to carefully remove the fat and connective tissue around the blood vessel without damaging the adventitia.

[0149] (2) Decellularization: The pretreated blood vessels were placed in a decellularization reagent containing 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt (8 mmol / L), EDTA (25 mmol / L), NaCl (0.12 mmol / L), NaOH (1 mol / L), and sterile deionized water. The cells were treated at 25°C for 30 h, with the solution changed every 8 h for a total of three times. After treatment, the cells were transferred to 0.9% (v / v) physiological saline and washed at 37°C for 40 min to obtain the decellularized blood vessels.

[0150] (3) Enzyme treatment: Place the blood vessels treated in the previous step in 0.9% (V / V) saline containing DNase I enzyme (20KU / L) and treat at 37°C for 48 hours, changing the solution every 8 hours, for a total of 5 times. After treatment, transfer to 0.9% (V / V) saline and wash at 37°C for 40 minutes. Then place in 0.9% (V / V) saline containing ficin (100KU / L) and treat at 37°C for 24 hours, changing the solution every 8 hours, for a total of 2 times. After treatment, transfer to 0.9% (V / V) saline and wash at 37°C for 40 minutes to obtain enzyme-treated blood vessels.

[0151] (4) Cross-linking treatment: Finally, the blood vessels treated with ficin were placed in a solution containing 3% (wt) dialdehyde starch at 25°C for 24 h, and then transferred to 0.9% (V / V) saline for washing for 40 min to obtain a biological artificial blood vessel.

[0152] (5) Covalent binding of heparin: 2000 mg of EDC and 1000 mg of Sulfo-NHS were dissolved in 20 ml of MES buffer. Similarly, 1500 mg of heparin was dissolved in 20 ml of MES buffer. The two were mixed in equal volumes and the pH was adjusted to 7 to obtain a heparin grafting reagent. The bioartificial blood vessel obtained in the previous step was then placed in the reagent and shaken in the dark for 3 h to obtain a heparin-coated bioartificial blood vessel.

[0153] (6) The blood vessels treated in step (5) were subjected to radial tensile and bursting pressure tests. The test results showed that the radial tensile strength was 2.06±0.37 N / mm (n=8) (as shown in Figure 13), and the bursting pressure strength was 2764±201 mmHg (n=8) (as shown in Figure 14); the test was carried out according to the method for determining the residual DNA content of animal-derived biological materials, and the DNA content was 39.31±1.8 ng / ml (n=8).

[0154] (7) The collagen and elastin content of the vascular wall of the bio-artificial blood vessel obtained in step (5) were quantitatively tested. The collagen content accounted for 72.31±10.03% (n=8) of the dry weight of the blood vessel, and the elastin content accounted for 0% (n=8) of the dry weight of the blood vessel.

[0155] Example 6

[0156] (1) Obtain a healthy, quarantined sheep inferior mesenteric artery with a diameter of 4-8 mm. Use medical scissors to carefully remove the fat and connective tissue surrounding the vessel without damaging the adventitia.

[0157] (2) Decellularization: The pretreated blood vessels were placed in a decellularization reagent prepared by mixing 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt (10 mmol / L), EDTA (30 mmol / L), NaCl (0.15 mmol / L), NaOH (1.2 mol / L), and sterile deionized water. The treatment was carried out at 25°C for 30 h, with the solution changed every 8 h for a total of 3 times. After treatment, the vessels were transferred to 0.9% (v / v) physiological saline and washed at 37°C for 40 min to obtain the decellularized blood vessels.

[0158] (3) Enzyme treatment: The treated blood vessels were placed in 0.9% (v / v) saline containing DNase I (50 kU / L) at 37°C for 48 h, with the solution changed every 8 h for a total of 5 times. After treatment, the vessels were transferred to 0.9% (v / v) saline and washed at 37°C for 40 min to obtain the enzyme-treated blood vessels.

[0159] (4) Cross-linking treatment: Finally, the enzyme-treated blood vessels were placed in a solution containing 6% (wt) dialdehyde starch at 25°C for 24 h, and then transferred to 0.9% (V / V) saline for washing for 40 min to obtain biological artificial blood vessels.

