Method for preparing biological valve material by copolymerization and crosslinking, biological valve material and application

Abstract

The application discloses a method for preparing a biological valve material by copolymerization and crosslinking, and the biological valve material and application, and the preparation method comprises the following steps: S110, contacting a biological material with an aldehyde group crosslinking agent solution to perform crosslinking; S120, soaking the biological material treated in the step S110 in a solution containing a first functional monomer to chemically react and introduce a first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and an oxirane group; S130, soaking the biological material treated in the step S120 in a solution containing a second functional monomer, the second functional monomer has a second carbon-carbon double bond and a functional group B; and S200, under the action of an initiator, performing a polymerization reaction on the carbon-carbon double bond to obtain the biological valve material. The method can introduce additional functional groups while introducing the carbon-carbon double bond for the second time, and can endow the biological material with new characteristics.

Description

Technical Field

[0001] This application relates to the field of interventional materials technology, specifically to a method for preparing biological valve materials through copolymer crosslinking, as well as the biological valve materials and their applications. Background Technology

[0002] Heart valve disease is a common valvular degeneration condition. Clinically, it manifests as regurgitation caused by narrowing of the valve opening or valvular insufficiency, which seriously endangers the patient's life.

[0003] Artificial heart valve replacement is the gold standard for treating valvular heart disease. It restores the normal opening and closing function of the valve by replacing the damaged heart valve with an artificial one. Artificial heart valves are divided into bioprosthetic valves and mechanical valves. Mechanical valves are made of synthetic materials and are implanted into the patient through open-chest surgery. Bioprosthetic valves are made by cross-linking animal tissue (porcine or bovine pericardium) with glutaraldehyde. They possess excellent hydrodynamic properties and are less thrombogenic than mechanical valves. Patients typically do not need lifelong anticoagulant medication after implantation. Furthermore, bioprosthetic valves can be replaced via a minimally invasive catheter procedure, reducing the surgical risks of valve replacement to some extent. Therefore, bioprosthetic valves are increasingly being chosen by patients and are gradually becoming the preferred artificial heart valve in heart valve replacement surgery.

[0004] Currently, most commercially available bioprosthetic valves are made from glutaraldehyde-crosslinked porcine or bovine pericardium. Glutaraldehyde can improve the mechanical strength of the pericardium and reduce the immunogenicity of exogenous pericardium by crosslinking the collagen matrix. However, the stability and degree of crosslinking of glutaraldehyde-crosslinked bioprosthetic valves are still not high. This leads to structural degradation and damage after implantation, further compromising their structural integrity and causing structural degeneration and failure. Furthermore, low crosslinking and stability mean that degradation of bioprosthetic valve components can induce mechanical damage, promoting calcification and decay, thereby impairing normal valve function and reducing its lifespan. Therefore, the stability and degree of crosslinking of bioprosthetic valves urgently need further improvement. Although bioprosthetic valves have lower thrombogenicity compared to mechanical valves, thrombosis still exists, which can impair normal valve function and lead to the risk of secondary valve replacement. On the other hand, calcification directly leads to the decay of bioprosthetic valves. Therefore, the degree of crosslinking, stability, antithrombotic properties, and anti-calcification performance of bioprosthetic valves still need to be improved.

[0005] Currently, glutaraldehyde-crosslinked bioprosthetic valves are still the most commonly used bioprosthetic valves in clinical practice. However, given that glutaraldehyde-crosslinked bioprosthetic heart valves still have problems such as instability, low crosslinking degree, thrombosis, and calcification, as well as the risk of structural degradation and failure caused by these problems, further modification of glutaraldehyde-crosslinked bioprosthetic heart valves is not only in line with the actual needs of real-world production but also has high scientific value.

[0006] The applicant of this application has long been committed to the research of biological heart valves. For example, in the early research, Chinese invention patent application document CN114748694A disclosed a co-crosslinked biological valve material and its preparation method and application. In the crosslinking treatment, the biological valve material is functionally modified by introducing functional monomers for co-crosslinking. In the biological valve preparation method disclosed in Chinese invention patent applications CN114748693A, CN114748697A, CN114748696A and CN114748695A, carbon-carbon double bonds are introduced by the functional monomers while adding functional monomers for co-crosslinking, as a basis for further crosslinking. The modification of the biological valve material is completed through two crosslinking processes.

[0007] In the studies described above, whether it is co-crosslinking modification by introducing functional monomers while crosslinking glutaraldehyde, or introducing carbon-carbon double bonds as the basis for further crosslinking during the co-crosslinking process, both involve introducing new modified substances to participate in the crosslinking reaction during the crosslinking process of glutaraldehyde. Summary of the Invention

[0008] This application provides a method for preparing bio-valve materials through copolymer crosslinking, as well as the bio-valve materials and their applications. After crosslinking with glutaraldehyde, carbon-carbon double bonds are introduced stepwise, providing a controllable crosslinking opportunity and range for the glutaraldehyde crosslinked membrane without changing the conventional glutaraldehyde crosslinking reaction process. At the same time, functional groups are introduced through functional monomers to further improve the various properties of the bio-valve materials.

