Dual-network hydrogel, composite blood vessel material, and preparation method and application of dual-network hydrogel and composite blood vessel material

Abstract

The invention discloses a dual-network hydrogel, a composite blood vessel material and a preparation method and application thereof, the dual-network hydrogel is a polymer formed by a first network and a second network, the first network is formed by diselenide modified gelatin, the second network is formed by polymerization of a photoinitiator and a monomer, and the photoinitiator is formed by polymerization of the first network and the second network. The monomer is at least one of an alcohol monomer and an acrylate monomer. The composite vascular material comprises an acellular extracellular matrix, and the acellular extracellular matrix is coated with a double-network hydrogel layer. According to the invention, the dual-network hydrogel and the acellular extracellular matrix are compounded, so that the composite blood vessel material has excellent mechanical strength, and diselenide contained in the dual-network hydrogel can effectively catalyze endogenous S-nitromercaptan in vivo to continuously generate NO, so that the smoothness and steady state of the blood vessel are maintained, thrombus formation is prevented, and the blood vessel has a good application prospect. The overgrowth of smooth muscles is inhibited, and the rapid recovery of natural endothelium is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogels, and particularly relates to a double-network hydrogel, a composite vascular material, a preparation method thereof and an application thereof. Background Art

[0002] Cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality globally. The most common cause of CVDs is atherosclerosis, which is the accumulation of fatty plaques within the arterial wall, leading to obstruction and reduced blood flow to downstream tissues. The current standard treatment is to surgically insert a vascular graft to bypass the blocked site. For this purpose, blood vessels harvested from the patient's own body (autografts), such as the great saphenous vein in the leg or the internal mammary artery in the chest wall, are usually preferred. Arteries have shown significant effects in long-term use, but their supply and length are very limited. In contrast, veins are prone to accelerating atherosclerosis due to mechanical property mismatches and are likely to fail after long-term use. In addition, due to diseases and other reasons, some patients do not have available autologous blood vessels, and surgeons can only rely on artificial substitutes or allografts.

[0003] Currently, the most common commercially available artificial grafts are made of expanded polytetrafluoroethylene (ePTFE) or polyethylene terephthalate (PET). These materials have high mechanical strength and strong chemical inertness and can be mass-produced. However, these synthetic materials fail to replicate the key properties of natural blood vessels and fail to regulate important biological mechanisms that determine the fate of the grafts. Commercial polymer grafts are much stiffer than natural blood vessels, with rough surfaces and high hydrophobicity, resulting in poor interaction with vascular cells and triggering the blood coagulation cascade. The lack of an intact endothelium in artificial grafts is at the core of their main failure mode. Synthetic materials tend to increase the blood coagulation rate, which is the main cause of acute failure of artificial grafts. Their rough and hydrophobic surfaces usually lead to higher blood cell interactions and protein adsorption, accelerating the formation of blood clots. The lack of endothelium also causes smooth muscle cells (SMCs) to migrate into the intima and proliferate excessively in the intima, a process called neointimal hyperplasia, which is the main cause of mid- to late-stage graft failure.

[0004] In recent years, due to the abundant availability and low cost of animal tissues and organs, xenografts have received extensive attention. Grafts can be easily obtained from a variety of animal sources, including mammals and non-mammals. However, xenografts may trigger adverse innate or adaptive immune responses, so they must first be decellularized. Decellularization methods include chemical, physical, and biological (enzymatic) methods, which are usually used alone or in combination. After successfully removing sufficient cell debris, the extracellular matrix (ECM) components and their three-dimensional ultrastructure can be well preserved, thus providing an ideal microenvironment for regulating the host response after implantation. Nevertheless, the long-term implantation results of decellularized xenografts are still not satisfactory, and the main failure reasons include early acute thrombosis, mid-term and late intimal hyperplasia and vascular calcification, and insufficient mechanical strength.

[0005] The information disclosed in this background section is only intended to enhance the overall understanding of the present invention and should not be regarded as an admission or any form of implication that this information constitutes prior art known to those of ordinary skill in the art. Summary of the Invention

[0006] An object of the present invention is to provide a double-network hydrogel, a composite vascular material, a preparation method thereof, and an application thereof. The composite vascular material has excellent mechanical strength and helps to maintain vascular patency and homeostasis.

[0007] To achieve the above object, the technical solutions provided by a specific embodiment of the present invention are as follows:

[0008] A double-network hydrogel, which is a polymer formed by a first network and a second network. The first network is formed by gelatin modified with diselenide ether, and the second network is formed by polymerization of a photoinitiator and a monomer, and the monomer is at least one of an alcohol monomer and an acrylate monomer.

[0009] In one or more embodiments of the present invention, the content of selenium element in the polymer is 0.01 wt% to 1 wt%.

[0010] In one or more embodiments of the present invention, the first network is formed by the ammonolysis reaction and oxidation reaction of gelatin and a lactone compound. The general formula of the lactone compound is: n is any integer from 1 to 3, and the mass ratio of gelatin to the lactone compound is 1:(0.0001 to 1).

[0011] In one or more embodiments of the present invention, the mass fraction of the second network in the polymer is less than or equal to 30%.

