An artificial blood vessel stent with anticoagulant properties and a method for constructing the same

Abstract

The application discloses an artificial blood vessel stent with anticoagulation performance and a construction method thereof. After a pig heart aorta blood vessel is subjected to decellularization treatment, a pre-gel solution is prepared by compounding polyvinyl alcohol and methacrylated gelatin, a designed model is printed into a hollow tubular mold by using 3D printing, the pre-gel solution is injected into the mold to obtain an artificial blood vessel stent with controllable shape and size after cross-linking, and finally, the artificial blood vessel stent loaded with heparin is obtained by loading the heparin on the prepared stent. The material for preparing the pre-gel solution is selected, and the advantages of the material functions can be complemented. The decellularized matrix is beneficial to cell adhesion, proliferation and differentiation; the compounding of polyvinyl alcohol and methacrylated gelatin improves the mechanical properties of the stent and improves the cell adhesion. The heparin is loaded on the artificial blood vessel stent to improve the anticoagulation performance of the stent and prevent the formation of thrombus. The stent serves as an excellent biomedical material and has a wide application prospect in the fields of cardiovascular tissue engineering and biology.

Description

Technical Field

[0001] The present invention belongs to the fields of tissue engineering, materials 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 methacrylated gelatin, and a construction method thereof. Background Art

[0002] Cardiovascular diseases have extremely high morbidity and mortality rates, and vascular bypass grafting therapy has attracted widespread attention by providing alternative conduits for narrowed, blocked, or traumatic 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] Currently, the main methods for constructing small-diameter artificial blood vessels are electrospinning, 3D printing, and microfluidics. Electrospinning and microfluidics are complex to operate, and the creation of vascular stents with controllable, complex macroscopic configurations remains a challenge. Furthermore, 3D printing often exhibits poor resolution and limited printability.

[0004] In response to the problems that small-diameter artificial blood vessels are prone to thrombosis and have 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 therefrom can more realistically simulate the in vivo cell microenvironment, which is conducive to cell adhesion, proliferation and differentiation. However, the mechanical properties of dECM are relatively poor, while GelMA retains the biocompatibility, degradability and cell adhesion of gelatin, and at the same time 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] To address 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 using white resin. The pre-gel solution is injected into the mold and cross-linked to obtain a tissue-engineered 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 this 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 degree of 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 first prepares a pregel solution based on natural porcine heart aorta decellularized matrix (dECM), combined with methacryloylated gelatin (GelMA) and polyvinyl alcohol (PVA); secondly, the designed model is printed into a hollow tubular mold via 3D printing; finally, the pregel solution is injected into the hollow tubular mold, cross-linked, and loaded with heparin (Hep) to obtain a tissue-engineered artificial vascular stent with anticoagulant function. The method specifically comprises the following steps:

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

[0010] Step (2) preparing a pre-gel solution by preparing 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. The freeze-thaw process is repeated 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 constant 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 irradiating it evenly under a UV lamp to crosslink the GelMA with the photoinitiator IHT-PI 659;

[0013] Step (5) placing the cross-linked hydrogel tube obtained in step (4) in phosphate buffered saline (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. It is then placed in a sodium dodecyl sulfate (SDS) solution and stirred for 24 to 72 hours. After washing the SDS solution remaining on its surface with a PBS solution, it is placed in a polyethylene glycol octylphenyl ether (Triton X-100) solution and stirred for 24 to 72 hours, and freeze-dried to obtain a pig heart aorta dECM. The dECM obtained after decellularization can effectively retain the protein and physicochemical properties of natural tissue, 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 vessel, and PVA serves as a supporting material to improve the mechanical properties of the artificial blood vessel.

[0017] Preferably, the dECM in the pregel solution in step (2) contains GAGs, which have a structure similar to Hep and have 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, the dECM concentration in the pre-gel solution in step (2) is 0.5-1.5 w / v%, the PVA concentration is 4-6 w / v%, and the GelMA concentration 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 hollow tubular molds (Y-shaped bifurcated, constant diameter, and variable diameter) are drawn using Rhino 7 and 3D Max software. STL files are generated and imported into a 3D printer. The hollow tubular molds are then 3D printed using non-toxic white resin. Printing parameters can be adjusted as needed to produce a hollow tubular mold with controllable shape and size.