[0009] A method for preparing bioprosthetic valve materials through copolymer crosslinking includes: Step S110: The biomaterial is cross-linked by contacting the aldehyde cross-linking agent solution; Step S120: Immerse the biomaterial treated in step S110 in a solution containing the first functional monomer, and react to attach the first carbon-carbon double bond. The first functional monomer has the first carbon-carbon double bond and an ethylene oxide group. Step S130: Immerse the biomaterial treated in step S120 in a solution containing a second functional monomer to physically infiltrate and introduce a second carbon-carbon double bond. The second functional monomer has a second carbon-carbon double bond and a functional group B. In step S200, carbon-carbon double bonds undergo a polymerization reaction under the action of an initiator to obtain a biological valve material.

[0010] Optionally, the aldehyde crosslinking agent is glutaraldehyde or formaldehyde.

[0011] Optionally, the biological material is animal tissue, which is selected from one or more of the pericardium, valves, intestinal membrane, meninges, pulmonary membrane, blood vessels, skin, and ligaments.

[0012] Optionally, the animal tissue may be fresh animal tissue or decellularized biological tissue.

[0013] In step S200: Optionally, the initiator can be added to the system treated in the previous step; or the biological material treated in the previous step can be taken out and directly or after washing before being immersed in a solution containing the initiator.

[0014] Optionally, the initiator may be a single initiator or a mixture of initiators.

[0015] Optionally, the mixed initiator is: The initiator is a mixture of ammonium persulfate and sodium bisulfite, or a mixture of ammonium persulfate and sodium bisulfite, or a mixture of sodium persulfate and sodium bisulfite, or a mixture of potassium persulfate and sodium bisulfite, or a mixture of potassium persulfate and sodium bisulfite, or a mixture of potassium persulfate and tetramethylethylenediamine, or a mixture of ammonium persulfate and tetramethylethylenediamine, or a mixture of sodium persulfate and tetramethylethylenediamine; the concentration of each component in the mixture is 1~100 mM.

[0016] Optionally, the single initiator may be any component of the mixed initiators.

[0017] Optionally, the double bond polymerization time is 3~24h.

[0018] Optionally, the first functional monomer is selected from at least one of allyl glycidyl ether, glycidyl methacrylate, and glycidyl acrylate.

[0019] Optionally, the second functional monomer is selected from one or more of polyethylene glycol diacrylate, 1,4-butanediol diacrylate, ethane-1,2-diyl diacrylate, ethyl acrylate, N-methyl-2-acrylamide, N-2,2-propenyl-2-acrylamide, N-ethylacrylamide, N,N'-vinylbisacrylamide, (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)diacrylate, N,N'-dimethylacrylamide, N,N-dimethylmethylacrylamide, and double-bonded polylysine.

[0020] Optionally, the functional group B is selected from at least one of hydroxyl, carboxyl, carboxycholine, sulfonic acid choline, phosphoric acid choline, pyrrolidone, sulfonic acid group, carboxylate ion, sulfonate, sulfoxide, amide group, and methoxy group.

[0021] When the second functional monomer has a functional group B, the second functional monomer is optionally selected from acrylamide, acrylic acid, sodium acrylate, methacrylic acid, sodium methacrylate, 2-(propionic-2-enoylamino)acetic acid, 2-acrylamido-2-methylpropanesulfonic acid, hydroxyethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, N-methyl-2-acrylamide, N-isopropylacrylamide, N-(hydroxymethyl)acrylamide, N-(2-hydroxyethyl)methacrylamide, 3-[N,N-dimethyl-[2-(2-methylpropionic-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, 2-methacryloyloxyethyl phosphocholine, N-(2-hydroxyethyl)acrylamide, N-(methoxymethyl)methacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and double-bonded hyaluronic acid.

[0022] In step S110: Optionally, the w / w concentration of the aldehyde crosslinking agent solution is 0.1%~5%; the crosslinking time is 0.5h-120h.

[0023] In step S120: Optionally, the w / w concentration of the first functional monomer in the solution containing the first functional monomer is 1% to 10%; the reaction time is 2 to 120 hours.

[0024] Optionally, the solution containing the first functional monomer contains only the first functional monomer and a solvent that does not participate in the chemical reaction.

[0025] Optionally, the solvent in the solution containing the first functional monomer is one or more of the following: an aqueous solution of methanol, ethanol, ethylene glycol, propanol, 1,2-propanediol, 1,3-propanediol, isopropanol, butanol, isobutanol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, glycerol, water, physiological saline, and pH neutral buffer.

