A double-layer small-diameter artificial blood vessel with in-situ modified anticoagulant coating and a preparation method thereof

Abstract

The application discloses a double-layer small-diameter artificial blood vessel with an in-situ modified anticoagulant coating and a preparation method. The double-layer small-diameter artificial blood vessel with an in-situ modified anticoagulant coating is prepared by combining the coaxial dry-jet wet spinning technology and the polydopamine-heparin modification technology. The double-layer small-diameter blood vessel obtained has sufficient mechanical strength and structural stability, the inner layer has good hydrophilicity and excellent anticoagulant function by the in-situ grafting of heparin through polydopamine, the activation of platelets and the adhesion of blood cells are reduced, the formation of short-term acute thrombus is prevented, and the endothelialization process of the small-diameter blood vessel is promoted. The method provided by the application simplifies the manufacturing and surface modification process of the small-diameter artificial blood vessel, avoids additional procedures such as secondary processing, the modification position and the grafting amount are controllable, the experimental stability is high, and the application provides a convenient and efficient preparation technical idea for the development of small-diameter artificial blood vessels.

Description

Technical Field

[0001] The present invention relates to a double-layer small-caliber artificial blood vessel and a preparation method thereof in the field of artificial blood vessel preparation technology, and in particular to a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating, a preparation method thereof, and a product thereof. Background Art

[0002] At present, large-caliber (inner diameter ≥ 6 mm) artificial blood vessels with expanded polytetrafluoroethylene (ePTFE) as the main material have been successfully commercialized and widely used in clinical practice. However, small-caliber (inner diameter < 6 mm) vascular grafts that can be used for coronary artery bypass grafting, hemodialysis arteriovenous fistula, peripheral vascular replacement surgery, etc. still use autologous blood vessels as the gold standard, and there are no clinically available artificial blood vessel products. Autologous blood vessels are limited in donor quality and availability, and the use of invasive collection techniques may lead to lesions in the donor site, and can no longer meet the current growing clinical needs. Therefore, the development of small-caliber artificial blood vessels has become an urgent problem to be solved.

[0003] Coaxial dry-jet wet spinning, based on phase separation, is an efficient, convenient, and easily controllable solution spinning technique widely used in the fabrication of thin film hollow tubes. The resulting hollow tubular structures exhibit high specific surface area, excellent permselectivity, and a typical porous structure, finding widespread application in biology and regenerative medicine. Through material selection and parameter manipulation, small-caliber, double-layer artificial blood vessels can be fabricated that mimic the structure and mechanical properties of natural blood vessels.

[0004] To ensure long-term patency, small-caliber artificial blood vessels must possess not only sufficient mechanical strength and good biocompatibility, but also excellent anticoagulant properties. Anticoagulant modification of the inner wall of small-caliber artificial blood vessels is particularly difficult. Traditional chemical modification requires the entire vessel to be immersed in a reaction solution for functionalization. This involves complex processing steps, and the location of the modification and the amount of grafting are difficult to control. This only provides a short-term anticoagulant effect on the surface of the substrate, failing to penetrate deeply into the artificial vessel in three dimensions. Blood components easily penetrate the inner wall of the vessel, inducing a coagulation cascade, leading to fibrin and platelet adhesion and, ultimately, thrombus formation.

[0005] Therefore, it is necessary to propose a double-layer small-caliber artificial blood vessel with in situ modified anticoagulant coating. Summary of the Invention

[0006] In response to the deficiencies of the above-mentioned prior art, the present invention combines coaxial dry-jet wet spinning technology with polydopamine-heparin modification technology to provide a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating, as well as a preparation method and application. The double-layer small-caliber blood vessel obtained by the present invention has an outer layer that provides sufficient mechanical strength and structural stability, and an inner layer that has good hydrophilicity and excellent anticoagulant function through in-situ grafting of heparin with polydopamine, which reduces platelet activation and blood cell adhesion, prevents the formation of short-term acute thrombosis, and promotes the endothelialization process of small-caliber blood vessels. The present invention realizes anticoagulant modification of the inner wall of the blood vessel through polydopamine-heparin combination during formation, and obtains a double-layer small-caliber artificial blood vessel with an inner wall in-situ modified with heparin anticoagulant coating at one time, avoiding additional procedures such as secondary processing, simplifying the manufacturing and surface modification process of the artificial blood vessel, and making the modification position and grafting amount controllable. The experimental stability is high, providing a convenient and efficient preparation technology idea for the development of small-caliber artificial blood vessels.

