Artificial blood vessel stent with anticoagulation performance and construction method of artificial blood vessel stent

Abstract

The construction method comprises the following steps: performing decellularization treatment on a pig heart aorta blood vessel, compounding the treated pig heart aorta blood vessel with polyvinyl alcohol and methacryloylated gelatin to prepare a pre-gel solution, printing a designed model into a hollow tubular mold by using white resin through 3D printing, and preparing the hollow tubular mold into the artificial blood vessel stent with the anticoagulation performance. Injecting the pre-gel solution into a mold for cross-linking to obtain an artificial blood vessel stent with controllable shape and size, and finally loading an anticoagulant drug heparin on the prepared stent to obtain the heparin-loaded artificial blood vessel stent. Materials for preparing the pre-gel solution are selected, and advantage complementation of material functions can be achieved. The acellular matrix is beneficial to adhesion, proliferation and differentiation of cells; the composite polyvinyl alcohol and methacrylated gelatin improve the mechanical properties of the scaffold and improve the cell adhesion. Heparin is loaded on the artificial blood vessel stent to improve the anticoagulation performance of the stent and prevent thrombus. As an excellent biomedical material, the stent has a wide application prospect in the related fields of cardiovascular tissue engineering and biology.

Description

Technical Field

[0001] The invention belongs to the fields of tissue engineering, material science and biology, and provides a tissue engineering artificial blood vessel stent with anticoagulant properties, which is composited with three materials: porcine aortic decellularized matrix, polyvinyl alcohol and methacryloyl gelatin, and a construction method thereof. Background Art

[0002] Cardiovascular diseases have extremely high morbidity and mortality rates. Vascular bypass grafting therapy has attracted widespread attention by providing alternative conduits for narrowed, blocked or traumatized blood vessels. Due to the limited availability of autologous blood vessels and certain requirements on the patient's physical condition, the demand for artificial blood vessels continues to increase. At present, large and medium diameter artificial blood vessels have been successfully used in clinical practice. However, the use of small diameter (<6mm) artificial blood vessels is subject to many limitations, such as donor site-related immunogenicity, thrombosis, intimal hyperplasia and insufficient mechanical properties. Tissue engineering combines cells, biomaterial scaffolds and bioactive molecules to repair the function and morphology of damaged tissues or organs in the human body, providing new possibilities for the clinical application of small diameter artificial blood vessels.

[0003] At present, the main methods for constructing small-diameter artificial blood vessels are electrospinning, 3D printing, and microfluidics. Among them, electrospinning and microfluidics are complex to operate, and it is still a challenge to manufacture vascular stents with controllable and complex macroscopic configurations; at the same time, 3D printing often exhibits poor resolution and limited printability.

[0004] In view of the problems that small-diameter artificial blood vessels are prone to thrombosis and poor mechanical properties, the present invention is based on the decellularized extracellular matrix (dECM) of natural porcine heart aorta, combined with methacrylated gelatin (GelMA) and polyvinyl alcohol (PVA) to prepare a pre-gel solution, wherein dECM effectively retains the extracellular matrix components, structure and biological activity of the tissue, and the scaffold constructed by it can more realistically simulate the cell microenvironment in the body, which is conducive to cell adhesion, proliferation and differentiation. However, the mechanical properties of dECM are poor, and GelMA retains the biocompatibility, degradability and cell adhesion of gelatin, and has a certain mechanical strength. PVA, as a non-toxic water-soluble polymer, has good biocompatibility, biodegradability and mechanical properties, and can optimize the mechanical properties of artificial blood vessels.

[0005] In view of the problems existing in the method of constructing small-diameter artificial blood vessels, the present invention uses 3D printing to print the designed model into a hollow tubular mold with white resin, and injects a pre-gel solution into the mold for cross-linking to obtain a tissue engineering artificial blood vessel with controllable shape and size. Summary of the invention

[0006] The present invention aims to establish a method for constructing an artificial vascular stent with anti-coagulant properties and its application in tissue engineering. The small-diameter tubular stent prepared by the method has good biocompatibility, sufficient biomechanical properties to withstand the pressure of blood flow, good anti-thrombotic properties to reduce the risk of thrombosis, and a certain biodegradability so that it can be gradually metabolized and absorbed by body tissues, thereby promoting the regeneration of new blood vessel tissue.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for constructing an artificial vascular stent with anticoagulant properties. The method firstly prepares a pre-gel solution based on natural porcine heart aorta vascular decellularized matrix (dECM), combined with methacryloyl gelatin (GelMA) and polyvinyl alcohol (PVA); secondly, prints the designed model into a hollow tubular mold by 3D printing; finally, injects the pre-gel solution into the hollow tubular mold, cross-links it, and loads heparin (Hep) to obtain a tissue engineering artificial vascular stent with anticoagulant function. Specifically, the method comprises the following steps:

[0009] Step (1) decellularizing the aorta on the surface of the pig heart to obtain a decellularized matrix of the aorta dECM;

[0010] Step (2) preparing a pre-gel solution by using porcine aorta decellularized matrix dECM, methacrylated gelatin GelMA and polyvinyl alcohol PVA for later use;

[0011] Step (3) is to 3D print a hollow tubular mold, pour the pre-gel in step (2) into the hollow tubular mold, completely freeze it at low temperature, and then thaw it naturally at room temperature, and repeat the freeze-thaw process to cross-link the PVA itself to obtain the final hydrogel tube; the hollow tubular mold includes three types: a Y-shaped bifurcated hollow tubular mold, a uniform diameter hollow tubular mold, and a variable diameter hollow tubular mold.