[0020] Preferably, the pregel solution in step (2) is injected into the hollow tubular mold in step (3) via a sterile syringe, and then frozen at -20°C to -40°C for 2 to 6 hours before being taken out and allowed to thaw naturally at room temperature. The PVA is then repeatedly frozen and thawed to crosslink the PVA. PVA can crosslink itself by generating hydrogen bonds through repeated freezing and thawing without the introduction of other toxic crosslinking agents, and the pore size and mechanical properties of the PVA hydrogel can be adjusted by varying the number of freeze-thaw cycles.

[0021] Preferably, in step (4), the hydrogel tube is separated from the mold and uniformly irradiated under UV light for 5 to 20 minutes to crosslink the GelMA. The double crosslinking of the UV crosslinking of the GelMA and the hydrogen bonding crosslinking of the PVA results in good mechanical properties for the artificial blood vessel. The uniform irradiation time under UV light 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 anti-coagulant performance and prevent thrombosis.

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

[0024] The tissue engineering artificial vascular 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 porcine heart aorta dECM, composite PVA, and GelMA to prepare an artificial vascular scaffold with anticoagulant function, achieving complementary material and functional advantages. Porcine hearts are widely available and low-cost. The dECM obtained after decellularization of the porcine heart aorta effectively retains the protein and physicochemical properties of the aorta, such as cell adhesion sites and corresponding biological signals. The scaffold 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, so that it has better mechanical properties. 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 mold combinations, thereby achieving various shapes and sizes of tissue-engineered artificial blood vessels. These mold designs can be customized according to clinical needs, enabling them to better simulate the structure of real small-diameter blood vessels, which is of great significance for vascular models in medical research and clinical applications.

[0029] (4) The combined effects of dECM and PVA used in the present invention can prolong clotting time, giving the artificial blood vessel 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 further improves the anticoagulant properties of the stent, effectively prevents thrombosis, and prolongs the time the artificial blood vessel stent remains 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 figure shows the design of the equal-diameter dEPG artificial blood vessel stent of Example 1; Figure 2 (a) is the design drawing of the equal-diameter mold using 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 shows a scanning electron microscope image of a cross section of the dEPG artificial vascular stent of Example 1; Figure 3 (a) Microscopic morphology of the cross section of the dEPG artificial vascular stent; Figure 3 (b) Figure 3 (a) Enlarged view within the dotted box;

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

[0034] Figure 5 The figure shows the structural diagram of the tensile performance of the dEPG artificial blood vessel stent in Example 1; Figure 5 (a) The natural state of the dEPG artificial vascular stent; Figure 5 (b) The stretching process of the 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 vascular stent of Example 1 are shown;

[0036] Figure 7 A schematic diagram of the compression performance structure of the dEPG artificial blood vessel stent in 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 dEPG artificial blood vessel stent compression performance of Example 1 is shown;

[0038] Figure 9 The degradation performance of the dEPG artificial vascular stent of Example 1 is shown;

[0039] Figure 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 the artificial vascular scaffold and L929 cells cultured for 4 days, a2-c2 represent the Hochest fluorescence images of the artificial vascular scaffold and L929 cells cultured for 4 days, a3-c3 represent the Calcein AM images of the 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 with 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] Figure 11The scanning electron micrographs of the dEPG artificial vascular scaffold and L929 cells of Example 1 are shown; Group a shows the microscopic morphology of L929 cells cultured on the artificial vascular scaffold for 4 days, specifically: a1-a3 show 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 magnified images in the dotted boxes of a1-a3, respectively, showing the structural morphology of the artificial vascular scaffold and L929 cells cultured for 4 days at a magnification of 1000 times, a1 and a4 show the microscopic morphology of the artificial vascular scaffold and L929 cells cultured for 4 days at a magnification of 0. The structural morphology of the 5d5P7G artificial vascular scaffold and L929 cells cultured for 4 days, a2 and a5 represent the structural morphology of the 1d6P5G artificial vascular scaffold and L929 cells cultured for 4 days, a3 and a6 represent the structural morphology of the 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] Figure 12 The anticoagulant performance (APTT) of the dEPG artificial vascular stent of Example 1 is shown;

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

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

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

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and specific examples. It should be understood that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention, and that those skilled in the art may make non-essential improvements and adjustments based on the above invention.