[0007] The double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating prepared by the present invention not only retains the original double-layer characteristics and microscopic pore structure, but also provides sufficient mechanical strength, and also ensures that the modification position and grafting amount of the polydopamine-sodium heparin anticoagulant coating are controllable. The preparation method provided is highly operable, has high experimental stability, and is easy to mass produce.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] 1. A method for preparing a double-layer small-caliber artificial blood vessel with in-situ modified anticoagulant coating

[0010] Step 1: Prepare the outer layer spinning solution, inner layer spinning solution, and spinning core solution containing sodium heparin, let them stand to remove bubbles, and then dry and seal them for storage. The standing time for removing bubbles is 24 hours.

[0011] Step 2: The spinning equipment includes a coaxial spinneret, several injection needles, several syringe pumps, and a curing tank. The outer layer spinning solution, inner layer spinning solution, and core solution containing sodium heparin are injected into the three injection needles of the spinning equipment, followed by dry-jet wet spinning. Once the polymer film hollow tube in the curing tank stabilizes, the double-layer small-diameter polyurethane blood vessel sample is removed from the curing tank.

[0012] Step 3: Soak the obtained double-layer small-caliber polyurethane blood vessel sample in a glycerol aqueous solution, then rinse with deionized water, and dry to obtain a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating.

[0013] In the step 1, the outer layer spinning solution is a first polyurethane solution A obtained by dissolving high-strength polyurethane transparent raw material particles in N-methylpyrrolidone.

[0014] In the step 1, the inner layer spinning solution is a second polyurethane solution B obtained by dissolving high-elasticity polyurethane transparent raw material particles and dopamine hydrochloride powder in N-methylpyrrolidone.

[0015] In the step 1, the spinning core solution containing sodium heparin is a third solution C obtained by dissolving sodium heparin in an alkaline Tris-HCl buffer.

[0016] The mass fraction of high-strength polyurethane in the first polyurethane solution A is 0.15-0.2 g / mL. The mass fraction of high-elasticity polyurethane in the second polyurethane solution B is 0.12-0.15 g / mL, and the concentration of dopamine hydrochloride is 0.5-5.0 g / L.

[0017] The tensile strength of the high-strength polyurethane is more than twice that of the high-elasticity polyurethane; and the Young's modulus of the high-elasticity polyurethane is less than 1 / 3 of the Young's modulus of the high-strength polyurethane.

[0018] The pH value of the Tris-HCl buffer is 8.0-10.0, and the concentration of heparin sodium in the third solution C is 5.0-30.0 g / L.

[0019] In step 2, the inner diameter of the inner needle tube of the spinneret is 0.8 mm, and the outer diameter is 1.1 mm; the inner diameter of the middle needle tube is 2.6 mm, and the outer diameter is 2.9 mm; and the inner diameter of the outer nozzle is 3.5 mm.

[0020] In the step 2, the distance between the outlets of the outer nozzle, the middle needle tube and the inner needle tube of the spinning equipment and the liquid surface of the solidification pool is 2-4 cm.

[0021] In the step 2, the extrusion rate of the outer layer spinning solution is 1.6-2.4 mL / min; the extrusion rate of the inner layer spinning solution is 2.0-2.8 mL / min; and the extrusion rate of the spinning core solution containing sodium heparin is in the range of 3.2-4.8 mL / min.

[0022] In the step 2, during the spinning process using the dry-jet wet spinning method, the temperature of the spinneret and the curing tank is maintained at 60-75°C.

[0023] In the step 2, the collection method is free fall, and the thin film hollow tube is immersed in the solidification tank for 24 hours.

[0024] In step 3, the volume fraction of the glycerol aqueous solution is 20%, the polymer film hollow tube is immersed in the glycerol aqueous solution for 24 hours, and the drying method is freeze-drying for 24 hours.

[0025] 2. A double-layer small-caliber artificial blood vessel with in situ modified anticoagulant coating

[0026] The double-layer small-caliber artificial blood vessel is prepared by the preparation method.