[0012] Step (4) separating the hydrogel tube obtained by the last freeze-thaw in step (3) from the hollow tubular mold, and placing it under a UV lamp for uniform irradiation to crosslink the GelMA with the photoinitiator IHT-PI 659;

[0013] Step (5) placing the cross-linked hydrogel tube obtained in step (4) in a phosphate buffered saline solution (PBS), wiping off excess water on the surface after swelling equilibrium, and freeze-drying to obtain a dEPG artificial vascular stent;

[0014] Step (6) soaking the dEPG artificial vascular stent obtained in step (5) in a heparin Hep solution, and taking it out after reaching swelling equilibrium to obtain a Hep-dEPG artificial vascular stent loaded with Hep.

[0015] Preferably, the pig heart aorta in step (1) is cut into small square pieces, soaked and cleaned, and then placed in a solution of trypsin and ethylenediaminetetraacetic acid (EDTA) for 4 to 8 hours, and then bathed in a DNA enzyme solution for 4 to 10 hours, and then placed in a sodium dodecyl sulfate (SDS) solution and stirred for 24 to 72 hours, and the SDS solution remaining on the surface is washed with a PBS solution, and then placed in a polyethylene glycol octylphenyl ether (Triton X-100) solution and stirred for 24 to 72 hours, and freeze-dried to obtain pig heart aorta dECM. The dECM obtained after decellularization can effectively retain the protein and physicochemical properties of natural tissues, such as cell adhesion sites and corresponding biological signals, so that the scaffold constructed by it can more realistically simulate the cell growth microenvironment in vivo.

[0016] Preferably, the GelMA in the pre-gel solution in step (2) retains the cell adhesion and biocompatibility of Gel, and can provide biological functions and signal molecules for the artificial blood vessels, and PVA serves as a supporting material to improve the mechanical properties of the artificial blood vessels.

[0017] Preferably, the dECM in the pre-gel solution in step (2) contains GAGs, which has a structure similar to Hep and has a certain anticoagulant effect. Furthermore, the combined effect of dECM and PVA can prolong the coagulation time of the prepared artificial vascular stent.

[0018] Preferably, in the pre-gel solution of step (2), the concentration of dECM is 0.5-1.5 w / v%, the concentration of PVA is 4-6 w / v%, and the concentration of GelMA is 5-9 w / v%. Experimental results show that the three groups of hydrogel tubes, 0.5% dECM / 5% PVA / 7% GelMA (abbreviated as 0.5d5P7G), 1% dECM / 6% PVA / 5% GelMA (abbreviated as 1d6P5G), and 1.5% dECM / 6% PVA / 7% GelMA (abbreviated as 1.5d6P7G), have good mechanical properties, degradability, anticoagulant properties and biocompatibility, among which 1.5d6P7G has the best anticoagulant effect.

[0019] Preferably, three types of hollow tubular molds, namely, Y-shaped bifurcated, equal diameter, and variable diameter, are drawn using modeling software Rhino 7 and 3d Max software, and an STL format file is generated. The file is imported into a 3D printer, and a hollow tubular mold is prepared by 3D printing using non-toxic white resin as a material. At the same time, the printing parameters can be changed according to the needs to prepare a hollow tubular mold with controllable shape and size.

[0020] Preferably, the pre-gel solution in step (2) is injected into the hollow tubular mold in step (3) through a sterile syringe, and the pre-gel solution is placed at -20°C to -40°C for 2 to 6 hours before being taken out and placed at room temperature to thaw naturally, and the PVA is cross-linked by repeated freezing and thawing. PVA can cross-link itself by generating hydrogen bonds through repeated freezing and thawing without introducing other toxic cross-linking agents, and the pore size and mechanical properties of the PVA hydrogel can be adjusted by changing the number of freeze-thaw cycles.

[0021] Preferably, in step (4), the hydrogel tube is separated from the mold and placed under a UV lamp for uniform irradiation for 5 to 20 minutes to crosslink the GelMA, and the prepared artificial blood vessel has good mechanical properties through double crosslinking of the UV crosslinking of the GelMA and the hydrogen bond crosslinking of the PVA. The uniform irradiation time under the UV lamp is preferably 10 minutes.

[0022] Preferably, in step (6), heparin Hep is directly loaded on the artificial vascular stent. Hep has good biocompatibility. Combining Hep with the artificial vascular stent material can improve its anticoagulant properties and prevent thrombus formation.

[0023] An artificial blood vessel stent with anti-coagulation performance is prepared by adopting the above construction method.

[0024] The tissue engineering artificial blood vessel stent with anticoagulant properties prepared by the above method can be applied to cardiovascular diseases such as hemorrhagic heart disease, occlusive arterial disease, myocardial infarction and stroke.

[0025] Compared with the prior art, the present invention has the following beneficial effects and advantages:

[0026] (1) The present invention uses pig heart aorta dECM, composite PVA, and GelMA to prepare an artificial vascular stent with anticoagulant function, realizing the complementary advantages of material functions. Pig hearts are widely available and low-cost. The dECM obtained after decellularization of pig heart aorta effectively retains the protein and physicochemical properties of the aorta, such as cell adhesion sites and corresponding biological signals. The stent constructed with it can more realistically simulate the growth microenvironment of vascular endothelial cells and fibroblasts.