[0012] In one or more embodiments of the present invention, the mass ratio of the photoinitiator to the monomer is 1:(100 to 10000).

[0013] The technical solution provided by another specific embodiment of the present invention is as follows:

[0014] A preparation method of the above double-network hydrogel, comprising the following steps:

[0015] S1. React the gelatin solution with a crosslinking agent at 0°C to 100°C for 1 h to 48 h to obtain a modified gelatin solution;

[0016] S2. Oxidize the modified gelatin solution to obtain a primary product;

[0017] S3. Immerse the primary product in a replacement solvent and soak it at 0°C to 40°C for 0.1 min to 1000 min to obtain a secondary product;

[0018] S4. Cure the secondary product under light irradiation to obtain a double-network hydrogel;

[0019] Wherein, the crosslinking agent is a lactone compound, and the general formula is: n is any integer from 1 to 3;

[0020] The replacement solvent includes a photoinitiator and a monomer, and the monomer is at least one of an alcohol monomer and an acrylate monomer.

[0021] The technical solution provided by another specific embodiment of the present invention is as follows:

[0022] A composite vascular material, comprising a decellularized extracellular matrix, and a double-network hydrogel layer is coated on the decellularized extracellular matrix, and the double-network hydrogel layer is formed by the above double-network hydrogel or a double-network hydrogel prepared by the preparation method of the above double-network hydrogel.

[0023] In one or more embodiments of the present invention, the thickness of the double-network hydrogel layer is 0.1 mm to 5 mm.

[0024] The technical solution provided by another specific embodiment of the present invention is as follows:

[0025] A preparation method of the above composite vascular material, comprising the following steps:

[0026] React the gelatin solution with a crosslinking agent at 0°C to 100°C for 1 h to 48 h to obtain a modified gelatin solution;

[0027] Coat the modified gelatin solution on the decellularized extracellular matrix, and then oxidize it to obtain a primary material;

[0028] Immerse the primary material in a replacement solvent and soak it at 0°C to 40°C for 0.1 min to 1000 min to obtain a secondary material;

[0029] The secondary material is cured under light irradiation to obtain a composite vascular material.

[0030] The technical solution provided by another specific embodiment of the present invention is as follows:

[0031] An application of the above composite vascular material in the field of bioengineering.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] 1. The present invention uses the ammonolysis lactone of gelatin with amino groups at the end groups and the ring-opening oxidation coupling method, and combines the methods of solvent replacement and photoinitiated polymerization to synthesize a double-network hydrogel. This hydrogel has the advantages of simple preparation conditions and a green synthesis process. At the same time, chemical crosslinking can effectively increase the sol-gel transition temperature of gelatin, making it possible to apply gelatin at physiological temperature.

[0034] 2. By changing the ratio of each raw material component in the synthesis process, the present invention can obtain a series of hydrogel materials with different mechanical properties, and the mechanical properties cover a wide range.

[0035] 3. By coating the prepared hydrogel material on the acellular extracellular matrix, and combining the good biocompatibility and vascular compliance of the extracellular matrix with the excellent mechanical properties of the hydrogel, the formed composite vascular material has the application prospect as an artificial blood vessel.

[0036] 4. The present invention introduces diselenide into the composite vascular material, and generates NO by reacting with endogenous S-nitrosothiol, preventing thrombus formation, inhibiting the excessive growth of smooth muscle, and promoting the rapid recovery of natural endothelium. Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is the preparation flow chart of the double-network hydrogel in an embodiment of the present invention;

[0039] Figure 2 It is the effect diagram of the composite vascular material in an embodiment of the present invention catalyzing the generation of NO from endogenous S-nitrosothiol in vivo;

[0040] Figure 3 It is the test result of the swelling performance of the hydrogel in an embodiment of the present invention;

[0041] Figure 4 The test results of the hydrogel stability in an embodiment of the present invention;

[0042] Figure 5 The test results of the mechanical properties of the composite vascular material in Examples 6-11 of the present invention;

[0043] Figure 6 The test results of the mechanical properties of the composite vascular material in Examples 12-17 of the present invention;

[0044] Figure 7 The test results of the mechanical properties of the composite vascular material in Examples 18-23 of the present invention;

[0045] Figure 8 The test results of the mechanical properties of the composite vascular material in Examples 24-29 of the present invention;

[0046] Figure 9 The test results of the mechanical properties of the composite vascular material in Examples 32-37 of the present invention;

[0047] Figure 10 The test results of the ability of the composite vascular material to catalytically release NO in an embodiment of the present invention. Detailed implementation manners

[0048] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0049] Nitric oxide (NO) is a key gas transfer molecule for maintaining vascular patency and homeostasis. Endogenous NO produced by endothelial cells can inhibit platelet aggregation and leukocyte adhesion, and reduce the occurrence of atherosclerosis. After vascular injury, NO can promote the regeneration of vascular endothelium and prevent subsequent intimal hyperplasia. In addition, NO also plays an important role in inhibiting the transdifferentiation of smooth muscle cells (SMCs) into osteoblasts, thereby preventing vascular calcification.

