Double-layer small-caliber artificial blood vessel with in-situ modified anticoagulant coating and preparation method

Abstract

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

Description

Technical Field

[0001] The present invention relates to a double-layer small-caliber artificial blood vessel and a preparation method in the technical field of artificial blood vessel preparation, and in particular to a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating, a preparation method and a product. 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 vascular products. Autologous blood vessels can no longer meet the current growing clinical needs due to limited donor quality and availability, and the use of invasive collection techniques may cause lesions in the donor site. Therefore, the development of small-caliber artificial blood vessels has become an urgent problem to be solved.

[0003] Coaxial dry-jet wet spinning technology based on phase separation is an efficient, convenient and easy-to-regulate solution spinning technology, which is widely used in the manufacture of thin film hollow tubes. The hollow tubular structure prepared by it has a large specific surface area, good selective permeability, and a typical porous structure. It is widely used in the fields of biology and regenerative medicine. Through material selection, parameter regulation and other means, double-layer small-caliber artificial blood vessels with the same structure and mechanical properties as natural blood vessels can be prepared.

[0004] In order to ensure long-term patency, small-caliber artificial blood vessels not only need to have sufficient mechanical strength and good biocompatibility, but also need to have excellent anticoagulant function. The anticoagulant modification of the inner wall of small-caliber artificial blood vessels is particularly difficult. Traditional chemical modification requires the entire blood vessel to be immersed in the reaction solution for functionalization. The processing steps are cumbersome, and the location of the modification and the amount of grafting are difficult to control. It only plays a short-term anticoagulant role on the surface of the substrate and cannot penetrate deeply into the artificial blood vessel in three dimensions. Blood components can easily penetrate into the inner wall of the blood vessel and induce a coagulation cascade reaction, causing fibrin and platelet adhesion, and then forming a thrombus.

[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 view of the deficiencies of the above prior art, the present invention combines the coaxial dry-jet wet spinning technology with the 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 sufficient mechanical strength and structural stability in the outer layer, and the inner layer has good hydrophilicity and excellent anticoagulant function through the in-situ grafting of heparin with polydopamine, which reduces the activation of platelets and the adhesion of blood cells, prevents the formation of short-term acute thrombus, and promotes the endothelialization process of small-caliber blood vessels; the present invention realizes the anticoagulant modification of the inner wall of the blood vessel through the combination of polydopamine-heparin during the forming process, and obtains a double-layer small-caliber artificial blood vessel with an in-situ modified heparin anticoagulant coating on the inner wall at one time, avoiding additional procedures such as secondary processing, simplifying the manufacturing and surface modification process of artificial blood vessels, and the modification position and grafting amount are controllable, and the experimental stability is high, which provides 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 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 modified position and grafting amount of the polydopamine-sodium heparin anticoagulant coating are controllable. The preparation method provided has strong operability, 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 spinning solution, the inner spinning solution and the spinning core solution containing sodium heparin, let them stand to remove bubbles, and dry and seal them for storage. The standing time to remove bubbles is 24 hours;

[0011] Step 2: The spinning equipment includes a coaxial spinning device, a plurality of injection needles, a plurality of injection pumps and a solidification tank. 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, and then the spinning is carried out by dry-jet wet spinning. When the shape of the polymer film hollow tube in the solidification tank is stable, the double-layer small-caliber polyurethane blood vessel sample is taken out from the solidification 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 after drying, 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 elastic 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] In the first polyurethane solution A, the mass fraction of high-strength polyurethane is 0.15-0.2 g / mL. In the second polyurethane solution B, the mass fraction of high-elasticity polyurethane 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-elastic polyurethane; and the Young's modulus of the high-elastic 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 device 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 curing tank for 24 hours.

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

[0029] The double-layer small-caliber artificial blood vessel with in-situ modified anticoagulant coating provided by the present invention has beautiful appearance, uniform and stable structural shape, and mechanical strength that meets the requirements of 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, excellent anticoagulant function, and significantly reduces 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 methods provided by the present invention all adopt mature technologies, do not require high operator experience, have high experimental stability, good repeatability, and are 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, realizes the anticoagulant modification of the inner wall of the blood vessel through polydopamine-heparin combination during the forming process, and avoids 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. Thus, on the basis of ensuring the strength of the artificial blood vessels, the compliance of the artificial blood vessels is improved, and thrombosis and intimal hyperplasia caused by the mismatch between the compliance of the artificial blood vessels and the real blood vessels at the anastomosis are prevented, thereby improving the long-term patency of the artificial blood vessels. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention and do not constitute a limitation of the present invention.