[0026] In step S130: Optionally, the v / v concentration of the second functional monomer in the solution containing the second functional monomer is 0.1%-20%; the soaking time is 0.5h-120h.

[0027] Furthermore, the v / v concentration of the second functional monomer in the solution containing the second functional monomer is 0.1%-6%.

[0028] Optionally, the second functional monomer enters the biomaterial through physical permeation.

[0029] The physical penetration can be understood as follows: when the biomaterial treated in step S120 is immersed in a solution containing the second functional monomer, the second functional monomer in the solution adheres to the surface of the biomaterial or is embedded in the gaps within the biomaterial. During this process, no chemical reaction occurs between the second functional monomer and the biomaterial.

[0030] Optionally, the solution containing the second functional monomer contains only the second functional monomer and a solvent that does not participate in the chemical reaction.

[0031] Optionally, the solvent in the solution containing the second functional monomer is one or a mixture of several of the following: water, physiological saline, ethanol, isopropanol, and pH neutral buffer solution.

[0032] This application also provides a biological valve material prepared by the method described above.

[0033] This application also provides a biological valve material, comprising: Step S110: The biomaterial is cross-linked by contacting the aldehyde cross-linking agent solution; Step S120: Immerse the biomaterial treated in step S110 in a solution containing the first functional monomer, and a chemical reaction is performed to introduce the first carbon-carbon double bond. The first functional monomer has the first carbon-carbon double bond and an ethylene oxide group. Step S130: Immerse the biomaterial treated in step S120 in a solution containing a second functional monomer, wherein the second functional monomer has a second carbon-carbon double bond and a functional group B. In step S200, carbon-carbon double bonds undergo a polymerization reaction under the action of an initiator to obtain a biological valve material.

[0034] This application also provides a bioprosthetic valve, including a stent and leaflets, wherein the leaflets are the bioprosthetic valve material described above.

[0035] Optionally, the bioprosthetic valve is a heart valve.

[0036] This application also provides an interventional system including a heart valve and a catheter assembly, wherein the heart valve is folded and delivered by the catheter assembly, and the heart valve includes a stent and leaflets, wherein the leaflets are the bio-valve material described above.

[0037] Compared with the prior art, this application has at least the following beneficial effects: (1) The method of this application modifies the glutaraldehyde cross-linked biovalve material. Double bonds are introduced into the glutaraldehyde cross-linked biovalve material by reacting with a double bond oxidizing agent. The resulting double bonded glutaraldehyde cross-linked biovalve material serves as a platform for functionalized copolymer cross-linking. Furthermore, functionalized copolymer cross-linking is achieved by initiating polymerization between the double bonds on the glutaraldehyde cross-linked biovalve material and the double bonds on the functional monomers to introduce functional monomer polymers as functionalized cross-linking networks. This can further improve the degree of cross-linking of the biovalve material and introduce functional groups. By increasing the degree of cross-linking, the stability of the biovalve material can be improved.

[0038] (2) In this application, after introducing double bonds into the glutaraldehyde crosslinked biovalve material, the polymerization between the double bonds on the double bonded biovalve material and the double bonds on the functional monomer is further initiated, thereby introducing a functional polymer crosslinking network into the biovalve material. This functional crosslinking network can act as a polymer barrier to reduce the contact and interaction between collagenase in vivo and the collagen matrix on the biovalve material to a certain extent, significantly reduce the degradation effect of collagenase on the collagen matrix on the biovalve material, improve the stability of the glutaraldehyde crosslinked biovalve material, and further reduce the risk of structural degradation of the biovalve caused by the structural degradation of the biovalve material.

[0039] (3) This application introduces a functional polymer crosslinking network by introducing double bonds into the glutaraldehyde crosslinked biovalve material and then initiating the polymerization between the double bonds on the glutaraldehyde crosslinked biovalve material and the double bonds on the functional monomers. This functional polymer crosslinking network can act as a polymer barrier to further reduce the binding of calcium ions with the mineralized areas on the biovalve material that are easily bound to calcium ions, thereby reducing the risk of calcification and thus playing an anti-calcification role.

[0040] (4) This application introduces double bonds into the glutaraldehyde-crosslinked biovalve material, and then further initiates polymerization between the double bonds on the double bonds of the double bonds on the glutaraldehyde-crosslinked biovalve material and the double bonds on the functional monomers to introduce a functional polymer crosslinking network. By introducing the functional polymer crosslinking network, the degree of crosslinking on the biovalve material increases, resulting in a more rigid structure of the biovalve material and thus increased elasticity. Furthermore, the functional polymer crosslinking network fills the gaps between the collagen matrix on the biovalve material to inhibit the deformation of collagen fibers, making the biovalve material relatively harder and improving its elasticity.