[0046] Example 1

[0047] (1) A fresh pig heart was purchased from the market, and the aorta on the surface of the pig heart was peeled off after washing. The fat on the blood vessels was removed and the blood vessels were cut into small square pieces with a length of 0.2 cm. The blood vessels were soaked and washed in 4°C distilled water for 24 hours; the small blood vessels were placed in a PBS solution of 0.15wt% trypsin and 1mM EDTA, stirred in a 20°C water bath for 4 hours, and then decellularized in a PBS solution of 50Unit / mL DNAse and 5mM MgCl2, stirred in a 20°C water bath for 4 hours, and the stirring speed was 500rpm to obtain a preliminary matrix;

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

[0049] (3) mixing the dECM obtained in step (2) with 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 added to the gelatin (Gel) precursor solution (mass-to-volume ratio of Gel to MA was 5:4) at a rate of 1 mL / min. The mixture was stirred in a 50°C water bath in the dark for 3 h. PBS solution was added to terminate the reaction. The mixture was transferred to an 8000-14000 kDa dialysis bag and dialyzed at 50°C for 7 days. The mixture was then centrifuged 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. After complete dissolution, a PVA solution with a mass concentration of 10% is obtained;

[0052] (6) 7 g of GelMA and 0.21 g of photoinitiator IHT-PI 659 were added to 50 mL of 10% PVA solution in a 50°C water bath and stirred evenly. After cooling to room temperature, 50 mL of 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, the printing parameters and appearance design of the 3D printing mold were performed. Then, a hollow tubular mold of equal diameter was 3D printed using white resin as the material. The dimensions of the mold were 4 mm in outer diameter, 2.5 mm in inner diameter, and 60 mm in length.

[0054] (8) The pregel solution was poured into a hollow tubular mold of equal diameter and frozen at -20°C for 4 h. The pregel solution was then taken out and allowed to thaw naturally at room temperature. The freeze-thaw process was repeated three times to crosslink the PVA. The hydrogel tube was then separated from the mold and uniformly irradiated under UV light for 5 min to crosslink the GelMA.

[0055] (9) The cross-linked hydrogel tube in step (8) was placed in PBS, and after swelling equilibrium, excess water on the surface was wiped off. The tube was placed in a -20°C refrigerator for 8 hours, and freeze-dried for 48 hours to obtain a dEPG artificial vascular scaffold;

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

[0057] Example 2

[0058] (1) A fresh pig heart was purchased from the market, and the aorta on the surface of the pig heart was peeled off after washing. The fat on the blood vessels was removed and the blood vessels were cut into small square pieces with a length of 0.5 cm. The blood vessels were soaked and washed in 4°C distilled water for 48 hours. The small blood vessels were placed in a PBS solution of 0.25wt% trypsin and 2mM EDTA, stirred in a 37°C water bath for 6 hours, and then decellularized in a PBS solution of 75Unit / mL DNAse and 10mM MgCl2, stirred in a 37°C water bath for 6 hours, and the stirring speed was 500rpm to obtain a preliminary matrix.

[0059] (2) The preliminary matrix obtained in step (1) was placed in a 1.5 wt% SDS solution and stirred for 48 h. The residual SDS solution on the surface was washed with PBS solution and then placed in a 2 wt% Triton X-100 solution and stirred for 48 h. Subsequently, the matrix was placed in a PBS solution and stirred at low speed (50 rpm) for 72 h. The PBS solution was replaced every 12 h. The matrix was freeze-dried for 48 h to obtain the 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 added dropwise to the gelatin (Gel) precursor solution (mass-to-volume ratio of Gel to MA was 5:4) at a rate of 1.5 mL / min. The mixture was stirred in a 50°C water bath in the dark for 5 h. PBS solution was added to terminate the reaction. The mixture was transferred to an 8000-14000 kDa dialysis bag and dialyzed at 50°C for 7 days. The mixture was then centrifuged 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. After complete dissolution, a PVA solution with a mass concentration of 12% is obtained;

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

[0064] (7) Using Rhino 7 and 3D Max software, the printing parameters and appearance design of the 3D printing mold were performed. Then, a hollow tubular mold with a variable diameter was 3D printed using white resin. The dimensions of the mold were: a thin top with an outer diameter of 3 mm and an inner diameter of 1 mm, a thick bottom with an outer diameter of 5 mm and an inner diameter of 3 mm, and a tube length of 60 mm.