[0160] (5) Covalent binding of heparin: 1600 mg of EDC and 800 mg of Sulfo-NHS were dissolved in 20 ml of MES buffer. Similarly, 1600 mg of heparin was dissolved in 20 ml of MES buffer. The two were mixed in equal volumes and the pH was adjusted to 7 to obtain a heparin grafting reagent. The bioartificial blood vessel obtained in the previous step was then placed in the reagent and shaken in the dark for 3 h to obtain a heparin-coated bioartificial blood vessel.

[0161] (6) The blood vessels treated in step (5) were subjected to radial tensile and bursting pressure tests according to the method in Example 2. The test results showed that the radial tensile strength was 2.36±0.07N / mm (n=8) (as shown in Figure 13) and the bursting pressure strength was 2412±105mmHg (n=8) (as shown in Figure 14); the biological artificial blood vessel specimens were tested according to the method for determining the residual DNA content of animal-derived biological materials, and the DNA content was 48.13±1.47ng / mg (n=8).

[0162] (7) The collagen and elastin content of the vascular wall of the bio-artificial blood vessel obtained in step (5) were quantitatively tested. The collagen content accounted for 76.19±9.32% (n=8) of the dry weight of the blood vessel, and the elastin content accounted for 0% (n=8) of the dry weight of the blood vessel.

[0163] Example 7

[0164] A commercial artificial blood vessel (control group) and a heparin-coated bioartificial blood vessel prepared in Example 1 (experimental group) were implanted in the corresponding areas of experimental pigs to create an artificial arteriovenous fistula model and a common carotid artery replacement model. The experimental pigs were male, weighing 80-100 kg. Five days prior to surgery, the pigs were given 100 mg / day of aspirin and 75 mg / day of Plavix. They were fasted for 12-24 hours before anesthesia.

[0165] During the operation, the animals were under general anesthesia, and their respiration, heart rate, and blood oxygen saturation were monitored in real time. The skin in the middle of the neck was shaved, disinfected, and covered with a three-layer drape. The skin was incised in the middle, hemostasis was performed layer by layer, and the right common carotid artery and right internal venous vein were isolated. The diameter, flow rate, and wall thickness of the arteries and veins were examined intraoperatively using an ultrasound probe. Heparin 200 U / kg was administered intravenously. Three minutes later, two dog-head clamps were used to clamp the artery. The right common carotid artery was longitudinally incised 8 mm. The lumen was flushed with heparin-papaverine solution, and 6-0 Prolene was used for continuous anastomosis. After completion, a bio-artificial blood vessel was clamped, and the anastomosis was checked for bleeding. Two dog-head clamps were used to clamp the internal jugular vein, the lumen was flushed with heparin-papaverine solution, and 6-0 Prolene was used for continuous suture. The dog-head clamp of the bio-artificial blood vessel was loosened with the last stitch to vent air, and the knot was tied. The bleeding was checked, as shown in Figure 15 (control group) and Figure 16 (experimental group). Postoperative ultrasound examination of the flow rate of the bio-artificial blood vessel and arteriovenous anastomosis was performed, as shown in Figures 17 and 18. The surgical field was carefully examined, and the incision was sutured layer by layer. Postoperative angiography examined the patency of the vascular graft in the experimental porcine arteriovenous fistula model, as shown in Figure 19. Following surgery, the animals recovered and were returned to the animal room for observation. Subsequent care and management were conducted in accordance with standard animal experimentation procedures. One week after surgery, 2.0 g of ceftriaxone sodium was administered twice daily. Aspirin 100 mg once daily and Plavix 75 mg once daily were also administered long-term postoperatively until the end of the three-month observation period.

[0166] In the experimental porcine right common carotid artery replacement model in Example 1, a heparin-coated bioprosthetic blood vessel was used, as shown in Figure 20 ; angiography performed immediately after surgery demonstrated vascular patency, as shown in Figure 21 . Postoperative ultrasound and DSA follow-up time points are shown in Table 3 . Follow-up angiography demonstrated patency of both the bioprosthetic blood vessel and the native blood vessel near the anastomosis, as shown in Figure 22 .

[0167] 1) Postoperative follow-up

[0168] Table 3 Ultrasound and DSA follow-up time points for arteriovenous fistula and common carotid artery replacement

[0169] 2) Postoperative complications and unexpected conditions: Observe neck hematoma, swelling, redness, abscess, pulmonary embolism, etc. after surgery.