[0041] (5) Compared with the glutaraldehyde modification method of the applicant in the previous study, which introduced carbon-carbon double bonds by adding functional monomers for co-crosslinking, the biological valve material modification process of this application first performs glutaraldehyde crosslinking treatment, and then the residual amino, hydroxyl, carboxyl and other active groups on the glutaraldehyde crosslinking membrane are chemically linked to the functional monomers with carbon-carbon double bonds. The functional monomers with carbon-carbon double bonds are connected to the amino, hydroxyl and carboxyl groups on the surface of the glutaraldehyde crosslinking membrane through ethylene oxide, so that the carbon-carbon double bonds are mainly connected to the surface of the biological valve material. In the process of glutaraldehyde crosslinking modification of biological valve material, no other substances that can participate in the crosslinking reaction are added, which can better protect the original fiber structure of the biological material. While effectively ensuring the mechanical properties of the membrane, the orientation direction of the original fibers of the biological material can be guaranteed, avoiding the problem that the addition of double-bonded functional monomers during crosslinking in the previous study may destroy the original fiber orientation of the biological material and increase the fiber disorder.

[0042] (6) Based on the chemical grafting of the first carbon-carbon double bond, the second carbon-carbon double bond is further introduced through physical infiltration. More carbon-carbon double bonds provide more cross-linking basis for secondary cross-linking, which can further improve the cross-linking degree of biological valve material and improve the mechanical properties of biological valve material.

[0043] (7) This application introduces double bonds into the glutaraldehyde-crosslinked biovalve material, and further initiates polymerization between the double bonds on the double bonds of the glutaraldehyde-crosslinked biovalve material and the double bonds on the functional monomers to introduce a functional polymer crosslinking network. Since the functional polymer crosslinking network also has functional groups, the introduction of the functional polymer crosslinking network not only realizes the re-crosslinking of the biovalve material, but also realizes the functionalization of the biovalve material. The crosslinked biovalve material after functionalized copolymerization has functional groups and exhibits the properties corresponding to the functional groups. The functional groups include hydroxyl, carboxyl, carboxylic acid choline, sulfonic acid choline, phosphoric acid choline, pyrrolidone, sulfonic acid group, carboxylate ion, sulfonate, sulfoxide, amide group, and methoxy group, which can bind water molecules through hydrogen bonds and ion hydration. This further enhances the hydrophilicity of the surface of the biovalve material, forming a certain hydration layer in the body to resist excessive adhesion of proteins and cells, and improves antithrombotic performance and biocompatibility. Attached Figure Description

[0044] Figure 1 This is a process flow diagram of the double bond post-functionalized copolymer crosslinking implementation method of this application; Figure 2 This is a schematic diagram illustrating the reaction principle of the double bond post-functionalization copolymer crosslinking implementation scheme of this application; Figure 3 Scanning electron micrograph of blood adhesion in control group 1 (glutaraldehyde-crosslinked porcine pericardium); Figure 4This is a scanning electron microscope image of blood adhesion in sample 1 of Example 1; Figure 5 This is a scanning electron microscope image of blood adhesion in sample 2 of Example 2; Figure 6 This is a scanning electron microscope image of blood adhesion in sample 7 of Example 7; Figure 7 Alizarin Red staining results of control group 1 (glutaraldehyde-crosslinked porcine pericardium) 30 days after subcutaneous implantation in rats; Figure 8 This is an image showing the results of Alizarin Red staining 30 days after Sample 1 was subcutaneously implanted in rats in Example 1. Figure 9 This is an image showing the results of Alizarin Red staining 30 days after Sample 2 was implanted subcutaneously in rats in Example 2. Figure 10 This is an image showing the results of Alizarin Red staining of Sample 8 from Example 8 after 30 days of subcutaneous implantation in rats; Figure 11 This is a schematic diagram of the heart valve structure in this application; Figure 12 This is a schematic diagram of the intervention system of this application. Detailed Implementation

[0045] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0047] To improve the function of conventional glutaraldehyde cross-linked membranes, this application, based on glutaraldehyde cross-linking, introduces carbon-carbon double bonds to initiate secondary cross-linking of these bonds, thereby improving the anticoagulant, anti-calcification, and elastic properties of glutaraldehyde-cross-linked bioprosthetic valves. Specifically, it provides a method for preparing a bioprosthetic valve material, comprising: In step S100, the first carbon-carbon double bond is attached to the first carbon-carbon double bond by chemical bonding through the first functional monomer (i.e., double bond-forming agent) using the amino group on the biomaterial, and at least an aldehyde crosslinking agent is present during the reaction process in step S100. In step S200, carbon-carbon double bonds undergo a polymerization reaction under the action of an initiator to obtain a biological valve material.