[0027] (2) The PVA in the pregel solution of the present invention generates hydrogen bond crosslinks through repeated freezing and thawing, and the pore size and mechanical properties of the PVA gel can be adjusted by changing the number of freeze-thaw cycles; the GelMA in the pregel solution is crosslinked by ultraviolet light to give it more excellent mechanical properties, and at the same time, GelMA retains the cell adhesion and biocompatibility of Gel. Therefore, the PVA and GelMA in the pregel can make the artificial vascular scaffold meet the requirements of cell growth and sufficient mechanical properties, while being close to the various aspects of the performance of natural blood vessels.

[0028] (3) The present invention uses 3D modeling software to adjust parameters and design a variety of different combination molds, thereby achieving various shapes and size requirements of tissue engineering artificial blood vessels. These mold designs can be customized according to clinical needs, so that they can better simulate the real small-diameter blood vessel structure, which is of great significance to the vascular model of medical research and clinical application.

[0029] (4) The combined effect of dECM and PVA selected in the present invention can prolong the coagulation time, so that the artificial blood vessel has a certain anticoagulant effect. Good anticoagulant properties can effectively prevent thrombosis and reduce the risk of complications caused by thrombosis in patients. Therefore, loading Hep on the artificial blood vessel stent can further improve the anticoagulant performance of the blood vessel stent, effectively prevent thrombosis, and prolong the time that the artificial blood vessel stent is unobstructed in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The macroscopic morphology of the dEPG artificial vascular stent of Example 1 is shown;

[0031] Figure 2 The design diagram of the equal-diameter dEPG artificial blood vessel stent of Example 1 is shown; Figure 2 (a) is the design drawing of equal diameter mold in Rhino 7 software; Figure 2 (b) Schematic diagram of the hydrogel tube dimensions; Figure 2 (c) Schematic diagram of the cross-section of the hydrogel tube size;

[0032] Figure 3 A scanning electron microscope image of a cross section of the dEPG artificial blood vessel stent of Example 1 is shown; Figure 3 (a) is the microscopic morphology of the cross section of the dEPG artificial vascular stent; Figure 3 (b) Figure 3 (a) The enlarged image in the dashed box;

[0033] Figure 4 The flexibility test diagram of the dEPG artificial blood vessel stent of Example 1 is shown; Figure (a) shows the dEPG artificial blood vessel stent being knotted; Figure (b) shows the dEPG artificial blood vessel stent being twisted; Figure (c) shows the dEPG artificial blood vessel stent being suspended with a heavy object, the weight of the suspended object being 40.60 g; Figure (d) shows the natural state of the dEPG artificial blood vessel stent, in which Ⅰ is the inner diameter of the stent and Ⅱ is the outer diameter of the stent;

[0034] Figure 5 The schematic diagram of the tensile performance structure of the dEPG artificial blood vessel stent in Example 1 is shown; Figure 5 (a) The natural state of the dEPG artificial vascular stent; Figure 5 (b) The stretching process of dEPG artificial vascular stent; Figure 5 (c) dEPG artificial vascular stent after stretching and fracture;

[0035] Figure 6 The tensile properties of the dEPG artificial blood vessel stent of Example 1 are shown;

[0036] Figure 7 A schematic diagram of the compression performance structure of the dEPG artificial blood vessel stent of Example 1 is shown; Figure 7 (a) The natural state of the dEPG artificial vascular stent; Figure 7 (b) The compression process of dEPG artificial vascular stent; Figure 7 (c) The dEPG artificial vascular stent returns to its natural state after being compressed by 80%;

[0037] Figure 8 The compression performance of the dEPG artificial blood vessel stent of Example 1 is shown;

[0038] Fig. 9 The degradation performance of the dEPG artificial blood vessel stent of Example 1 is shown;

[0039] Fig.10The fluorescence images of the live and dead staining of L929 cells on the dEPG artificial vascular scaffold of Example 1 are shown; Group a represents the fluorescence images of Calcein AM, Hochest 33342 and PI mixed staining of L929 cells cultured on the artificial vascular scaffold for 4 days, specifically: a1-a6 represent the fluorescence images of 0.5d5P7G artificial vascular scaffold and L929 cells cultured for 4 days, b1-b6 represent the fluorescence images of 1d6P5G artificial vascular scaffold and L929 cells cultured for 4 days, c1-c6 represent the fluorescence images of 1.5d6P7G artificial vascular scaffold and L929 cells cultured for 4 days, a1-c1 represent the Brightfield images of artificial vascular scaffold and L929 cells cultured for 4 days, a2-c2 represent the Hochest fluorescence images of artificial vascular scaffold and L929 cells cultured for 4 days, a3-c3 represent the Calcein AM images of artificial vascular scaffold and L929 cells cultured for 4 days. AM fluorescence images, a4-c4 represent PI fluorescence images of artificial vascular scaffolds and L929 cells cultured for 4 days, a5-c5 represent Merge fluorescence images of artificial vascular scaffolds and L929 cells cultured for 4 days, a6-c6 represent 3D fluorescence images of artificial vascular scaffolds and L929 cells cultured for 4 days; Group b represents Calcein AM, Hochest Fluorescence images of mixed staining of 33342 and PI, specifically: a1-a6 represent fluorescence images of 0.5d5P7G artificial vascular scaffold and L929 cells cultured for 7 days, b1-b6 represent fluorescence images of 1d6P5G artificial vascular scaffold and L929 cells cultured for 7 days, c1-c6 represent fluorescence images of 1.5d6P7G artificial vascular scaffold and L929 cells cultured for 7 days, a1-c1 represent Brightfield images of artificial vascular scaffold and L929 cells cultured for 7 days, a2-c2 represent Hochest fluorescence images of artificial vascular scaffold and L929 cells cultured for 7 days, a3-c3 represent Calcein AM fluorescence images of artificial vascular scaffold and L929 cells cultured for 7 days, a4-c4 represent PI fluorescence images of artificial vascular scaffold and L929 cells cultured for 7 days, a5-c5 represent Merge fluorescence images of artificial vascular scaffold and L929 cells cultured for 7 days, and a6-c6 represent 3D fluorescence images of artificial vascular scaffold and L929 cells cultured for 7 days;