[0050] Inspired by the multiple biological functions of NO, the present invention designs and fabricates a composite biological vascular graft by combining acellular extracellular matrix with a diselenide-modified double-network hydrogel with NO-releasing properties, so as to partially simulate the physiological functions of natural endothelium. The hydrogel is a double-network hydrogel composed of a polymer formed by a diselenide-modified gelatin and a polymer of an alcohol monomer or a polymer of an acrylate monomer. The diselenide can catalyze the continuous production of NO from endogenous S-nitrosothiols in the body (such as S-nitroalbumin and S-nitroglutathione). The double-network structure improves the mechanical strength of the composite graft. This composite biological vascular graft can withstand the flushing of blood, prevent thrombosis, inhibit the excessive growth of smooth muscle, and promote the rapid recovery of natural endothelium.

[0051] A specific embodiment of the present invention provides a double-network hydrogel, including a polymer formed by a first network and a second network. The first network is formed by a diselenide-modified gelatin, and the second network is formed by polymerizing a photoinitiator with an alcohol monomer or a photoinitiator with an acrylate monomer.

[0052] Specifically, the content of selenium element in the double-network hydrogel is 0.01 wt% to 1 wt%. The selenium element will form diselenide bonds in the double-network hydrogel. The diselenide bonds can catalyze the production of NO to prevent restenosis of artificial blood vessels and can promote the rapid recovery of natural endothelium. In addition, by controlling the content of selenium element, the effective function of the double-network hydrogel can be further ensured.

[0053] Furthermore, the first network is formed by the ammonolysis reaction and oxidation reaction of gelatin and a lactone compound. The general formula of the lactone compound is: n is any integer from 1 to 3. The mass ratio of gelatin to the lactone compound is 1:(0.0001 - 1), preferably the mass ratio is 1:(0.001 - 0.1). By ammonolyzing the lactone and ring-opening oxidative coupling of gelatin with an amino group at the end group, a first network containing diselenide can be formed.

[0054] Furthermore, the mass fraction of the second network in the polymer is less than or equal to 30%. By controlling the mass fraction of the second network in the polymer, the mechanical properties of the double-network hydrogel can be improved.

[0055] Furthermore, the mass ratio of the photoinitiator to the monomer is 1:(100 - 10,000), preferably 1:(500 - 1,000). The photoinitiator is 2,4,6-trimethylbenzoyl diphenylphosphine oxide (TPO), 2-hydroxy-2-methyl-1-phenylpropanone, or 1-hydroxycyclohexyl phenyl ketone. The alcohol monomer is at least one of polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 800, glycerol, ethylene glycol, triethylene glycol, tetraethylene glycol, and pentaethylene glycol. The acrylate monomer is at least one of polyethylene glycol dimethacrylate 200, polyethylene glycol dimethacrylate 400, polyethylene glycol dimethacrylate 600, and polyethylene glycol dimethacrylate 800. Selecting the above types of photoinitiators and monomers can promote the formation of the second network, interpenetrate with the first network, and enhance the mechanical strength of the double-network hydrogel.

[0056] Another specific embodiment of the present invention provides a method for preparing a double-network hydrogel, including steps 1 - 4.

[0057] Step 1: React the gelatin solution with the crosslinking agent at 0°C - 100°C for 1 h - 48 h to obtain a modified gelatin solution.

[0058] Specifically, the gelatin solution is an aqueous solution of gelatin or a phosphate buffer solution of gelatin. The pH value of the phosphate buffer solution is 7.0 - 8.0, preferably 7.4. In the gelatin solution, the concentration of gelatin is 3 wt% - 30 wt%, preferably 10 wt% - 20 wt%, and more preferably 10 wt%.

[0059] Furthermore, the crosslinking agent is a lactone compound, and its general formula is n is any integer from 1 to 3. The mass ratio of gelatin to the crosslinking agent is 1:(0.0001 - 1), preferably 1:(0.001 - 0.1). The appropriate mass ratio of gelatin to the crosslinking agent can further improve the stability and mechanical properties of the double-network hydrogel.

[0060] Furthermore, the reaction in step 1 is carried out in a nitrogen atmosphere or an argon atmosphere. The reaction temperature is preferably 40°C - 60°C, and the reaction time is preferably 12 h - 24 h.

[0061] Step 2: Oxidize the modified gelatin solution to obtain a primary product.

[0062] Specifically, the aminolysis reaction between the crosslinking agent and the gelatin amino group in step 1 introduces selenol groups into the hydrogel, and then through the oxidation reaction in step 2, the selenol groups are oxidized and coupled to form disulfide bonds.

[0063] Further, the oxidation reaction is carried out in air, or oxidized using any one of oxygen, sodium hypochlorite, and hydrogen peroxide. Preferably, air or oxygen is used for oxidation. Further preferably, the amount of air or oxygen is in excess.

[0064] Step 3: Immerse the primary product in a displacement solvent and soak it at 0°C to 40°C for 0.1 min to 1000 min to obtain a secondary product.