[0027] 3. One product

[0028] The product includes the double-layer small-caliber artificial blood vessel with the in-situ modified anticoagulant coating.

[0029] The double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating provided by the present invention has an aesthetically pleasing appearance, a uniform and stable structural shape, and mechanical strength that meets the requirements for vascular transplantation; the inner wall modification is controllable, and the heparinization density is high; the coating improves the hydrophilicity of the material surface, has high blood compatibility, and has excellent anticoagulant function, significantly reducing the adhesion of platelets and whole blood cells on the sample surface; the inner surface with anticoagulant function is conducive to the proliferation and spreading of vascular endothelial cells, and is conducive to the formation of a long-term stable vascular endothelium.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The double-layer small-caliber artificial blood vessel provided by the present invention has controllable size, stable structural shape and sufficient mechanical strength; the preparation method provided by the present invention adopts mature technology, does not require high operator experience, has high experimental stability, good repeatability, and is easy to mass produce.

[0032] 2. The preparation method of the double-layer small-caliber artificial blood vessel with in situ modified anticoagulant coating provided by the present invention simplifies the manufacturing and surface modification process of the artificial blood vessel. The anticoagulant modification of the inner wall of the blood vessel is achieved through the combination of polydopamine and heparin during the formation, avoiding additional procedures such as secondary processing; the modification position and grafting amount are controllable.

[0033] 3. The double-layer small-caliber artificial blood vessel with in situ modified anticoagulant coating provided by the present invention has excellent blood compatibility, significantly avoids platelet activation and reduces the adhesion of whole blood cells on the sample surface; and has a good endothelialization effect.

[0034] 4. The polyurethane in the outer layer spinning solution and the inner layer spinning solution provided by the present invention are respectively of high strength and high elasticity. In this way, on the basis of ensuring the strength of the artificial blood vessel, the compliance of the artificial blood vessel is improved, and thrombosis and intimal hyperplasia caused by the mismatch between the compliance of the artificial blood vessel and the real blood vessel at the anastomosis are prevented, thereby improving the long-term patency of the artificial blood vessel. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in describing the embodiments. Obviously, the drawings are used to provide a further understanding of the present invention and constitute part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention.

[0036] Figure 1 The actual image and schematic diagram of the double-layer small-caliber artificial blood vessel with in-situ modified anticoagulant coating in Examples 1-3 of the present invention are shown;

[0037] Figure 2 Schematic diagram of the device principle for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating by combining the coaxial dry-jet wet spinning technology with the polydopamine-heparin modification technology in Examples 1-3 of the present invention;

[0038] Figure 3 This is a scanning electron microscope image of a cross section of a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating in Example 1 of the present invention;

[0039] Figure 4 This is the axial tensile stress-strain curve of the double-layer small-caliber artificial blood vessel with in-situ modified anticoagulant coating in Example 1 of the present invention;

[0040] Figure 5 This is a comparison of the heparinization density of a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating and an artificial blood vessel directly soaked in heparin in Example 1 of the present invention;

[0041] Figure 6 This is a comparison chart of the hydrophilicity and hydrophobicity of the inner and outer layers of a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating and an artificial blood vessel without surface modification in Example 1 of the present invention;

[0042] Figure 7 This is a scanning electron microscopic comparison of whole blood cell adhesion in vitro on the inner surface of a double-layer small-caliber artificial blood vessel with an in situ modified anticoagulant coating and an artificial blood vessel without anticoagulant coating modification in Example 1 of the present invention;

[0043] Figure 8 This is the in vitro cytocompatibility evaluation of the double-layer small-caliber artificial blood vessel with in situ modified anticoagulant coating in Example 1 of the present invention, that is, the fluorescent staining images of human umbilical vein endothelial cells cultured on the inner surface on the first and fifth days. DETAILED DESCRIPTION

[0044] The following will be combined with the accompanying drawings in 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, rather than all the embodiments. Based on the embodiments of the present invention, ordinary technicians in this field should understand that these specific implementation methods and embodiments are used to illustrate the present invention, not to limit the present invention.

[0045] Unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0046] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0047] The apparatus for preparing the anticoagulant artificial blood vessel used in the following examples includes a spinning device, three injection needles, three injection pumps and a solidification tank. Figure 2 shown.