[0040] Fig.11The scanning electron microscope images of the dEPG artificial vascular scaffold and L929 cells of Example 1 are shown; Group a represents the microscopic morphology of L929 cells cultured on the artificial vascular scaffold for 4 days, specifically: a1-a3 represent the structural morphology of the artificial vascular scaffold and L929 cells cultured for 4 days at a magnification of 600 times, a4-a6 correspond to the enlarged images in the dotted boxes of a1-a3, respectively, representing the structural morphology of the artificial vascular scaffold and L929 cells cultured for 4 days at a magnification of 1000 times, a1 and a4 represent 0. The structural morphology of 5d5P7G artificial vascular scaffold and L929 cells cultured for 4 days, a2 and a5 represent the structural morphology of 1d6P5G artificial vascular scaffold and L929 cells cultured for 4 days, a3 and a6 represent the structural morphology of 1.5d6P7G artificial vascular scaffold and L929 cells cultured for 4 days; Group b represents the microscopic morphology of L929 cells cultured on the artificial vascular scaffold for 7 days, and the specific meanings of b1-b6 are the same as a1-a6 in Group a, except that the culture time is changed to 7 days;

[0041] Fig.12 The anticoagulant performance (APTT) of the dEPG artificial vascular stent of Example 1 is shown;

[0042] Fig.13 The anticoagulant performance (PT) of the dEPG artificial vascular stent of Example 1 is shown;

[0043] Fig.14 The infrared spectra of dECM, GelMA, PVA and dEPG artificial vascular stents of Example 1 are shown; Fig.14 (a) is the FTIR image of dEPG hydrogel; Fig.14 (b) Fig.14 (a) Enlarged view in the dashed box;

[0044] Fig.15 The macro structures of the Y-shaped bifurcation of Example 2 and the variable diameter artificial vascular stent of Example 3 are shown; Fig.15 (a) is a Y-shaped bifurcated dEPG artificial vascular stent; Fig.15 (b) is the tube cross section of the Y-shaped bifurcated dEPG artificial vascular stent; Fig.15 (c) is a variable diameter dEPG artificial vascular stent; Fig.15 (d) is the cross section of the variable diameter dEPG artificial vascular stent. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention, and those skilled in the art in this field can make some non-essential improvements and adjustments based on the above invention content.

[0046] Example 1

[0047] (1) Purchasing fresh pig hearts from the market, washing them, peeling off the aorta on the surface of the pig hearts, removing the fat on the blood vessels, cutting them into small square pieces of blood vessels with a length of 0.2 cm, and soaking and cleaning them in 4°C distilled water for 24 hours; placing the small pieces of blood vessels in a PBS solution of 0.15wt% trypsin and 1mM EDTA, stirring in a water bath at 20°C for 4 hours, and then placing them in a PBS solution of 50Unit / mL DNA enzyme and 5mM MgCl2, stirring in a water bath at 20°C for 4 hours for decellularization to obtain a preliminary matrix, with a stirring speed of 500rpm;

[0048] (2) The preliminary matrix obtained in step (1) was placed in a 1wt% SDS solution and stirred for 24 hours, and the SDS solution remaining on the surface was washed with a PBS solution and then placed in a 1wt% Triton X-100 solution and stirred for 24 hours, and then placed in a PBS solution and stirred at a low speed (50 rpm) for 48 hours, and the PBS solution was replaced every 12 hours. Freeze-dried for 48 hours to obtain dECM;

[0049] (3) mixing the dECM obtained in step (2) and pepsin at a ratio of 1:10 mg / mg, placing the mixture in a 0.02 M HCl solution, and stirring and dissolving the mixture at room temperature to obtain a dECM solution;

[0050] (4) Methacrylic anhydride (MA) was slowly dripped into the gelatin (Gel) precursor solution (the mass volume ratio of Gel and MA was 5:4) at a rate of 1 mL / min, and the mixture was stirred in a 50°C water bath in the dark for 3 h. PBS solution was added to terminate the reaction, and the mixture was transferred to a 8000-14000 kDa dialysis bag and dialyzed at 50°C for 7 days, followed by centrifugation at 2500 rpm for 5 min. The supernatant was freeze-dried for 48 h to obtain a white sponge-like GelMA solid;

[0051] (5) Accurately weigh a certain amount of PVA solid, add 70°C distilled water and place in a 70°C constant temperature water bath and stir for 3 h to obtain a PVA solution with a mass concentration of 10% after complete dissolution;