[0065] Specifically, the displacement solvent includes a photoinitiator and a monomer. The monomer is at least one of an alcohol monomer and an acrylate monomer. In Step 3, through solvent displacement, the water in the gelatin hydrogel on the decellularized extracellular matrix is replaced with the displacement solvent to form a second network through subsequent photoinitiated polymerization.

[0066] Further, in the displacement solvent, the mass ratio of the photoinitiator to the monomer is 1:(100 - 10000). Preferably, the mass ratio of the photoinitiator to the monomer is 1:(500 - 1000). The soaking temperature is preferably 20°C to 30°C, and the soaking time is preferably 5 min to 100 min.

[0067] Step 4: Cure the secondary product under light irradiation to obtain a double-network hydrogel.

[0068] Specifically, during the photocuring process, the light source is 365 nm to 450 nm, the power is 100 mW / cm 2 , and the photocuring time is 5 min to 60 min, preferably 10 min to 20 min. In Step 4, the photoinitiator and the monomer polymerize under light irradiation to form a second network. The second network intersects with the first network formed by gelatin to form an interpenetrating double-network hydrogel.

[0069] Further, as Figure 1 shown, the amino group contained in gelatin reacts with selenolactone to introduce a selenol group into the gelatin backbone, and then through oxidation, an interchain diselenide bond is formed to form the first network. Then, through solvent displacement, the water in the first network is replaced with a photoinitiator and a monomer, and through photopolymerization, the photoinitiator and the monomer form a second network in the first network, thereby forming a double-network gel.

[0070] Another specific embodiment of the present invention provides a composite vascular material, including a decellularized extracellular matrix, and a double-network hydrogel layer is coated on the decellularized extracellular matrix. The double-network hydrogel layer is formed by the above double-network hydrogel or the double-network hydrogel prepared by the preparation method of the above double-network hydrogel.

[0071] Specifically, the acellular extracellular matrix is the acellular vascular matrix of the rat abdominal aorta or the rat carotid aorta, preferably the acellular vascular matrix of the rat abdominal aorta, and its preparation is to perform acellular treatment on the rat abdominal aorta using (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. Specifically, the abdominal aorta is taken from an adult rat (250-300 g body weight) and carefully dissected under a microscope to remove the perivascular fat and connective tissue in the blood vessel. Then it is thoroughly washed in 1M NaCl, and then on a stirrer at 37 °C, a solution is prepared with (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS, Sigma) (8 mM CHAPS, 1M NaCl, 25 mM EDTA), and it is placed in an oscillator for treatment 3 times, 2 hours each time.

[0072] Furthermore, the thickness of the double-network hydrogel layer is 0.1 mm to 5 mm, preferably 0.1 mm to 1 mm. By controlling the thickness of the network hydrogel layer, it can ensure that the composite vascular material has better mechanical strength, and at the same time, the diselenide bonds contained can also effectively play their roles.

[0073] Another specific embodiment of the present invention provides a preparation method of a composite vascular material, including the following steps: reacting a gelatin solution with a crosslinking agent at 0 °C to 100 °C for 1 h to 48 h to obtain a modified gelatin solution; coating the modified gelatin solution on the acellular extracellular matrix, and then performing oxidation to obtain a primary material; soaking the primary material in a replacement solvent at 0 °C to 40 °C for 0.1 min to 1000 min to obtain a secondary material; curing the secondary material under light irradiation to obtain the composite vascular material.

[0074] Specifically, the coating process is carried out at a temperature of 30 °C to 100 °C, preferably 40 °C to 60 °C, and the coating thickness is 0.1 mm to 5 mm, preferably 0.1 mm to 1 mm.

[0075] Another specific embodiment of the present invention provides the application of the above composite vascular material or the composite vascular material prepared by the preparation method of the above composite vascular material in the field of bioengineering.

[0076] Specifically, the diselenide bonds contained in the composite vascular material can catalyze the production of NO in the body, which can further promote the regeneration of vascular endothelium, and it has excellent mechanical properties and has broad application prospects as an artificial blood vessel for transplantation.

[0077] Such as Figure 2As shown, a composite vascular material is formed by combining a double-network hydrogel with the acellular vascular matrix of the rat abdominal aorta. After the composite vascular material is transplanted into the body, it can catalyze the continuous production of NO from endogenous S-nitrosothiols (such as S-nitroalbumin and S-nitroglutathione) in the body, thereby helping to maintain vascular patency and homeostasis.

[0078] The present invention will be further described in detail below with reference to specific embodiments.

[0079] The gelatin used in the present invention is purchased from Yuanye Bio-Technology Co., Ltd., with the product number S22176; the selenolactone used is γ-selenobutyrolactone. Unless otherwise specified, the raw materials used in the present invention can be obtained commercially.

[0080] Examples of double-network hydrogels

[0081] Example 1

[0082] Under argon protection, selenolactone (1 g) was added dropwise to an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, i.e., selenol-modified gelatin. The polymer was directly used in the next step without separation and purification.

[0083] The polymer was fully oxidized in air for 12 h to obtain a primary product.

[0084] According to the mass ratio of TPO to polyethylene glycol diacrylate 400 of 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol diacrylate 400 (50 g) to obtain a replacement solvent. The primary product was completely immersed in the replacement solvent and replaced at 25 °C for 10 min to obtain a secondary product.