[0048] The first carbon-carbon double bond introduced through chemical bonding undergoes a polymerization reaction under the action of an initiator, further forming a cross-linked network, which improves the anticoagulant, anti-calcification, elasticity, and other properties of the glutaraldehyde-based bioprosthetic valve.

[0049] In step S100, the first functional monomer needs to participate in the chemical bonding reaction. The first functional monomer also has an ethylene oxide group as an active group, which participates in the chemical bonding.

[0050] In this application, the use of amino groups on biomaterials is understood as at least a portion of the amino groups on biomaterials participating in the chemical reaction that introduces the first carbon-carbon double bond. In actual operation, step S100 may include multiple sub-steps. The raw materials involved in the reaction system of step S100 participate in at least one of the sub-steps, but are not strictly limited to participating in the reactions of all sub-steps.

[0051] Currently, almost all bioprosthetic valve products used clinically are made from bioprosthetic valve materials cross-linked with glutaraldehyde. Glutaraldehyde reacts with the collagen matrix in bioprosthetic valve materials to cross-link the collagen, further reducing the immunogenicity of the bioprosthetic valve material and increasing its mechanical strength. However, even after glutaraldehyde cross-linking, bioprosthetic valve materials still suffer from insufficient cross-linking and face the risk of structural degradation. This directly leads to component degradation after implantation, disrupting structural integrity and causing structural deterioration and failure. Furthermore, the degradation of bioprosthetic valve components further promotes mechanical damage to the valve leaflet structure and induces calcification, affecting normal valve opening and closing movements and reducing the lifespan of the bioprosthetic valve as a result of structural degradation. Although bioprosthetic valves have lower thrombogenicity compared to mechanical valves, thrombosis still exists, which can impair normal valve function and lead to the risk of secondary valve replacement. On the other hand, calcification directly leads to the failure of the bioprosthetic valve.

[0052] Therefore, the cross-linking degree, stability, antithrombotic properties, and anti-calcification performance of bioprosthetic valves still need improvement. Currently, bioprosthetic valves prepared by glutaraldehyde cross-linking are still the most commonly used bioprosthetic valves in clinical practice. However, given that glutaraldehyde-crosslinked bioprosthetic heart valves still have problems such as instability, low cross-linking degree, thrombosis, and calcification, as well as the risks of structural degradation and failure caused by these problems, a series of functional post-cross-linking processes on glutaraldehyde-crosslinked bioprosthetic heart valves not only meet the practical needs of real-world production but also have high scientific value.

[0053] This application describes a method for crosslinking biological heart valve materials based on glutaraldehyde. By treating the glutaraldehyde-crosslinked biovalve material with double bonds, a carbon-carbon double bond is introduced as a platform for functionalized copolymer crosslinking. This is achieved by initiating a copolymerization reaction between the double bonds in the double-bonded glutaraldehyde-crosslinked biovalve material and the double bonds of the functional monomers, introducing a functional polymer crosslinking network into the glutaraldehyde-crosslinked biovalve material. This further expands the crosslinking network, achieving post-functionalized copolymer crosslinking of the biovalve material after double bond formation. Specifically, based on the second method, the second functional monomer also contains a functional group B. This will increase the degree of crosslinking of the glutaraldehyde-crosslinked biovalve material membrane and enhance its structural stability. The introduction of the functional polymer crosslinking network functionalizes the biovalve material, further improving its anti-calcification, anti-thrombotic, and biocompatibility properties. The introduction of the functional polymer crosslinking network increases the degree of crosslinking of the biovalve material and fills the gaps between collagen matrices to inhibit collagen fiber deformation, further hardening the biovalve material while improving its elasticity.

[0054] Specifically, including (see Figure 1 ): Step S110: Immerse the bioprosthetic valve material in an aldehyde-based crosslinking agent solution for crosslinking; prepare glutaraldehyde-crosslinked bioprosthetic valve material; In step S120, the glutaraldehyde-crosslinked biovalve material prepared in step S110 is immersed in a solution of a double-bonding reagent (first functional monomer) for double-bonding modification to prepare a double-bonded biovalve material; the double-bonding reagent (first functional monomer) has at least one first carbon-carbon double bond and an ethylene oxide group.

[0055] Step S130: The double-bonded bio-valve material obtained in step S120 is soaked in a second functional monomer solution, wherein the second functional monomer has at least one second carbon-carbon double bond and at least one functional group B. In step S200, an initiator is added to the solution after soaking in step S130, so that it comes into contact with the bio-valve material and the functional monomer solution to initiate double bond polymerization.