[0052] (6) 7 g GelMA and 0.21 g photoinitiator IHT-PI 659 were added to 50 mL 10% PVA solution in a 50°C water bath and stirred evenly. After cooling to room temperature, 50 mL 1% dECM solution was added and stirred overnight to obtain a 0.5% dECM / 5% PVA / 7% GelMA (0.5d5P7G) pregel solution;

[0053] (7) Using Rhino 7 and 3d Max software to design the printing parameters and appearance of the 3D printed mold, a hollow tubular mold of equal diameter was 3D printed using white resin as the material, with dimensions of 4 mm outer diameter, 2.5 mm inner diameter, and 60 mm tube length;

[0054] (8) The pre-gel solution was poured into a hollow tubular mold of equal diameter, and then frozen at -20°C for 4 hours, and then taken out and allowed to thaw naturally at room temperature. The freeze-thaw process was repeated 3 times to cross-link the PVA. The hydrogel tube was then separated from the mold and evenly irradiated under a UV lamp for 5 minutes to cross-link the GelMA.

[0055] (9) placing the cross-linked hydrogel tube in step (8) in PBS, wiping off excess water on the surface after swelling equilibrium, placing it in a -20°C refrigerator for 8 hours, and freeze-drying it for 48 hours to obtain a dEPG artificial vascular stent;

[0056] (10) Soak the dEPG artificial vascular stent obtained in step (9) in a 0.5% Hep solution for 8 hours, and take it out after reaching swelling equilibrium to obtain a dEPG-Hep artificial vascular stent loaded with Hep.

[0057] Example 2

[0058] (1) Purchasing fresh pig hearts from the market, washing them, peeling off the aorta on the surface of the pig hearts, removing the fat on the blood vessels, cutting them into small square pieces of blood vessels with a length of 0.5 cm, and soaking and cleaning them in 4°C distilled water for 48 hours; placing the small pieces of blood vessels in a PBS solution of 0.25wt% trypsin and 2mM EDTA, stirring in a 37°C water bath for 6 hours, and then placing them in a PBS solution of 75Unit / mL DNA enzyme and 10mM MgCl2, stirring in a 37°C water bath for 6 hours to perform decellularization treatment to obtain a preliminary matrix, with a stirring speed of 500rpm;

[0059] (2) The preliminary matrix obtained in step (1) was placed in a 1.5 wt% SDS solution and stirred for 48 h, and the SDS solution remaining on the surface was washed with a PBS solution and then placed in a 2 wt% Triton X-100 solution and stirred for 48 h, and then placed in a PBS solution and stirred at a low speed (50 rpm) for 72 h, and the PBS solution was replaced every 12 h. Freeze-dried for 48 h to obtain dECM;

[0060] (3) mixing the dECM obtained in step (2) and pepsin at a ratio of 1:15 mg / mg, placing the mixture in a 0.02 M HCl solution, and stirring and dissolving the mixture at room temperature to obtain a dECM solution;

[0061] (4) Methacrylic anhydride (MA) was slowly dripped into the gelatin (Gel) precursor solution (the mass volume ratio of Gel and MA was 5:4) at a rate of 1.5 mL / min, and the mixture was stirred in a 50°C water bath in the dark for 5 h. PBS solution was added to terminate the reaction, and the mixture was transferred to a 8000-14000 kDa dialysis bag and dialyzed at 50°C for 7 days, followed by centrifugation at 3000 rpm for 10 min. The supernatant was freeze-dried for 48 h to obtain a white sponge-like GelMA solid;

[0062] (5) Accurately weigh a certain amount of PVA solid, add 90°C distilled water and place in a 90°C constant temperature water bath and stir for 2 h to obtain a PVA solution with a mass concentration of 12% after complete dissolution;

[0063] (6) Add 5 g GelMA and 0.15 g photoinitiator IHT-PI 659 to 50 mL 12% PVA solution in a 50°C water bath and stir evenly. After cooling to room temperature, add 50 mL 2% dECM solution and stir overnight to obtain a 1% dECM / 6% PVA / 5% GelMA (1d6P5G) pregel solution;

[0064] (7) Using Rhino 7 and 3d Max software to design the printing parameters and appearance of the 3D printed mold, a hollow tubular mold with a variable diameter was 3D printed using white resin as the material. The size of the mold was 3 mm in outer diameter and 1 mm in inner diameter at the thin top, 5 mm in outer diameter and 3 mm in inner diameter at the thick bottom, and the tube length was 60 mm.

[0065] (8) The pre-gel solution was poured into a hollow tubular mold of equal diameter, and then frozen at -30°C for 3 hours, and then taken out and allowed to thaw naturally at room temperature. The freeze-thaw process was repeated 3 times to cross-link the PVA. The hydrogel tube was then separated from the mold and evenly irradiated under a UV lamp for 10 minutes to cross-link the GelMA.

[0066] (9) placing the cross-linked hydrogel tube in step (8) in PBS, wiping off excess water on the surface after swelling equilibrium, placing it in a -20°C refrigerator for 8 hours, and freeze-drying it for 48 hours to obtain a dEPG artificial vascular stent;

[0067] (10) Soak the dEPG artificial vascular stent obtained in step (9) in a 1% Hep solution for 10 h, and take it out after reaching swelling equilibrium to obtain a dEPG-Hep artificial vascular stent loaded with Hep.