[0085] The secondary product was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain a double-network hydrogel.

[0086] Example 2

[0087] Under argon protection, selenolactone (0.1 g) was added dropwise to an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, i.e., selenol-modified gelatin. The polymer was directly used in the next step without separation and purification.

[0088] The polymer was fully oxidized in air for 12 h to obtain a primary product.

[0089] According to the mass ratio of TPO to polyethylene glycol diacrylate 400 of 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol diacrylate 400 (50 g) to obtain a replacement solvent. The primary product was completely immersed in the replacement solvent and replaced at 25 °C for 10 min to obtain a secondary product.

[0090] The secondary product was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain the double-network hydrogel.

[0091] Example 3

[0092] Under argon protection, selenolactone (0.01 g) was added dropwise to an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, i.e., gelatin modified with selenol. The polymer was directly used in the next step without separation and purification.

[0093] The polymer was fully oxidized in air for 12 h to obtain the primary product.

[0094] According to the mass ratio of TPO to polyethylene glycol diacrylate 400 being 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol diacrylate 400 (50 g) to obtain a replacement solvent. The primary product was completely immersed in the replacement solvent and replaced at 25 °C for 10 min to obtain the secondary product.

[0095] The secondary product was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain the double-network hydrogel.

[0096] Example 4

[0097] Under argon protection, selenolactone (1 g) was added dropwise to an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, i.e., gelatin modified with selenol. The polymer was directly used in the next step without separation and purification.

[0098] The polymer was fully oxidized in air for 12 h to obtain the primary product.

[0099] According to the mass ratio of TPO to polyethylene glycol diacrylate 200 being 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol diacrylate 200 (50 g) to obtain a replacement solvent. The primary product was completely immersed in the replacement solvent and replaced at 25 °C for 10 min to obtain the secondary product.

[0100] The secondary product was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain the double-network hydrogel.

[0101] Example 5

[0102] Under argon protection, selenolactone (1 g) was added dropwise to an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, i.e., gelatin modified with selenol. The polymer was directly used in the next step without separation and purification.

[0103] The polymer was fully oxidized in air for 12 h to obtain a primary product.

[0104] According to the mass ratio of TPO to polyethylene glycol diacrylate 600 being 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol diacrylate 600 (50 g) to obtain a replacement solvent. The primary product was completely immersed in the replacement solvent and replaced at 25 °C for 10 min to obtain a secondary product.

[0105] The secondary product was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain a double-network hydrogel.

[0106] Examples of composite vascular materials

[0107] Examples 6 - 11

[0108] Examples 6 - 11 were all prepared into composite vascular materials by the following method:

[0109] Under argon protection, selenolactone (1 g) was dropped into an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, i.e., gelatin modified with selenol. The polymer was directly used in the next step without separation and purification.

[0110] At 60 °C, the decellularized vascular matrix of rat abdominal aorta was immersed in the above polymer, taken out and quickly immersed in liquid nitrogen for 1 min, repeated 3 times to control the coating thickness, and fully oxidized in air for 12 h to obtain a primary material.

[0111] According to the mass ratio of TPO to polyethylene glycol diacrylate 400 being 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol diacrylate 400 (50 g) to obtain a replacement solvent. The primary material was completely immersed in the replacement solvent and replaced at 25 °C for a certain time to obtain a secondary material.

[0112] The secondary material was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain a composite vascular material.

[0113] The difference between Examples 6 - 11 was only the replacement time of the primary material in the replacement solvent, as shown in Table 1 specifically:

[0114] Table 1 Replacement time of the primary material in the replacement solvent in Examples 6 - 11

[0115] Group Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Replacement time 10min 20min 30min 40min 50min 60min

[0116] Examples 12 - 17

[0117] Examples 12 - 17 were all prepared into composite vascular materials by the following method:

[0118] Under argon protection, selenolactone (0.1 g) was dropped into an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, namely gelatin modified with selenol. The polymer was directly used in the next step without separation and purification.

[0119] At 60 °C, the acellular vascular matrix of rat abdominal aorta was immersed in the above polymer, taken out and quickly immersed in liquid nitrogen for 1 min, repeated 3 times to control the coating thickness, and fully oxidized in air for 12 h to obtain the primary material.

[0120] According to the mass ratio of TPO to polyethylene glycol diacrylate 400 being 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol diacrylate 400 (50 g) to obtain a replacement solvent. The primary material was completely immersed in the replacement solvent and replaced at 25 °C for a certain time to obtain the secondary material.

[0121] The secondary material was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain the composite vascular material.

[0122] The difference between Examples 12 - 17 was only the replacement time of the primary material in the replacement solvent, as shown in Table 2 specifically:

[0123] Table 2 Replacement time of the primary product in the replacement solvent in Examples 12 - 17

[0124]

[0125]

[0126] Examples 18 - 23

[0127] Examples 18 - 23 were all prepared into composite vascular materials by the following method:

[0128] Under argon protection, selenolactone (0.01 g) was dropped into an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, namely gelatin modified with selenol. The polymer was directly used in the next step without separation and purification.