[0065] (8) The pregel solution was poured into a hollow tubular mold of equal diameter, frozen at -30°C for 3 h, and then taken out and allowed to thaw naturally at room temperature. The freeze-thaw process was repeated three times to crosslink the PVA. The hydrogel tube was then separated from the mold and uniformly irradiated under UV light for 10 min to crosslink the GelMA.

[0066] (9) The cross-linked hydrogel tube in step (8) was placed in PBS, and after swelling equilibrium, excess water on the surface was wiped off. The tube was placed in a -20°C refrigerator for 8 hours, and freeze-dried for 48 hours to obtain a dEPG artificial vascular scaffold;

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

[0068] Example 3

[0069] (1) A fresh pig heart was purchased from the market, and the aorta on the surface of the pig heart was peeled off after washing. The fat on the blood vessels was removed and the blood vessels were cut into small square pieces with a length of 1 cm. The blood vessels were soaked and washed in 4°C distilled water for 72 hours. The small blood vessels were placed in a 0.3wt% trypsin and 5mM EDTA PBS solution and stirred in a 40°C water bath for 8 hours. Subsequently, the blood vessels were decellularized in a 100Unit / mL DNase and 20mM MgCl2 PBS solution and stirred in a 40°C water bath for 10 hours. The stirring speed was 500rpm.

[0070] (2) The preliminary matrix obtained in step (1) was placed in a 2 wt% SDS solution and stirred for 72 h. The residual SDS solution on the surface was washed with PBS solution and then placed in a 2.5 wt% Triton X-100 solution and stirred for 72 h. Subsequently, the matrix was placed in a PBS solution and stirred at low speed (50 rpm) for 72 h. The PBS solution was replaced every 12 h. The matrix was freeze-dried for 48 h to obtain the 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 added to the gelatin (Gel) precursor solution (mass-to-volume ratio of Gel to MA was 5:4) at a rate of 2 mL / min. The mixture was stirred in a 50°C water bath in the dark for 7 h. PBS solution was added to terminate the reaction. The mixture was transferred to an 8000-14000 kDa dialysis bag and dialyzed at 50°C for 7 days. The mixture was then centrifuged 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 of GelMA and 0.21 g of photoinitiator IHT-PI 659 were added to 50 mL of 12% PVA solution in a 50°C water bath and stirred evenly. After cooling to room temperature, 50 mL of 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, the printing parameters and appearance design of the 3D printing mold were performed. Then, a Y-shaped hollow tubular mold was 3D printed using white resin as the material. The dimensions of the mold were 6 mm in outer diameter, 3 mm in inner diameter, and 42 mm in length.

[0076] (8) The pregel solution was poured into a hollow tubular mold of equal diameter, 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 three times to crosslink the PVA. The hydrogel tube was then separated from the mold and uniformly irradiated under UV light for 20 min to crosslink the GelMA.

[0077] (9) The cross-linked hydrogel tube in step (8) was placed in PBS, and after swelling equilibrium, excess water on the surface was wiped off. The tube was placed in a -20°C refrigerator for 8 hours, and freeze-dried for 48 hours to obtain a dEPG artificial vascular scaffold;

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

[0079] Performance testing and characterization

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

[0081] The macromorphology of artificial vascular stents is determined by Figure 1 As shown, the cross section of the tubular support is as follows Figure 2 shown.

[0082] Scanning electron microscope (SEM) was used to observe its microstructure: the freeze-dried artificial vascular stent was cut into small sections of 3 mm in length with a blade, placed in a high vacuum coating apparatus and sprayed with gold for 120 s, and 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 in 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 transport 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 was used to simulate the various forces that the artificial vascular stent may withstand in the human body, and its deformation ability under different stress conditions was evaluated, such as Figure 4 shown.

[0085] Figure 4 The results showed that the artificial vascular stent could be easily knotted and twisted, and then essentially returned to its original shape without damage, demonstrating the hydrogel tube's good elasticity. Furthermore, the artificial vascular stent was able to hang a 40.60g weight without any damage, indicating that the stent has a certain load-bearing capacity.