[0068] Example 3

[0069] (1) Purchasing fresh pig hearts from the market, washing them, peeling off the aorta on the surface of the pig hearts, removing the fat on the blood vessels, cutting them into small square pieces of blood vessels with a length of 1 cm, and soaking and cleaning them in 4°C distilled water for 72 hours; placing the small pieces of blood vessels in a PBS solution of 0.3wt% trypsin and 5mM EDTA, stirring in a 40°C water bath for 8 hours, and then placing them in a PBS solution of 100Unit / mL DNA enzyme and 20mM MgCl2, stirring in a 40°C water bath for 10 hours to perform decellularization treatment to obtain a preliminary matrix, with a stirring speed of 500rpm;

[0070] (2) The preliminary matrix obtained in step (1) was placed in a 2wt% SDS solution and stirred for 72 hours, and the SDS solution remaining on the surface was washed with a PBS solution and then placed in a 2.5wt% Triton X-100 solution and stirred for 72 hours, and then placed in a PBS solution and stirred at a low speed (50 rpm) for 72 hours, and the PBS solution was replaced every 12 hours. Freeze-dried for 48 hours to obtain dECM;

[0071] (3) mixing the dECM obtained in step (2) and pepsin at a ratio of 1:20 mg / mg, placing the mixture in a 0.02 M HCl solution, and stirring and dissolving the mixture at room temperature to obtain a dECM solution;

[0072] (4) Methacrylic anhydride (MA) was slowly dripped into the gelatin (Gel) precursor solution (the mass volume ratio of Gel and MA was 5:4) at a rate of 2 mL / min, and the mixture was stirred in a 50°C water bath in the dark for 7 h. PBS solution was added to terminate the reaction, and the mixture was transferred to a 8000-14000 kDa dialysis bag and dialyzed at 50°C for 7 days, followed by centrifugation at 3500 rpm for 10 min. The supernatant was freeze-dried for 48 h to obtain a white sponge-like GelMA solid;

[0073] (5) Accurately weigh a certain amount of PVA solid, add 95°C distilled water and place in a 95°C constant temperature water bath and stir for 1 h. After complete dissolution, a PVA solution with a mass concentration of 12% is obtained;

[0074] (6) 7 g GelMA and 0.21 g photoinitiator IHT-PI 659 were added to 50 mL 12% PVA solution in a 50°C water bath and stirred evenly. After cooling to room temperature, 50 mL 3% dECM solution was added and stirred overnight to obtain a 1.5% dECM / 6% PVA / 7% GelMA (1.5d6P7G) pregel solution;

[0075] (7) Using Rhino 7 and 3d Max software to design the printing parameters and appearance of the 3D printed mold, a Y-shaped hollow tubular mold was then 3D printed using white resin as the material, with dimensions of 6 mm outer diameter, 3 mm inner diameter, and 42 mm tube length;

[0076] (8) The pre-gel solution was poured into a hollow tubular mold of equal diameter, and then frozen at -40°C for 2 h, and then taken out and allowed to thaw naturally at room temperature. The freeze-thaw process was repeated 3 times to cross-link the PVA. The hydrogel tube was then separated from the mold and evenly irradiated under a UV lamp for 20 min to cross-link the GelMA.

[0077] (9) placing the cross-linked hydrogel tube in step (8) in PBS, wiping off excess water on the surface after swelling equilibrium, placing it in a -20°C refrigerator for 8 hours, and freeze-drying it for 48 hours to obtain a dEPG artificial vascular stent;

[0078] (10) Soak the dEPG artificial vascular stent obtained in step (9) in a 2% Hep solution for 12 hours, and take it out after reaching swelling equilibrium to obtain a dEPG-Hep artificial vascular stent loaded with Hep.

[0079] Performance Testing and Characterization

[0080] (1) Macroscopic and microscopic morphology of the dEPG artificial vascular stent obtained in Example 1

[0081] The macroscopic morphology of artificial vascular stents is Figure 1 As shown, the cross section of the tubular support is Figure 2 shown.

[0082] Scanning electron microscope (SEM) was used to observe its microscopic morphology: the freeze-dried artificial vascular stent was cut into small sections with a length of 3 mm using a blade, and placed in a high vacuum coating apparatus for gold spraying for 120 s. The cross-sectional microstructure of the artificial vascular stent was observed by SEM. Figure 3 As shown. Figure 3 It can be seen that the artificial vascular stent of Example 1 of the present application is a porous structure, which is similar to the ECM structure of natural vascular tissue, and is conducive to the transportation and exchange of nutrients and oxygen, promotes cell growth and metabolism, and maintains normal physiological functions of cells.

[0083] (2) Mechanical properties of the dEPG artificial vascular stent prepared in Example 1

[0084] The method of knotting and twisting the artificial vascular stent obtained in Example 1 is used to simulate various forces that the artificial vascular stent may withstand in the human body, and its deformation capacity under different stress conditions is evaluated, such as Figure 4 shown.

[0085] Figure 4 The results show that the artificial blood vessel stent can be easily knotted and twisted, and can basically return to its original shape without damage, proving that the hydrogel tube has good elasticity. At the same time, the artificial blood vessel stent has no damage after hanging a 40.60g object, indicating that the stent has a certain ability to bear heavy objects.