[0129] At 60 °C, the acellular vascular matrix of rat abdominal aorta was immersed in the above polymer, taken out and quickly immersed in liquid nitrogen for 1 min, repeated 3 times to control the coating thickness, and fully oxidized in air for 12 h to obtain the primary material.

[0130] According to the mass ratio of TPO to polyethylene glycol diacrylate 400 being 1:1000, dissolve TPO (0.05 g) in polyethylene glycol diacrylate 400 (50 g) to obtain a displacement solvent. Immerse the primary material completely in the displacement solvent and displace for a certain time at 25 °C to obtain a secondary material.

[0131] Cure the secondary material under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain a composite vascular material.

[0132] The difference between Examples 18 - 23 lies only in the different displacement times of the primary product in the displacement solvent, as shown in Table 3 specifically:

[0133] Table 3 Displacement times of the primary product in the displacement solvent in Examples 18 - 23

[0134] Group Example 18 Example 19 Example 20 Example 21 Example 22 Example 23 Replacement time 10min 20min 30min 40min 50min 60min

[0135] Examples 24 - 29

[0136] Examples 24 - 29 are all prepared to obtain a composite vascular material by the following method:

[0137] Under argon protection, drop selenium lactone (1 g) into an aqueous gelatin solution (100 g, 10 wt%) and react at 60 °C for 48 h to obtain a polymer, namely gelatin modified with selenol. The polymer is directly used in the next step without separation and purification.

[0138] At 60 °C, immerse the decellularized vascular matrix of the rat abdominal aorta into the above polymer, take it out and quickly immerse it in liquid nitrogen for 1 min, repeat 3 times to control the coating thickness, and oxidize it fully in the air for 12 h to obtain a primary material.

[0139] According to the mass ratio of TPO to polyethylene glycol diacrylate 200 being 1:1000, dissolve TPO (0.05 g) in polyethylene glycol diacrylate 200 (50 g) to obtain a displacement solvent. Immerse the primary material completely in the displacement solvent and displace for a certain time at 25 °C to obtain a secondary material.

[0140] Cure the secondary material under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain a composite vascular material.

[0141] The difference between Examples 24 - 29 lies only in the different displacement times of the primary product in the displacement solvent, as shown in Table 4 specifically:

[0142] Table 4 Displacement times of the primary product in the displacement solvent in Examples 24 - 29

[0143] Group Example 24 Example 25 Example 26 Example 27 Example 28 Example 29 Replacement time 10min 20min 30min 40min 50min 60min

[0144] Example 30

[0145] Under argon protection, selenium lactone (1 g) was dropped into an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, i.e., gelatin modified with selenol. The polymer was directly used in the next step without separation and purification.

[0146] At 60 °C, the acellular vascular matrix of the rat abdominal aorta was immersed in the above polymer, taken out and quickly immersed in liquid nitrogen for 1 min, and repeated 3 times to control the coating thickness, and then fully oxidized in air for 12 h to obtain the primary material.

[0147] According to the mass ratio of TPO to polyethylene glycol diacrylate 600 of 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol diacrylate 600 (50 g) to obtain a displacement solvent. The primary material was completely immersed in the displacement solvent and displaced at 25 °C for 10 min to obtain the secondary material.

[0148] The secondary material was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain the composite vascular material.

[0149] Example 31

[0150] Under argon protection, selenium lactone (1 g) was dropped into an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, i.e., gelatin modified with selenol. The polymer was directly used in the next step without separation and purification.

[0151] At 60 °C, the acellular vascular matrix of the rat abdominal aorta was immersed in the above polymer, taken out and quickly immersed in liquid nitrogen for 1 min, and repeated 3 times to control the coating thickness, and then fully oxidized in air for 12 h to obtain the primary material.

[0152] According to the mass ratio of TPO to polyethylene glycol 400 of 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol 400 (50 g) to obtain a displacement solvent. The primary material was completely immersed in the displacement solvent and displaced at 25 °C for a certain time to obtain the secondary product.

[0153] The secondary material was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain the composite vascular material.

[0154] Examples 32 - 37

[0155] Examples 32 - 37 were all prepared by the following method to obtain the composite vascular material:

[0156] Under argon protection, selenolactone (1 g) was dropped into an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain gelatin modified with polymeric selenol. The polymer was directly used in the next step without separation and purification.

[0157] At 60 °C, a glass rod treated with piranha solution (a mixture of concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3) was immersed in the above polymer, then quickly immersed in liquid nitrogen for 1 min after being taken out, and this was repeated 3 times to control the coating thickness, followed by full oxidation in air for 12 h to obtain the primary material.

[0158] According to the mass ratio of TPO to polyethylene glycol diacrylate 400 being 1:1000, TPO (0.05 g) was dissolved in polyethylene glycol diacrylate 400 (50 g) to obtain a replacement solvent. The primary material was completely immersed in the replacement solvent and replaced at 25 °C for a certain time to obtain the secondary material.

[0159] The secondary material was cured under ultraviolet light (405 nm, 100 mW / cm 2 ) for 10 min to obtain the composite vascular material.