[0056] In this application, the biomaterial first undergoes a cross-linking reaction with an aldehyde cross-linking agent (step S110), then reacts with the active group of the first functional monomer to introduce a first carbon-carbon double bond (step S120), and then introduces a second carbon-carbon double bond through physical infiltration of a second functional monomer (step S130). During the preparation process, an aldehyde cross-linking agent is first added, which reacts with some of the amino groups in the biomaterial. Then, the first functional monomer is added, utilizing the remaining amino groups and other groups (e.g., hydroxyl and carboxyl groups) on the biomaterial to directly introduce the first carbon-carbon double bond through reaction with the active groups on the first functional monomer. In this scheme, the active group of the first functional monomer is ethylene oxide. Besides the remaining amino groups participating in the reaction, the hydroxyl and carboxyl groups on the biomaterial can also react with the ethylene oxide group to participate in the chemical reaction. Based on the introduction of the first carbon-carbon double bond through chemical reaction, a second carbon-carbon double bond is introduced again through physical infiltration of the second functional monomer. The second functional monomer introduces a functional group B simultaneously with the introduction of the second carbon-carbon double bond. Finally, the first carbon-carbon double bond introduced through the chemical reaction undergoes a polymerization reaction under the action of an initiator. The reaction principle of this application: In this double-bond crosslinking scheme, after the bio-valve material is crosslinked with glutaraldehyde, a first carbon-carbon double bond is introduced by using a double-bonding reagent (first functional monomer) solution. The double-bonding of the bio-valve material with glutaraldehyde is used as a platform for secondary crosslinking. The double-bonding reagent (first functional monomer) used has both carbon-carbon double bonds and ethylene oxide, and the second functional monomer has a second carbon-carbon double bond and a functional group B.

[0057] To facilitate understanding of the chemical principles involved in this scheme, as follows: Figure 2 To further illustrate, the following example illustrates the modification of glutaraldehyde-crosslinked biovalve materials using a double-bonding reagent (first functional monomer). This involves a ring-opening reaction between the ethylene oxide in the double-bonding reagent (first functional monomer) and the hydroxyl, carboxyl, and residual amino groups on the glutaraldehyde-crosslinked biovalve material, thereby introducing carbon-carbon double bonds into the glutaraldehyde-crosslinked biovalve material. Further, a second functional monomer introduces carbon-carbon double bonds and functional group B through physical permeation. Even further, polymerization is initiated between the double bonds on the double-bonded glutaraldehyde-crosslinked biovalve material and the double bonds on the functional monomer, introducing a functional polymer crosslinking network and achieving further secondary crosslinking. This completes the post-functionalization copolymerization crosslinking treatment of the biovalve material.

[0058] Because more functional groups (hydroxyl and carboxyl groups other than amino groups) on bioprosthetic valve materials are used for crosslinking, and the crosslinking network of bioprosthetic valve materials is further expanded by introducing polymers of functional monomers as crosslinking networks through copolymerization with functional monomers, the degree of crosslinking of bioprosthetic valve materials treated with double bond functionalized copolymer crosslinking will be significantly improved, and their structural stability and anti-calcification performance will also be significantly improved with the introduction of functional polymer crosslinking networks.

[0059] After crosslinking with glutaraldehyde, carbon-carbon double bonds are introduced. These carbon-carbon double bonds are mainly attached to the surface of the bio-valve material. During the process of crosslinking and modifying the bio-valve material with glutaraldehyde, no other substances that can participate in the crosslinking reaction are added, which can better protect the original fiber structure of the biomaterial. This can effectively ensure the mechanical properties of the membrane while maintaining the orientation of the original fibers of the biomaterial. It avoids the problem that the direct addition of double-bonded functional monomers during crosslinking in previous studies may have damaged the original fiber orientation of the biomaterial and increased fiber disorder.

[0060] Based on the chemical grafting of the first carbon-carbon double bond, a second carbon-carbon double bond is further introduced through physical infiltration. More carbon-carbon double bonds provide more cross-linking basis for secondary cross-linking, which can further improve the cross-linking degree of biological valve materials and improve the mechanical properties of biological valve materials.

[0061] Furthermore, the second functional monomer also carries functional groups, making the bioprosthetic valve material rich in functional groups, thereby endowing the bioprosthetic valve material with the properties corresponding to the functional groups; the functional group B can be selected from hydroxyl, carboxyl, carboxylic acid choline, sulfonic acid choline, phosphoric acid choline, pyrrolidone, sulfonic acid group, carboxylate ion, sulfonate, sulfoxide, amide group, methoxy group. These groups can bind water molecules with water molecules through hydrogen bonds and ion hydration, which further enhances the hydrophilicity of the surface of the bioprosthetic valve material, forming a certain hydration layer on the bioprosthetic valve to resist excessive adhesion of proteins and cells in the body, and improve antithrombotic performance and biocompatibility.