[0086] Tensile properties: Use a vernier caliper to accurately measure and record the inner and outer diameters of the hydrogel tube. Fix both ends of the hydrogel tube on a universal testing machine equipped with a 100N load cell, record the initial length of the hydrogel tube to be tested, and clamp the artificial vascular stent on a static universal material testing machine, such as Figure 5 As shown, the material is stretched at a crosshead rate of 5 mm / min until it breaks, and the stretching length L at the moment before the break is recorded. The elongation at break of the material is calculated from the displacement (L)-force (F)-time (t) curve during the stretching process. The test results are shown in Figure 5 , Figure 6 shown.

[0087] Figure 6 The elongation at break of the dEPG artificial vascular stent is shown. The elongation at break of the 1d6P5G group is the highest, reaching 204.78±14.83%.

[0088] Compression performance: The artificial vascular stent is made into a flat cylindrical shape, and its bottom diameter and height are measured with a vernier caliper. The compression performance of the material is tested using a universal testing machine, with the maximum pressure set to 100N, the compression speed set to 5mm / min, and the compression amount set to 80%. The compression stress-strain curve is directly read from the tested compression curve. The stress corresponding to the compression modulus of 80% is the yield limit. The test results are as follows: Figure 7 , Figure 8 shown.

[0089] Figure 8 The compressive yield strength of the dEPG artificial vascular stent is shown to be 0.34±0.02MPa, 1.60±0.09MPa and 0.93±0.06MPa, respectively. It can be seen that the compressive yield limit of the 1d6P5G group is significantly higher than that of the other two groups (p<0.001), indicating that 1d6P5G has the best anti-compression ability and the highest strength.

[0090] (3) Degradability of the dEPG artificial vascular stents prepared in Examples 1, 2 and 3

[0091] The enzymatic hydrolysis method was used to evaluate the in vitro degradation of the dEPG artificial vascular stents prepared in Example 1, Example 2, and Example 3. The artificial vascular stents were dried to constant weight and weighed as M0. Then the stents were immersed in a PBS buffer solution containing 1 mg / mL lysozyme (pH=7.4) and placed in a 37°C incubator to simulate the in vivo environment. A group of hydrogel tubes were taken out every 1, 3, 5, 7, 14, and 28 days, and the weight was recorded as M1 after freeze-drying. The in vitro degradation rate (D) formula is: The results are as follows Fig. 9 shown. Fig. 9It was shown that the artificial vascular stents after 28 days of degradation could still maintain structural integrity without damage. The degradation rates of the three groups of artificial vascular stents were 51.88±4.49%, 43.06±4.23%, and 49.41±0.83%, respectively.

[0092] (4) Biocompatibility of the dEPG artificial vascular stents prepared in Example 1, Example 2, and Example 3

[0093] The dEPG artificial vascular stents prepared in Example 1, Example 2, and Example 3 were immersed in 75% ethanol solution for 12 hours, irradiated under ultraviolet light for 6 hours, washed with PBS and dried naturally for 3 hours. Mouse fibroblasts (L929) were cultured using high-glucose complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin). All cells were cultured in an incubator at 37°C and 5% carbon dioxide.

[0094] Cell death and viability fluorescence staining: 10 μL of the cell culture medium with a density of 5×10 6 Cell suspension of 10 cells / mL was inoculated on the surface and inside of the scaffold and cultured in a cell culture incubator. The culture medium was replaced every two days. On the 4th and 7th days of culture, the artificial vascular scaffold was fluorescently stained with Calcein AM, Hochest 33342 and PI mixed dye (1mL PBS+15μL Hoechst+2μL AM+1μL PI). After incubation in the incubator in the dark for 0.5h, the cell growth status was observed under a laser confocal microscope. Fig.10 As shown, green fluorescence (Calcein AM) indicates live cells, and red fluorescence (PI) indicates dead cells. Fig.10 The fluorescence image of the cross section of the artificial vascular scaffold shows that as the culture time increases, the cells grow and proliferate on the artificial vascular scaffold.

[0095] SEM: 10 μL at a density of 2 × 10 6 Cell suspensions of 10 cells / mL were inoculated on the surface and inside of the scaffolds and cultured in an incubator at 37°C with 5% carbon dioxide, with the culture medium renewed every two days. On the 4th and 7th days, the scaffolds were fixed in PBS containing 2.5% glutaraldehyde for 4 hours. They were then placed in 50%, 70%, 90%, and 100% ethanol for gradient dehydration, with the solution replaced every 30 minutes. After gold spraying, the adhesion and growth of cells on the scaffolds were observed under a scanning electron microscope, as shown in Figure 2. Fig.11 As shown. Fig.11 It can be observed that as the culture time increases, the number of cells on the artificial vascular scaffold increases significantly, the cells aggregate into cell clusters, and grow and migrate into the pores of the scaffold.

[0096] Fluorescence images of cells seeded on artificial vascular scaffolds ( Fig.10 ) and SEM images ( Fig.11 ) showed that the scaffold had good biocompatibility.

[0097] (5) Anticoagulant performance of dEPG artificial vascular stents prepared in Example 1, Example 2, and Example 3

[0098] Activated partial thromboplastin time (APTT): Fresh pig blood containing sodium citrate solution (anticoagulant and whole blood ratio is 1:9) is centrifuged at 3000rpm for 10min to obtain platelet-poor plasma (PPP). Artificial blood vessels are cut into 5mm-long tube segments and placed in a 24-well plate. 100μL PPP and 100μL APTT reagent (37℃) are added to the well plate. After incubation at 37℃ for 5min, 0.1mL is taken and added to 0.025MCaCl2 (37℃) solution. The coagulation time at the initial stage of turbidity is recorded. Fig.12 Prothrombin time (PT): Replace APTT reagent with PT reagent, and the other steps are the same as above, such as Fig.13 shown.