[0160] The difference between Examples 32 - 37 is only the replacement time of the primary product in the replacement solvent, as shown in Table 5 specifically:

[0161] Table 5 Replacement time of the primary product in the replacement solvent in Examples 32 - 37

[0162] Group Example 32 Example 33 Example 34 Example 35 Example 36 Example 37 Replacement time 10min 20min 30min 40min 50min 60min

[0163] Comparative Example 1

[0164] Gelatin was prepared into an aqueous solution of 10 wt%.

[0165] At 60 °C, a glass rod treated with piranha solution (a mixture of concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3) was immersed in the gelatin solution, then quickly immersed in liquid nitrogen for 1 min after being taken out, and this was repeated 3 times to control the coating thickness, followed by full oxidation in air for 12 h to obtain the hydrogel.

[0166] Comparative Example 2

[0167] Under argon protection, selenolactone (1 g) was dropped into an aqueous gelatin solution (100 g, 10 wt%) and reacted at 60 °C for 48 h to obtain a polymer, namely gelatin modified with selenol. The polymer was directly used in the next step without separation and purification.

[0168] At 60 °C, the glass rod treated with piranha solution (a mixture of concentrated sulfuric acid and 30% hydrogen peroxide with a volume ratio of 7:3) was immersed in the above polymer, quickly immersed in liquid nitrogen for 1 min after being taken out, and repeated 3 times to control the coating thickness, and fully oxidized in air for 12 h to obtain the composite vascular material.

[0169] The composite vascular materials in the examples and the hydrogels in the comparative examples were tested by the following test methods:

[0170] (1) The rheological properties of the formed hydrogel were measured on a HAAKE rheometer (RS 6000). All rheological measurements were carried out at 37 °C to simulate the human body environment. When using a 25 mm parallel plate for measurement, the distance between the rotor and the sample stage was 1 mm. Strain sweep settings: frequency was 1 Hz, and the strain sweep was set from 1 to 1000%; frequency sweep settings: strain was 1.0%, and the frequency sweep was set from 1 to 100 Hz. Time sweep measurement was carried out at a frequency of 1 Hz, a strain of 1%, and a sweep time of 300 s.

[0171] (2) Swelling property test of the hydrogel

[0172] First, the hydrogels in Examples 1-3 and the hydrogel in Comparative Example 1 were made into thin slices of 5 mm * 5 mm * 2 mm and freeze-dried until the weight no longer changed. The samples were immersed in 10 mL of phosphate buffer saline (PBS) solution (10 mmol / L, pH = 7.4), swollen at room temperature, and the samples were taken out at regular intervals to weigh and calculate the swelling ratio until the swelling equilibrium was reached. The swelling ratio was calculated by the following formula: Ws represents the mass of the hydrogel after swelling for a certain time, and W0 represents the initial mass of the hydrogel. All samples were tested three times and the average value was taken. The test results are as Figure 1 shown.

[0173] Combined Figure 3 , the swelling ratio of the hydrogel is related to the cross-linking density of the hydrogel. The more the content of diselenide bonds in the hydrogel in the examples, the higher the cross-linking density of the hydrogel, and the smaller the maximum swelling ratio of the hydrogel. In addition, gelatin can reach equilibrium swelling after diselenide cross-linking and solvent replacement, while the pure gelatin in Comparative Example 1 is very unstable during the swelling process, disintegrates into extremely small fragments over time, resulting in a decrease rather than an increase in the swelling ratio of the hydrogel, and finally leading to a large test error.

[0174] (3) Stability test of the hydrogel

[0175] The hydrogels in Examples 1-5 and Comparative Example 1 were taken, and the rheological properties of the hydrogels at different temperatures were measured on a HAAKE rheometer (RS 6000). The results are asFigure 2 As shown Figure 2 The ordinate in [figure] is the shear modulus

[0176] From Figure 4 it can be seen that, compared with Comparative Example 1, the hydrogel in the embodiment of the present invention has a higher shear modulus, showing better stability and the ability to resist deformation

[0177] (4) Mechanical property test

[0178] In this experiment, the storage modulus and loss modulus of the composite vascular material were tested by rheological tests

[0179] Frequency sweep tests the response of the mechanical properties of the material to frequency under a constant strain. The strain was set to 1%, the frequency ranged from 1 to 10 Hz, a flat plate fixture with a diameter of 25 mm was used, and the test temperature was 37°C. The samples were swollen to a solid content of 10% with a PBS buffer solution with a pH of 7.4 to control the dosage. The average value was taken from at least three test results of each sample

[0180] According to ASTM standard F2150, the elastic modulus and elongation at break of the hydrogel were studied by tensile tests using a universal testing machine (Tinius Olsen model H10KS) at 37°C. The crosshead speed of 10 mm·min –1 was used to test the samples (the tensile machine was equipped with a 5000 N load cell) and determine the elongation at break