[0062] Regarding the introduced functional group B: Hydroxyl group: As a hydrophilic group, it enhances the surface hydrophilicity of biomaterials to achieve an anticoagulant effect; Carboxyl group: As a hydrophilic group, it enhances the surface hydrophilicity of biomaterials to achieve an anticoagulant effect; Carboxylate ions and sulfonic acid groups: enhance the surface hydrophilicity of biomaterials through ion hydration to achieve anticoagulant effects; Sulfoxides and pyrrolidones: As hydrophilic groups, they enhance the surface hydrophilicity of biomaterials to achieve anticoagulant effects; Amphoteric ions: enhance the surface hydrophilicity of biomaterials through ion hydration to achieve anticoagulant effects; facilitate the formation of electrically neutral surfaces on bio-valve membranes, thereby reducing the adsorption of calcium ions and achieving anti-calcification effects; Polyethylene glycol: As a hydrophilic group, it enhances the surface hydrophilicity of biomaterials; it increases the steric hindrance between calcium ions and collagen, thus enhancing the surface hydrophilicity of bioprosthetic valve materials. Carbamate group: As a hydrophilic group, it enhances the surface hydrophilicity of biomaterials to achieve an anticoagulant effect.

[0063] Amides: As a hydrophilic group, they enhance the surface hydrophilicity of biomaterials to achieve anticoagulant effects; as a toughening group, they can dynamically adjust the elasticity of biomaterials to improve their utilization rate, and the valves prepared with them have excellent hydrodynamic properties.

[0064] Optionally, in step S120 of this application, non-condensation chemical bonding is used to connect the first carbon-carbon double bond.

[0065] Optionally, in step S110, the biomaterial has not undergone any chemical reaction involving any other reagents before being treated with the aldehyde crosslinking agent.

[0066] Further optionally, the first carbon-carbon double bond is provided in the reaction system of step S120 by a first functional monomer with an active group, and the reaction raw materials in steps S110 and S120 include only the biomaterial, the first functional monomer and the aldehyde crosslinking agent.

[0067] In step S110: The crosslinking agent used in this application is an aldehyde-based crosslinking agent used in current mainstream crosslinking methods. Optionally, the aldehyde-based crosslinking agent can be selected from glutaraldehyde or formaldehyde.

[0068] The concentration of the glutaraldehyde solution can be 0.1% to 5% (w / w); the crosslinking time can be any time between 0.5h and 120h.

[0069] The biomaterials used in this application are conventional biomaterials used in existing glutaraldehyde crosslinking processes, and the collagen content of the biomaterials is 60% to 90%. Further, the biomaterials are animal tissues, derived from pigs, cattle, horses, or sheep, including one or more of the following: pericardium, valves, intestinal membranes, meninges, pulmonary membranes, blood vessels, skin, and ligaments.

[0070] Optionally, the animal tissue may be fresh animal tissue or decellularized biological tissue.

[0071] Optionally, in the decellularization step, the biological tissue is treated with a surfactant as follows: Decellularization of biological tissues using ionic surfactants; or Nonionic surfactants are used to decellularize biological tissues.

[0072] The ionic surfactants are mainly used to lyse cells, while the nonionic surfactants are mainly used to remove lipids (such as phospholipids).

[0073] Optionally, the ionic surfactant is at least one of sodium deoxycholate, potassium fatty acid soap, sodium dodecyl sulfate, sodium cholate, hexadecyltrimethylammonium bromide, potassium fatty acid salt, and alkyl dimethyl sulfopropyl betaine.

[0074] Optionally, the nonionic surfactant is at least one of Triton and Tween.

[0075] In step S120: Optionally, the double-bonding agent, i.e. the first functional monomer, is selected from at least one of allyl glycidyl ether, glycidyl methacrylate, and glycidyl acrylate.

[0076] Optionally, the concentration of the double-bonding reagent in the solution containing the first functional monomer, i.e., the double-bonding reagent, is 1% to 10% (w / w); the reaction time for double-bonding modification is 2 to 120 hours.

[0077] Optionally, the solvent in the solution containing the first functional monomer, i.e. the double-bonding reagent, is one or more of the following: water, physiological saline, pH neutral buffer, or aqueous solutions of methanol, ethanol, ethylene glycol, propanol, 1,2-propanediol, 1,3-propanediol, isopropanol, butanol, isobutanol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, and glycerol.

[0078] Optionally, the biofilm material treated in step S110 can be removed, cleaned, or placed directly in a solution containing a double-bonding reagent (first functional monomer).

[0079] In step S130: The bioprosthetic valve material processed in step S120 is directly or after cleaning immersed in the second functional monomer solution.

[0080] Optionally, the second functional monomer has at least one second carbon-carbon double bond and at least one functional group B.