[0048] The specific operation method of the above device is:

[0049] (1) Assemble the coaxial spinneret, heat and dry it, and then suspend and fix it so that the outlet at the bottom of the spinneret remains horizontal;

[0050] (2) Injecting the first polyurethane solution A, the second polyurethane solution B, and the third solution C into three injection needles, respectively, installing the injection needles on three microinjection pumps, and connecting the outlets of the injection needles to the three inlets of the spinneret through hoses; adjusting the height of the spinneret outlet and the liquid level of the curing tank;

[0051] (3) According to preset parameters, three microinjection pumps are used to control the injection speed of the injection needle. The first polyurethane solution A, the second polyurethane solution B, and the third solution C are simultaneously injected into different channels of the spinneret. The solution is extruded from the spinneret outlet, passes through the air section, and enters the solidification tank. When the shape of the polymer film hollow tube is stable, a double-layer small-diameter polyurethane blood vessel sample is obtained from the solidification tank;

[0052] (4) The artificial blood vessel is solidified and formed according to the preset soaking time, and the artificial blood vessel is taken out for further solidification; after cleaning and drying, a small-caliber double-layer artificial blood vessel with an anticoagulant coating is obtained.

[0053] The high-strength polyurethane used in the following examples is thermoplastic polyurethane ARC-55D of aromatic polycarbonate, and the high-elasticity polyurethane is thermoplastic polyurethane ARC-80A of aromatic polycarbonate.

[0054] In the following examples, a polyurethane double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating was prepared by combining coaxial dry-jet wet spinning technology with polydopamine-heparin modification technology.

[0055] Example 1

[0056] The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating provided in this embodiment comprises the following steps:

[0057] (1) 20 g of ARC-55D thermoplastic polyurethane elastomer rubber polymer solute transparent raw material particles were dispersed in 78 mL of N-methylpyrrolidone, and stirred for 24 hours under 50° C. water bath heating to uniformly dissolve the polymer to obtain a first polyurethane solution A with a solute mass fraction of 20%. After standing for 12 hours to remove bubbles in the solution, the solution was dried and sealed for storage; 15 g of ARC-80A thermoplastic polyurethane elastomer rubber polymer solute transparent raw material particles were dispersed in 83 mL of N-methylpyrrolidone, and stirred for 24 hours under 50° C. water bath heating to uniformly dissolve the polymer to obtain a polyurethane solution with a solute mass fraction of 15%. 0.5 g of dopamine hydrochloride was added and stirred for 4 hours to completely dissolve the dopamine hydrochloride to obtain a second polyurethane solution B. After standing for 12 hours to remove bubbles in the solution, the solution was dried and sealed for storage; 0.5 g of heparin sodium was dissolved in Tris-HCl buffer solution with a pH of 9.0 to obtain a third solution C with a heparin sodium concentration of 10 g / L;

[0058] (2) Build the equipment, assemble the spinneret, heat and dry it, and then suspend and fix it so that the outlet at the bottom of the spinneret remains horizontal. Inject the first polyurethane solution A, the second polyurethane solution B, and the third solution C into three injection needles, respectively. Install the injection needles on three microinjection pumps, and connect the outlets of the injection needles to the three inlets of the spinneret through hoses. Adjust the height between the spinneret outlet and the liquid level of the curing tank to 3.0 mm.

[0059] (3) The injection speed of the injection needle was controlled by three micro-injection pumps, and the first polyurethane solution A, the second polyurethane solution B, and the third solution C were injected into the spinneret at the same time. The solution was extruded from the spinneret outlet and entered the solidification tank after passing through the air section. The flow rates of the micro-injection pumps were 2.0 mL / min, 2.8 mL / min, and 4.0 mL / min, respectively. During the preparation process, the temperature of the spinneret and the solidification tank was maintained at 75°C. The collection method was free fall, and the thin film hollow tube was immersed in the solidification tank for 24 h.

[0060] (4) After the shape of the polymer film hollow tube is stabilized, a double-layer small-diameter polyurethane blood vessel sample is obtained from the solidification tank, and the obtained artificial blood vessel is immersed in a 20% glycerol aqueous solution for 24 hours; the completely solidified double-layer small-diameter artificial blood vessel is taken out, rinsed with deionized water three times, and freeze-dried in a freeze dryer for 24 hours to obtain a finished double-layer small-diameter artificial blood vessel with an in-situ modified anticoagulant coating, such as Figure 1 shown.