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

[0037] Figure 2 This is a schematic diagram of the principle of a device 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 The axial tensile stress-strain curve of the double-layer small-caliber artificial blood vessel with in-situ modified anticoagulation coating in Example 1 of the present invention;

[0040] Figure 5 This is a comparison diagram of the heparinization density between the double-layer small-caliber artificial blood vessel with in-situ modified anticoagulant coating and the 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 1st and 5th 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 described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, ordinary technicians in this field should understand that these specific implementation methods and embodiments are used to illustrate the present invention, rather than to limit the present invention.

[0045] Unless otherwise specified, the terms used herein should be understood as the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes 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 by existing methods.

[0047] The device 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) assembling a coaxial spinneret, heating and drying the spinneret, and then suspending and fixing the spinneret 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 micro-injection pumps respectively, and connecting the outlets of the injection needles to the three inlets of the spinneret respectively 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 respectively, and the first polyurethane solution A, the second polyurethane solution B and the third solution C are injected into different channels of the spinneret at the same time, extruded from the spinneret outlet, and enter the solidification tank after passing through the air section. 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 immersion 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-elastic 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 the coaxial dry-jet wet spinning technology with the 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, stirred for 24 hours under 50° C. water bath heating to uniformly dissolve the polymer, and 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, stirred for 24 hours under 50° C. water bath heating to uniformly dissolve the polymer, and obtain a polyurethane solution with a solute mass fraction of 15%. 0.5 g of dopamine hydrochloride was added, and the solution was stirred for 4 hours to completely dissolve the dopamine hydrochloride, and 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 at pH=9.0 to obtain a third solution C with a heparin sodium concentration of 10 g / L;

[0058] (2) Building equipment, assembling the spinneret, and suspending and fixing it after heating and drying so that the outlet at the bottom of the spinneret remains horizontal, respectively 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 micro-injection pumps, and connecting the outlets of the injection needles to the three inlets of the spinneret through hoses, respectively, and adjusting the height between the outlet of the spinneret and the liquid level of the curing tank to 3.0 mm;

[0059] (3) The injection speed of the injection needle is controlled by three micro-injection pumps, and the first polyurethane solution A, the second polyurethane solution B and the third solution C are injected into the spinneret at the same time, extruded from the spinneret outlet, and enter the curing tank after passing through the air section. The flow rates of the micro-injection pumps are 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 curing tank is maintained at 75°C; the collection method is free fall, and the immersion time of the thin film hollow tube in the curing tank is 24 h;

[0060] (4) After the appearance of the polymer film hollow tube is stable, 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% by volume glycerol aqueous solution for 24 hours; the completely solidified double-layer small-diameter artificial blood vessel is taken out, rinsed with deionized water for 3 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 at pH=8.0 to obtain a third solution C with a heparin sodium concentration of 5 g / L;

[0064] (2) Building equipment, assembling the spinneret, and suspending and fixing it after heating and drying so that the outlet at the bottom of the spinneret remains horizontal, respectively 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 micro-injection pumps, and connecting the outlets of the injection needles to the three inlets of the spinneret through hoses, respectively, and adjusting 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 is controlled by three micro-injection pumps, and the first polyurethane solution A, the second polyurethane solution B and the third solution C are injected into the spinneret at the same time, extruded from the spinneret outlet, and enter the curing tank after passing through the air section. The flow rates of the micro-injection pumps are 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 curing tank is maintained at 60°C; the collection method is free fall, and the immersion time of the thin film hollow tube in the curing tank is 24 h;

[0066] (4) After the appearance of the polymer film hollow tube is stabilized, a double-layer small-diameter polyurethane blood vessel sample is obtained from the curing pool, and the obtained artificial blood vessel is immersed in a 20% by volume glycerol aqueous solution for 24 hours; the completely cured double-layer small-diameter artificial blood vessel is taken out, rinsed with deionized water for 3 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 at pH=10.0 to obtain a third solution C with a heparin sodium concentration of 30 g / L;

[0070] (2) Building equipment, assembling the spinneret, and suspending and fixing it after heating and drying so that the outlet at the bottom of the spinneret remains horizontal, respectively 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 micro-injection pumps, and connecting the outlets of the injection needles to the three inlets of the spinneret through hoses, respectively, and adjusting 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 is controlled by three micro-injection pumps, and the first polyurethane solution A, the second polyurethane solution B and the third solution C are injected into the spinneret at the same time, extruded from the spinneret outlet, and enter the curing tank after passing through the air section. The flow rates of the micro-injection pumps are 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 curing tank is maintained at 75°C; the collection method is free fall, and the immersion time of the thin film hollow tube in the curing tank is 24 h;