[0170] 3) Animal Sacrifice and Sampling: At the end of the observation period, the experimental animals were euthanized according to the standard operating procedures for laboratory animal experiments, and a comprehensive examination was performed, including the animal's overall condition and major organs (heart, liver, spleen, lungs, and kidneys). A midline skin incision was made, and the right common carotid artery and right internal jugular vein were isolated. Heparin 200 U / kg was administered. Three minutes later, two dog-head clamps were used to clamp the right common carotid artery and internal jugular vein. A 2-cm section of blood vessels near the arterial anastomosis and a 5-cm section of blood vessels near the venous anastomosis were retained. The "worker"-shaped specimen with the artificial blood vessel was removed, washed with saline, and fixed in 4% paraformaldehyde. After the autopsy and sample collection were completed, the animal's body was initially processed according to the standard operating procedures and then transported to a body disposal plant for harmless incineration.

[0171] 4) Experimental Evaluation: Ultrasound and DSA were used to evaluate the patency and degree of vascular stenosis. Sections were stained and the percentage of neointima in the lumen area at the arterial anastomosis, the mid-section of the graft, the venous anastomosis, and the proximal and distal ends of the vein was calculated.

[0172] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a nanostructured low-immunogenicity biological artificial blood vessel, characterized in that: The following steps are involved: S1) performing a decellularization treatment on the pretreated animal blood vessels to obtain decellularized blood vessels; S2) treating the decellularized blood vessel in an enzyme solution to obtain an enzyme-treated blood vessel; the enzyme solution comprises nuclease and / or biological enzyme; S3) cross-linking the blood vessel after the enzyme treatment with a cross-linking agent to obtain a nanostructured low-immunogenicity bio-artificial blood vessel.

2. The preparation method according to claim 1, characterized in that: The animal blood vessel is selected from the artery or vein of a large animal; the large animal is selected from pigs, sheep, dogs, cattle or horses; The animal blood vessel is selected from the group consisting of aorta, pulmonary artery, superior vena artery, inferior vena artery, common carotid artery, internal jugular vein, external jugular vein, femoral artery, femoral vein, iliac artery, iliac vein, superior mesenteric artery, inferior mesenteric artery, rectal artery, median sacral artery or lower limb artery; The decellularization reagent used in the decellularization treatment in step S1) includes a detergent or a detergent and a chelating agent; the concentration of the detergent in the decellularization reagent is 0.01 to 500 mmol / L; the concentration of the chelating agent in the decellularization reagent is 0.01 to 500 mmol / L; The temperature of the decellularization treatment is 10° C. to 38° C.; the time of the decellularization treatment is 2 to 72 hours; and the decellularization reagent is replaced every 1 to 24 hours during the decellularization treatment.

3. The preparation method according to claim 2, characterized in that: When the decellularization reagent includes a detergent, the detergent is selected from one or more of a nonionic detergent, an anionic detergent, a cationic detergent, and a zwitterionic detergent; When the decellularization reagent includes a detergent and a chelating agent, the detergent is selected from one or more of a nonionic detergent, an anionic detergent, a cationic detergent and a zwitterionic detergent; The nonionic detergent is selected from one or more of polyethylene glycols, polyethylene glycol octylphenyl ethers, polyols, polyoxyethylene fatty alcohol ethers and polyoxyethylene alkylphenol ethers; The anionic detergent is selected from one or more of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecyl sulfonate, sodium cholate and sodium deoxycholate; The cationic detergent is selected from benzalkonium bromide and / or cetyltrimethylammonium bromide; The zwitterionic detergent is selected from 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonic acid inner salt and / or or n-tetradecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate; The chelating agent is an inorganic chelating agent and / or an organic chelating agent; The inorganic chelating agent is selected from one or more of sodium tripolyphosphate, sodium hexametaphosphate and sodium pyrophosphate; The organic chelating agent is selected from one or more of aminotriacetic acid, ethylenediaminetetraacetic acid, ethylene glycol ditetraacetic acid, ethylenediaminediacetic acid, cyclohexanediaminetetraacetic acid, S,S-ethylenediaminedisuccinic acid, diethyltriacetic acid, diethylenetriaminepentaacetic acid and its salts, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid and dihydroxyethylglycine; The decellularization reagent also includes sodium chloride, sodium hydroxide and water; The concentration of sodium chloride in the decellularization reagent is 0.01-1.0 mmol / L; The concentration of sodium hydroxide in the decellularization reagent is 0.1-2.0 mol / L.