[0061] Example 2

[0062] The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating provided in this embodiment comprises the following steps:

[0063] (1) 15 g of ARC-55D thermoplastic polyurethane elastomer rubber polymer solute transparent raw material particles were dispersed in 83 mL of N-methylpyrrolidone, and stirred for 24 hours under 50° C. water bath heating to uniformly dissolve the polymer to obtain a first polyurethane solution A with a solute mass fraction of 15%. After standing for 12 hours to remove bubbles in the solution, the solution was dried and sealed for storage; 15 g of ARC-80A thermoplastic polyurethane elastomer rubber polymer solute transparent raw material particles were dispersed in 83 mL of N-methylpyrrolidone, and stirred for 24 hours under 50° C. water bath heating to uniformly dissolve the polymer to obtain a polyurethane solution with a solute mass fraction of 15%. 0.05 g of dopamine hydrochloride was added and stirred for 4 hours to completely dissolve the dopamine hydrochloride to obtain a second polyurethane solution B. After standing for 12 hours to remove bubbles in the solution, the solution was dried and sealed for storage; 0.25 g of heparin sodium was dissolved in Tris-HCl buffer solution with a pH of 8.0 to obtain a third solution C with a heparin sodium concentration of 5 g / L;

[0064] (2) Build the equipment, assemble the spinneret, heat and dry it, and then suspend and fix it so that the outlet at the bottom of the spinneret remains horizontal. Inject the first polyurethane solution A, the second polyurethane solution B, and the third solution C into three injection needles, respectively. Install the injection needles on three microinjection pumps, and connect the outlets of the injection needles to the three inlets of the spinneret through hoses. Adjust the height between the spinneret outlet and the liquid level of the curing tank to 4.0 mm.

[0065] (3) The injection speed of the injection needle was controlled by three micro-injection pumps, and the first polyurethane solution A, the second polyurethane solution B, and the third solution C were injected into the spinneret at the same time. The solution was extruded from the spinneret outlet and entered the solidification tank after passing through the air section. The flow rates of the micro-injection pumps were 1.6 mL / min, 2.4 mL / min, and 4.0 mL / min, respectively. During the preparation process, the temperature of the spinneret and the solidification tank was maintained at 60°C. The collection method was free fall, and the thin film hollow tube was immersed in the solidification tank for 24 h.

[0066] (4) After the shape of the polymer film hollow tube stabilizes, a double-layer small-diameter polyurethane blood vessel sample is obtained from the solidification tank, and the obtained artificial blood vessel is immersed in a 20% glycerol aqueous solution for 24 hours; the completely solidified double-layer small-diameter artificial blood vessel is taken out, rinsed with deionized water three times, and freeze-dried in a freeze dryer for 24 hours to obtain a finished double-layer small-diameter artificial blood vessel with an in situ modified anticoagulant coating.

[0067] Example 3

[0068] The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating provided in this embodiment comprises the following steps:

[0069] (1) 20 g of ARC-55D thermoplastic polyurethane elastomer rubber polymer solute transparent raw material particles were dispersed in 78 mL of N-methylpyrrolidone, and stirred for 24 hours under 50° C. water bath heating to uniformly dissolve the polymer to obtain a first polyurethane solution A with a solute mass fraction of 20%. After standing for 12 hours to remove bubbles in the solution, the solution was dried and sealed for storage; 12 g of ARC-80A thermoplastic polyurethane elastomer rubber polymer solute transparent raw material particles were dispersed in 86 mL of N-methylpyrrolidone, and stirred for 24 hours under 50° C. water bath heating to uniformly dissolve the polymer to obtain a polyurethane solution with a solute mass fraction of 12%. 0.5 g of dopamine hydrochloride was added and stirred for 4 hours to completely dissolve the dopamine hydrochloride to obtain a second polyurethane solution B. After standing for 12 hours to remove bubbles in the solution, the solution was dried and sealed for storage; 1.5 g of heparin sodium was dissolved in Tris-HCl buffer solution with a pH of 10.0 to obtain a third solution C with a heparin sodium concentration of 30 g / L;

[0070] (2) Build the equipment, assemble the spinneret, heat and dry it, and then suspend and fix it so that the outlet at the bottom of the spinneret remains horizontal. Inject the first polyurethane solution A, the second polyurethane solution B, and the third solution C into three injection needles, respectively. Install the injection needles on three microinjection pumps, and connect the outlets of the injection needles to the three inlets of the spinneret through hoses. Adjust the height between the spinneret outlet and the liquid level of the curing tank to 2.0 mm.