[0181] The mechanical property test results are as Figures 5 - 9 shown, where Figures 5 - 9 0 min in [figure] all refers to Comparative Example 1. Compared with the comparative example, the mechanical properties of the composite vascular material prepared in the example can be adjusted within a large range. At the same time, the shear modulus and tensile strength of the hydrogel increase with the increase of the disulfide bond, increase with the increase of the replacement time, increase with the increase of the molecular weight of the replaced polyethylene glycol, and photo-crosslinking can significantly enhance the mechanical properties of the hydrogel. The elastic modulus and elongation at break of the hydrogel first increase and then decrease with the increase of the replacement time, proving that with the increase of the replacement time, the vascular graft gradually becomes harder and more brittle

[0182] In addition, the shear modulus and tensile strength of the composite vascular material in Example 30 are better than those in Example 31, which also shows that with the increase of the molecular weight of the replaced monomer, the mechanical properties of the hydrogel increase accordingly, thus enhancing the mechanical properties of the composite vascular material

[0183] (5) Test on the ability of the composite vascular material to catalytically release NO

[0184] An improved Griess test (urinary nitrite test) was used to quantify the generation of NO in the composite vascular material

[0185] The Griess reagent was prepared by mixing equal volumes of sulfanilic acid and N-(1-naphthyl)ethylenediamine dihydrochloride solution. 50 mm 3 (2 mm × 5 mm × 5 mm) composite vascular materials were placed into a 96-well plate, and 50 μL of GSH (glutathione) (500 μg / mL), 50 μL of GSNO (S-nitrosoglutathione) (500 μg / mL), 20 μL of Griess reagent, and 130 μL of deionized water were added. Additionally, a blank group was set up in the 96-well plate where only 180 μL of deionized water was added without the composite vascular materials. Then, the reaction was carried out for 30 min in a dark environment at room temperature. The absorbance at 548 nm was measured using a multimode microplate reader.

[0186] Figure 10 It is a comparison chart of the catalytic release of NO from GSNO in Example 6, Example 12, Example 18, Example 32, Comparative Example 1, Comparative Example 2, and the blank group. The experiment found that the diselenide in the composite vascular material is the key to catalyzing the production of NO. In the blank group without diselenide and Comparative Example 1, it was almost impossible to catalyze the production of NO from GSNO and GSH, while Example 6, 12, 18, 32 containing diselenide and Comparative Example 2 could all significantly catalyze the release of NO. Moreover, comparing Example 6, 12, and 18, the higher the content of diselenide, the more NO was catalytically released. Comparing Example 6, 32, and Comparative Example 2, the release content of NO was only related to the concentration of diselenide and had nothing to do with other components.

[0187] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

[0188] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A double network hydrogel, characterized in that: The double network hydrogel is a polymer formed by a first network and a second network, wherein the first network is formed by diselenide-modified gelatin, and the second network is formed by polymerization of a photoinitiator and a monomer, wherein the monomer is at least one of an alcohol monomer and an acrylate monomer.

2. The double network hydrogel according to claim 1, characterized in that The content of selenium in the polymer is 0.01wt% to 1wt%.

3. The double network hydrogel according to claim 1, characterized in that The first network is formed by gelatin and a lactone compound through an aminolysis reaction and an oxidation reaction, and the lactone compound has a general formula of: n is any integer from 1 to 3; The mass ratio of the gelatin to the lactone compound is 1:(0.0001-1).

4. The double network hydrogel according to claim 1, characterized in that The mass fraction of the second network in the polymer is less than or equal to 30%.

5. The double network hydrogel according to claim 1, characterized in that: The mass ratio of the photoinitiator to the monomer is 1:(100-10000).

6. A method for preparing a double network hydrogel, characterized in that: The following steps are involved: reacting the gelatin solution with the cross-linking agent at 0°C to 100°C for 1h to 48h to obtain a modified gelatin solution; The modified gelatin solution is oxidized to obtain a primary product; The primary product is immersed in a replacement solvent at 0°C to 40°C for 0.1 min to 1000 min to obtain a secondary product; The secondary product is cured under light irradiation to obtain a double network hydrogel; Wherein, the cross-linking agent is a lactone compound, and the general formula is: n is any integer from 1 to 3; The displacement solvent comprises a photoinitiator and a monomer, wherein the monomer is at least one of an alcohol monomer and an acrylate monomer.

7. A composite vascular material, characterized in that: It comprises a decellular extracellular matrix, wherein the decellular extracellular matrix is ​​coated with a double network hydrogel layer, and the double network hydrogel layer is formed by the double network hydrogel according to any one of claims 1 to 5 or by the double network hydrogel prepared by the preparation method of the double network hydrogel according to claim 6.

8. The composite vascular material according to claim 7, characterized in that: The thickness of the double network hydrogel layer is 0.1 mm to 5 mm.

9. A method for preparing the composite vascular material according to claim 7, characterized in that: The steps include: reacting the gelatin solution with the cross-linking agent at 0°C to 100°C for 1h to 48h to obtain a modified gelatin solution; The modified gelatin solution was coated on the decellularized extracellular matrix and then oxidized to obtain the primary material; The primary material is immersed in a displacement solvent at 0°C to 40°C for 0.1 min to 1000 min to obtain a secondary material; The secondary material is cured under light irradiation to obtain a composite vascular material.

10. Use of the composite vascular material according to claim 7 in the field of bioengineering.

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