[0072] (4) After the appearance of the polymer film hollow tube is stabilized, a double-layer small-diameter polyurethane blood vessel sample is obtained from the curing pool, and the obtained artificial blood vessel is immersed in a 20% by volume glycerol aqueous solution for 24 hours; the completely cured double-layer small-diameter artificial blood vessel is taken out, rinsed with deionized water for 3 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 dimensions of the double-layer artificial blood vessel of the embodiment of the present invention can be specifically customized by adjusting the corresponding parameters. The cross-sectional morphology of the double-layer small-caliber artificial blood vessel with in-situ modified anticoagulant coating of Test Example 1 is as follows: Figure 3 The small-caliber artificial blood vessel prepared by the present invention has an obvious double-layer structure, and the cross section has a typical porous structure.

[0077] The axial tensile strength of the double-layer small-caliber artificial blood vessel with the in-situ modified anticoagulation coating of Example 1 was tested. Figure 4 As shown: the tensile strength of the double-layer (55D-80A) artificial blood vessel is between the strength of the artificial blood vessel with a single outer layer (55D) of 3.52±0.22MPa and the strength of the artificial blood vessel with a single inner layer (80A) of 1.61±0.14MPa, reaching 2.99±0.24MPa, showing excellent mechanical properties.

[0078] The heparinization density of the double-layer small-caliber artificial blood vessel with the in-situ modified anticoagulant coating of Example 1 was tested. Figure 5 In the in-situ modified anticoagulant coating prepared by the present invention, the heparin density is 0.83±0.09μg / mm 2 , which is much higher than the heparin density modified by the immersion method (0.42±0.03μg / mm 2 This result shows that compared with the traditional modification method, the in situ modification anticoagulant coating method has a higher heparin grafting amount and is more conducive to long-term anticoagulation.

[0079] The hydrophilicity and hydrophobicity of the inner and outer surfaces of the double-layer small-caliber artificial blood vessel with the in-situ modified anticoagulation coating of Example 1 were tested. Figure 6 As shown, the contact angles of the inner and outer surfaces of the double-layer small-caliber artificial blood vessels with in situ modified anticoagulant coating were 22.43±4.24° and 25.74±3.05°, respectively; compared with the artificial blood vessels without surface modification, the inner and outer surfaces of the double-layer small-caliber artificial blood vessels with in situ modified anticoagulant coating were more hydrophilic, which proves the effectiveness of in situ modified anticoagulant coating from another perspective.

[0080] The whole blood cell adhesion on the inner surface of the double-layer small-caliber artificial blood vessel with the in situ modified anticoagulant coating of Example 1 was tested. Figure 7 As shown. Figure 7It 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 Example 1 was tested. Figure 8 As shown. Figure 8 It can be seen that the inner layer of the artificial blood vessel exhibits good biocompatibility; when the endothelial cells are cultured on the inner surface of the artificial blood vessel on the first day, the morphology is mostly spherical or ellipsoidal. When cultured to the fifth day, the endothelial cells have obvious adhesion and spreading phenomenon on the inner surface of the artificial blood vessel, showing an elongated spindle shape; 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 implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope proposed by the present invention, which should be covered by the protection scope 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: Inject the outer spinning solution, the inner spinning solution and the spinning core solution containing sodium heparin into three injection needles of the spinning device respectively, and then use the dry-jet wet spinning method to spin. When the shape of the polymer film hollow tube in the solidification tank is stable, take out the double-layer small-diameter polyurethane blood vessel sample from the solidification tank; Step 3: Soak the obtained double-layer small-caliber polyurethane blood vessel sample in a glycerol aqueous solution, then rinse with deionized water, and after drying, obtain a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating.

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 step 1, the outer spinning solution is a first polyurethane solution A obtained by dissolving high-strength polyurethane particles in N-methylpyrrolidone; the inner spinning solution is a second polyurethane solution B obtained by dissolving high-elastic polyurethane particles and dopamine hydrochloride powder in N-methylpyrrolidone.

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 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.

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

5. The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating according to claim 2, 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.

6. The method for preparing a double-layer small-caliber artificial blood vessel with an in-situ modified anticoagulant coating according to claim 3, 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.

7. 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.

8. 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.

9. 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.

10. A product, characterized in that The product comprises a double-layer small-caliber artificial blood vessel with an in situ modified anticoagulation coating according to claim 9.

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