4. The preparation method according to claim 1, characterized in that: The concentration of the nuclease in the enzyme solution is 1 to 50,000 KU / L; and / or the concentration of the biological enzyme in the enzyme solution is 1 to 50,000 KU / L; The enzyme solution also includes 1 to 50 wt% human serum or animal serum; The enzyme solution also includes physiological saline or buffer; The temperature of the enzyme treatment in step S2) is 36° C. to 37° C.; the time of the enzyme treatment is 2 to 72 hours; and the enzyme solution is replaced every 1 to 24 hours during the enzyme treatment.

5. The preparation method according to claim 1, characterized in that: After the enzyme treatment in step S2), the blood vessel is further treated with a solution containing a biological enzyme to obtain an enzyme-treated blood vessel; The nuclease is selected from DNA enzyme and / or RNA enzyme; the biological enzyme is selected from one or more of pepsin, lipase, trypsin, cathepsin, papain, ficin and subtilisin; the concentration of the biological enzyme in the solution containing the biological enzyme is 1 to 50000 KU / L; the solvent of the solution containing the biological enzyme is physiological saline, buffer or special solution for enzyme preparation; The temperature for treating with the solution containing biological enzyme is 36° C. to 37° C.; the treatment time is 0.25 to 48 hours; and the solution containing biological enzyme is replaced every 1 to 24 hours during the treatment process.

6. The preparation method according to claim 1, characterized in that: The cross-linking agent is selected from one or more of glutaraldehyde solution, oxidized starch solution, dialdehyde starch solution and Jeffamine buffer; The concentration of the glutaraldehyde solution is 0.1 to 30 wt %; The concentration of the oxidized starch solution is 0.01 to 10 wt % The concentration of the dialdehyde starch solution is 0.01 to 10 wt %; The concentration of the Jeffamine buffer is 0.01-1.0 mol / L.

7. The preparation method according to claim 1, characterized in that: After the cross-linking treatment in step S3), heparin is covalently bound using a chemical method to obtain a biological artificial blood vessel.

8. The preparation method according to claim 7, characterized in that: The solution used for chemically covalently binding heparin is a MES buffer solution containing carbodiimide, N-hydroxysulfosuccinimide and heparin; the concentration of heparin in the solution used for chemically covalently binding heparin is 1-200 mg / ml.

9. The nanostructured low-immunogenicity bioartificial blood vessel prepared by the preparation method of any one of claims 1 to 8, characterized in that: The biological artificial blood vessel comprises vascular collagen or a nanofiber structure formed by vascular collagen and elastin.

10. The nanostructured low immunogenic biological artificial blood vessel according to claim 9, characterized in that: The vascular collagen includes type I collagen, type III collagen and / or type IV collagen; When the biological artificial blood vessel includes vascular collagen, the content of the vascular collagen is 30% to 85% of the dry weight of the biological artificial blood vessel; When the biological artificial blood vessel comprises vascular collagen and elastin, the content of the vascular collagen is 20% to 55% of the dry weight of the biological artificial blood vessel, and the content of the elastin is 20% to 55% of the dry weight of the biological artificial blood vessel.

11. Use of the nanostructured low-immunogenicity bioartificial blood vessel prepared by any one of the preparation methods of claims 1 to 8 or the nanostructured low-immunogenicity bioartificial blood vessel described in claim 9 or 10 in the preparation of vascular graft materials for hemodialysis vascular access for chronic renal failure, vascular graft materials for lower limb artery trauma, vascular graft materials for peripheral arterial disease bypass surgery, or vascular graft materials for coronary artery bypass surgery.

12. Use of the nanostructured low-immunogenicity bioartificial blood vessel prepared by any one of the preparation methods of claims 1 to 8 or the nanostructured low-immunogenicity bioartificial blood vessel described in claim 9 or 10 in the preparation of vascular graft materials for vascular access in chronic dialysis patients; the bioartificial blood vessel is used as a replacement repair graft material after chronic dialysis autologous arteriovenous fistula or artificial vascular fistula has failed, infected or formed aneurysm.

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