[0071] (3) The injection speed of the injection needle was controlled by three micro-injection pumps, and the first polyurethane solution A, the second polyurethane solution B, and the third solution C were injected into the spinneret at the same time. The solution was extruded from the spinneret outlet and entered the solidification tank after passing through the air section. The flow rates of the micro-injection pumps were 2.0 mL / min, 2.4 mL / min, and 4.0 mL / min, respectively. During the preparation process, the temperature of the spinneret and the solidification tank was maintained at 75°C. The collection method was free fall, and the thin film hollow tube was immersed in the solidification tank for 24 h.

[0072] (4) After the shape of the polymer film hollow tube stabilizes, a double-layer small-diameter polyurethane blood vessel sample is obtained from the solidification tank, and the obtained artificial blood vessel is immersed in a 20% glycerol aqueous solution for 24 hours; the completely solidified double-layer small-diameter artificial blood vessel is taken out, rinsed with deionized water three times, and freeze-dried in a freeze dryer for 24 hours to obtain a finished double-layer small-diameter artificial blood vessel with an in situ modified anticoagulant coating.

[0073] The cross-sectional dimensions of the double-layer small-caliber artificial blood vessels prepared in Examples 1-3 were tested, and the results are shown in Table 1:

[0074] Table 1 shows the cross-sectional dimensions of the double-layer small-caliber artificial blood vessels obtained in Examples 1-3.

[0075] Group Outer diameter / (mm) Inner diameter / (mm) Wall thickness / (mm) Example 1 2.45±0.02 1.71±0.07 0.37±0.03 Example 2 2.18±0.11 1.70±0.13 0.24±0.01 Example 3 2.43±0.18 1.83±0.20 0.30±0.01

[0076] As shown in Table 1, the cross-sectional size of the double-layer artificial blood vessel of the embodiment of the application can be specifically customized by adjusting the corresponding parameters. The cross-sectional morphology of the double-layer small-diameter artificial blood vessel with in-situ modified anticoagulant coating of Test Example 1 is shown in FIG. 2. The double-layer structure of the small-diameter artificial blood vessel prepared by the application is obvious, and the cross-section has a typical porous structure. Figure 3

[0077] The axial tensile strength of the double-layer small-diameter artificial blood vessel with in-situ modified anticoagulant coating of Test Example 1 is shown in FIG. 3. The tensile strength of the double-layer (55D-80A) artificial blood vessel is between the strength of the artificial blood vessel with only the outer layer (55D) of 3.52±0.22 MPa and the strength of the artificial blood vessel with only the inner layer (80A) of 1.61±0.14 MPa, reaching 2.99±0.24 MPa, which shows excellent mechanical properties. Figure 4

[0078] The heparinization density of the double-layer small-diameter artificial blood vessel with in-situ modified anticoagulant coating of Test Example 1 is shown in FIG. 4. In the in-situ modified anticoagulant coating prepared by the application, the heparin density is 0.83±0.09 μg / mm 2 , which is much higher than the heparin density of 0.42±0.03 μg / mm 2 modified by the immersion method. This result shows that, compared with the traditional modification method, the in-situ modified anticoagulant coating method has a higher heparin grafting amount and is more conducive to long-term anticoagulation. Figure 5 The hydrophilicity and hydrophobicity of the inner and outer surfaces of the double-layer small-diameter artificial blood vessel with in-situ modified anticoagulant coating of Test Example 1 are shown in FIG. 5. The contact angles of the inner and outer surfaces of the double-layer small-diameter artificial blood vessel with in-situ modified anticoagulant coating are 22.43±4.24° and 25.74±3.05°, respectively. Compared with the artificial blood vessel without surface modification, the double-layer small-diameter artificial blood vessel with in-situ modified anticoagulant coating has higher hydrophilicity of the inner and outer surfaces, which proves the effectiveness of the in-situ modified anticoagulant coating modification from another angle.