[0099] Fig.12 The APTT values ​​of the blank group, 0.5d5P7G, 1d6P5G, and 1.5d6P7G were 22.67±1.15s, 26.00±2.00s, 28.00±1.00s, and 30.33±2.52s, respectively. Compared with the blank group, the APTT of 1d6P5G and 1.5d6P7G was significantly prolonged (p<0.01), indicating that the artificial vascular stent prolonged the coagulation time to a certain extent.

[0100] Fig.13 The PT values ​​of the blank group, 0.5d5P7G, 1d6P5G, and 1.5d6P7G were 11.33±1.15s, 12.33±0.58s, 13.33±0.58s, and 15.00±1.00s, respectively. The PT value of 1.5d6P7G was significantly different from that of the blank group (p<0.01), indicating that the artificial vascular stent prolonged the coagulation time to a certain extent.

[0101] Fig.14 The results showed that the successful introduction of dECM, PVA and GelMA into artificial vascular scaffolds successfully prepared a double cross-linked hydrogel network of hydrogen bond cross-linking and photo-cross-linking. This double cross-linked network can make the molecular connections of the material tighter, thereby giving the material better mechanical properties.

[0102] Fig.15 The prepared Y-shaped bifurcated and variable diameter artificial vascular stent is shown to meet the small diameter vascular tissue structure with different needs.

[0103] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for constructing an artificial blood vessel stent with anticoagulant properties, characterized in that: The construction method comprises the following steps: Step (1) decellularizing the aorta on the surface of the pig heart to obtain a decellularized matrix of the aorta dECM; Step (2) preparing a pre-gel solution by preparing porcine aorta decellularized matrix dECM, methacrylated gelatin GelMA and polyvinyl alcohol PVA; Step (3) 3D printing a hollow tubular mold, pouring the pre-gel in step (2) into the hollow tubular mold, freezing it completely at low temperature and then allowing it to melt naturally, repeating the freeze-thaw process to cross-link the PVA itself, and obtaining the final hydrogel tube; Step (4) separating the hydrogel tube obtained by the last freeze-thaw in step (3) from the hollow tubular mold, and placing it under a UV lamp for uniform irradiation to crosslink the GelMA with the photoinitiator IHT-PI 659; Step (5) placing the hydrogel tube cross-linked in step (4) in a phosphate buffered saline solution (PBS), wiping off excess water on the surface after swelling equilibrium, and freeze-drying to obtain a dEPG artificial vascular stent; Step (6) soaking the dEPG artificial vascular stent obtained in step (5) in a heparin Hep solution, taking it out after reaching swelling equilibrium, and obtaining a Hep-dEPG artificial vascular stent loaded with heparin Hep.

2. The method for constructing an artificial blood vessel stent with anticoagulant properties according to claim 1, characterized in that: The decellularization process in step (1) is as follows: cutting the pig heart aorta, soaking and cleaning it, placing it in a trypsin and ethylenediaminetetraacetic acid (EDTA) solution for 4 to 8 hours, and bathing it in a DNA enzyme solution for 4 to 10 hours, then placing it in a sodium dodecyl sulfate (SDS) solution and stirring it for 24 to 72 hours, washing the SDS solution remaining on its surface with a PBS solution, placing it in a polyethylene glycol octylphenyl ether solution and stirring it for 24 to 72 hours, and freeze-drying it to obtain the pig heart aorta dECM.

3. The method for constructing an artificial blood vessel stent with anticoagulant properties according to claim 1, characterized in that: In step (2), the concentration of dECM in the pre-gel solution is 0.5-1.5 w / v%, the concentration of PVA is 4-6 w / v%, and the concentration of GelMA is 5-9 w / v%.

4. The method for constructing an artificial blood vessel stent with anticoagulant properties according to claim 1, characterized in that: In the step (3), the hollow tubular mold includes three types: a Y-shaped bifurcated hollow tubular mold, a constant diameter hollow tubular mold, and a variable diameter hollow tubular mold.

5. The method for constructing an artificial blood vessel stent with anticoagulant properties according to claim 1, characterized in that: In the step (3), the freeze-thaw process is as follows: the pre-gel solution is injected into the hollow tubular mold through a sterile syringe, and the pre-gel solution is placed at -20°C to -40°C for 2 to 6 hours before being taken out and placed at room temperature for natural melting, and the pre-gel solution is repeatedly frozen and thawed for multiple times to cross-link the PVA.

6. The method for constructing an artificial blood vessel stent with anticoagulant properties according to claim 1, characterized in that: In the step (4), the uniform irradiation time of the ultraviolet lamp is 5 to 20 minutes.

7. An artificial vascular stent with anticoagulant properties, characterized in that: The artificial blood vessel stent is prepared by the construction method described in any one of claims 1-66.

8. An application of the artificial vascular stent with anticoagulant properties as claimed in claim 7, characterized in that: The artificial blood vessel stent is a tissue engineering artificial blood vessel stent with anti-coagulation performance.

9. The use of an artificial blood vessel stent with anticoagulant properties according to claim 7, characterized in that: The artificial blood vessel stent is used for cardiovascular diseases.

Images