[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 stretched 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 80% compression modulus 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 stents was 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 was significantly higher than that of the other two groups (p<0.001), indicating that 1d6P5G has the best compression resistance 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 Examples 1, 2, and 3. The artificial vascular stents were dried to a constant weight and weighed as M0. The stents were then immersed in a PBS buffer solution (pH = 7.4) containing 1 mg / mL lysozyme and incubated in a 37°C incubator to simulate the in vivo environment. A group of hydrogel tubes were removed after 1, 3, 5, 7, 14, and 28 days, freeze-dried, and weighed as M1. The in vitro degradation rate (D) formula is: The results are as follows Figure 9 shown. Figure 9The results showed that the artificial vascular stents could still maintain structural integrity without damage after 28 days of degradation. 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 Examples 1, 2, and 3

[0093] The dEPG artificial vascular scaffolds prepared in Examples 1, 2, and 3 were immersed in 75% ethanol solution for 12 hours, irradiated under UV light for 6 hours, washed with PBS, and naturally dried for 3 hours. Mouse fibroblasts (L929) were cultured in 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 live fluorescence staining: 10 μL of the cell culture medium was diluted with 5 × 10 6 Cell suspensions of 10 cells / mL were inoculated on the surface and interior of the scaffold and cultured in a cell culture incubator, with the culture medium replaced every two days. On the 4th and 7th days of culture, the artificial vascular scaffold was fluorescently stained using a mixed stain of Calcein AM, Hochest 33342, and PI (1 mL PBS + 15 μL Hoechst + 2 μL AM + 1 μL PI). After incubation in the dark in an incubator for 0.5 h, the cell growth status was observed under a laser confocal microscope, as shown in Figure 5. Figure 10 As shown, green fluorescence (Calcein AM) indicates live cells, and red fluorescence (PI) indicates dead cells. Figure 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 interior of the scaffolds and cultured in an incubator at 37°C containing 5% carbon dioxide, with the culture medium refreshed 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 dehydrated in a gradient of 50%, 70%, 90%, and 100% ethanol, 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. Figure 11 As shown. Figure 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 ( Figure 10 ) and SEM images ( Figure 11 ) showed that the scaffold had good biocompatibility.

[0097] (5) Anticoagulant properties of the dEPG artificial vascular stents prepared in Examples 1, 2, and 3

[0098] Activated partial thromboplastin time (APTT): Fresh porcine blood containing sodium citrate solution (anticoagulant to whole blood ratio of 1:9) was centrifuged at 3000 rpm for 10 minutes to obtain platelet-poor plasma (PPP). Artificial blood vessels were cut into 5 mm long segments and placed in a 24-well plate. 100 μL of PPP and 100 μL of APTT reagent (37°C) were added to the plate. After incubation at 37°C for 5 minutes, 0.1 mL was taken and added to 0.025 M CaCl2 (37°C) solution. The coagulation time at the onset of turbidity was recorded. Figure 12 Prothrombin time (PT): Replace APTT reagent with PT reagent, and the other steps are the same as above, such as Figure 13 shown.

[0099] Figure 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 values ​​of 1d6P5G and 1.5d6P7G were significantly prolonged (p<0.01), indicating that the artificial vascular stent prolonged the clotting time to some extent.

[0100] Figure 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 clotting time to some extent.

[0101] Figure 14 The results showed that the successful introduction of dECM, PVA and GelMA into artificial vascular scaffolds successfully prepared a double-crosslinked hydrogel network of hydrogen bond crosslinking and photocrosslinking. This double-crosslinked network can make the intermolecular connection of the material tighter, thereby giving the material better mechanical properties.

[0102] Figure 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 basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing an artificial vascular 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 pig heart 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, placing it in a low temperature and completely freezing it, and then allowing it to melt naturally, repeating the freeze-thaw process to cross-link the PVA itself, to obtain 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 irradiating it evenly under a UV lamp 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, and taking it out after reaching swelling equilibrium to obtain a Hep-dEPG artificial vascular stent loaded with heparin Hep; 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 then 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.

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

3. The method for constructing an artificial vascular 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.

4. The method for constructing an artificial vascular 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 a hollow tubular mold through a sterile syringe, and the pre-gel solution is placed in a -20°C to -40°C freezer for 2 to 6 hours, and then taken out and placed at room temperature to melt naturally, and the pre-gel solution is repeatedly frozen and thawed to cross-link the PVA.

5. The method for constructing an artificial vascular 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.

6. 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 1-5.

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