[0079] Figure 6 The whole blood cell adhesion of the inner surface of the double-layer small-diameter artificial blood vessel with in-situ modified anticoagulant coating of Test Example 1 is shown in FIG. 6. From

[0080] Figure 7 Figure 7 ​​​​​It can be seen that the platelets on the inner surface of the artificial blood vessels without surface modification are activated, and a large number of blood cells aggregate to form microthrombi; while the inner surface of the double-layer small-caliber artificial blood vessels with in situ modified anticoagulant coating has only a small amount of unactivated platelets and no obvious blood cell aggregation; this result shows that the double-layer small-caliber artificial blood vessels with in situ modified anticoagulant coating have excellent anticoagulant function, which provides a favorable guarantee for the long-term patency of the artificial blood vessels.

[0081] The in vitro endothelial cell culture on the inner surface of the double-layer small-caliber artificial blood vessel with the in situ modified anticoagulant coating of Test Example 1 was as follows: Figure 8 As shown. Figure 8 It can be seen that the inner layer of the artificial blood vessel exhibits good biocompatibility; on the first day of culture on the inner surface of the artificial blood vessel, the endothelial cells are mostly spherical or ellipsoidal in shape. On the fifth day of culture, the endothelial cells have obvious adhesion and spreading on the inner surface of the artificial blood vessel, showing an elongated spindle-shaped morphology; this shows that the inner layer of the blood vessel of the present invention has good biocompatibility, supports the growth and proliferation of endothelial cells, and can promote the formation of the endothelial layer.

[0082] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope proposed by the present invention, can make equivalent replacements or changes based on the technical solution and inventive concept of the present invention, which should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating, characterized in that: The preparation method comprises the following steps: Step 1: preparing an outer layer spinning solution, an inner layer spinning solution and a spinning core solution containing sodium heparin; Step 2: The outer layer spinning solution, the inner layer spinning solution, and the spinning core solution containing sodium heparin are respectively injected into the three injection needles of the spinning equipment, followed by spinning using a dry-jet wet spinning method. When the shape of the polymer film hollow tube in the solidification tank stabilizes, the double-layer small-diameter polyurethane blood vessel sample is removed from the solidification tank; Step 3: Soaking the obtained double-layer small-caliber polyurethane blood vessel sample in a glycerol aqueous solution, then rinsing it with deionized water, and drying it to obtain a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating; In step 1, the outer layer spinning solution is a first polyurethane solution A obtained by dissolving high-strength polyurethane particles in N-methylpyrrolidone; the inner layer spinning solution is a second polyurethane solution B obtained by dissolving high-elasticity polyurethane particles and dopamine hydrochloride powder in N-methylpyrrolidone; In the step 1, the spinning core solution containing sodium heparin is a third solution C obtained by dissolving sodium heparin in an alkaline Tris-HCl buffer.

2. The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating according to claim 1, characterized in that: In the first polyurethane solution A, the mass fraction of polyurethane is 0.15-0.2 g / mL.

3. The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating according to claim 1, characterized in that: In the second polyurethane solution B, the mass fraction of polyurethane is 0.12-0.15 g / mL, and the concentration of dopamine hydrochloride is 0.5-5.0 g / L.

4. The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating according to claim 1, characterized in that: The pH value of the Tris-HCl buffer is 8.0-10.0, and the concentration of heparin sodium in the third solution C is 5.0-30.0 g / L.

5. The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating according to claim 1, characterized in that: In the step 2, the extrusion rate of the outer layer spinning solution is 1.6-2.4 mL / min; the extrusion rate of the inner layer spinning solution is 2.0-2.8 mL / min; and the extrusion rate of the spinning core solution containing sodium heparin is in the range of 3.2-4.8 mL / min.

6. The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating according to claim 1, characterized in that: In the step 2, during the spinning process using the dry-jet wet spinning method, the temperature of the spinneret and the curing tank is maintained at 60-75°C.

7. A double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating, characterized in that: The double-layer small-caliber artificial blood vessel is prepared by the preparation method according to claim 1.

8. A product, characterized in that The product comprises a double-layer small-caliber artificial blood vessel with an in situ modified anticoagulant coating according to claim 7.

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