[0081] Optionally, the second functional monomer is one or more of acrylamide, acrylic acid, sodium acrylate, methacrylic acid, sodium methacrylate, 2-(propionic-2-enoylamino)acetic acid, 2-acrylamido-2-methylpropanesulfonic acid, hydroxyethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, N-methyl-2-acrylamide, N-isopropylacrylamide, N-(hydroxymethyl)acrylamide, N-(2-hydroxyethyl)methacrylamide, 3-[N,N-dimethyl-[2-(2-methylpropionic-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, 2-methacryloyloxyethyl phosphocholine, N-(2-hydroxyethyl)acrylamide, N-(methoxymethyl)methacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, and double-bonded hyaluronic acid (which can be prepared by the method described above).

[0082] Optionally, the concentration of the second functional monomer solution is 0.1% to 6% (v / v).

[0083] Optionally, the solvent for the second functional monomer solution is one or a mixture of several of the following: water, physiological saline, ethanol, isopropanol, or a pH-neutral buffer solution.

[0084] Optionally, the soaking time in the second functional monomer solution is 0.5h-120h.

[0085] In step S200: After the biological valve material is treated in step S120, it is washed with deionized water and then immersed in the initiator solution for the treatment in step S200, or the initiator is directly added to the reaction system of step S120 to initiate the polymerization reaction, the latter being commonly known as the one-pot method.

[0086] Optionally, the solvent in the initiator-containing solution is water, physiological saline, or a pH neutral buffer.

[0087] As mentioned above, the concentration of the initiator in the one-pot process can be understood as the concentration of the initiator in the solution contained in the reaction system in step S120, and in the stepwise process, the concentration can be understood as the concentration in the solution containing the initiator.

[0088] Optionally, the initiator is a mixture of ammonium persulfate and sodium bisulfite, or a mixture of ammonium persulfate and sodium sulfite, or a mixture of sodium persulfate and sodium sulfite, or a mixture of potassium persulfate and sodium sulfite, or a mixture of sodium persulfate and sodium bisulfite, or a mixture of potassium persulfate and sodium bisulfite, or potassium persulfate and tetramethylethylenediamine, or ammonium persulfate and tetramethylethylenediamine, or sodium persulfate and tetramethylethylenediamine; the concentration of each component in the mixture is 1~100 mM.

[0089] The reaction time for step S200 is 3 to 24 hours.

[0090] In this application, all reaction processes in S100 and S200 can be carried out at 0~50°C unless otherwise specified. Preferably, the temperature does not need to be specially controlled and room temperature is acceptable, but it is preferable to keep it below the temperature that the human body can adapt to, and preferably at 36~37°C.

[0091] In this application, all reactions in S100 and S200 can be either static or dynamic unless otherwise specified. Dynamic reactions can be carried out under the action of a device that can circulate the solution, such as a peristaltic pump, or by shaking at a speed of 10 rpm to 150 rpm. The peristaltic circulation or shaking can be carried out continuously or intermittently.

[0092] Optionally, this application also includes dehydration and drying treatment after double bond polymerization to prepare a dry membrane. After double bond polymerization, the bioprosthetic valve material is routinely cleaned and softened before dehydration and drying.

[0093] The cleaning solution can be one or a mixture of several of the following: water, physiological saline, ethanol, isopropanol, or a pH-neutral buffer solution. The pH can be adjusted to between 5.0 and 9.5 before and during use, or it can be left unadjusted.

[0094] Optionally, the dehydration process involves exposing the double-bonded polymerized membrane or the valve formed by sewing the membrane to a dehydration solution.

[0095] Optionally, the dehydration solution is a mixture of an alcohol solution and water, with the alcohol solution accounting for 20-90% (v / v). The alcohol reagent can be one or a mixture of two of ethanol and isopropanol.

[0096] Optionally, the drying process involves exposing the dehydrated membrane or valve to a fabric softener solution for 20 minutes to 10 hours.

[0097] Optionally, the main component of the fabric softener solution is a mixture of one or two of glycerin and polyethylene glycol, with a glycerin concentration of 10-100% (v / v), and other components are one or more of water, ethanol, and isopropanol, accounting for 0-90% (v / v).

[0098] Optionally, the valves after drying can be sterilized using either ethylene oxide sterilization or electron beam sterilization.

[0099] The bioprosthetic valve material prepared by the above method can be used for interventional bioprosthetic valves, such as through minimally invasive intervention; it can also be used for surgical bioprosthetic valves, such as through surgical implantation.

[0100] like Figure 11As shown, in one embodiment, an artificial heart valve is provided, including a stent 1 and leaflets 2 connected within the stent 1. The stent is cylindrical in shape, with a hollowed-out mesh structure on the sidewalls. The stent has a blood flow channel inside, and multiple leaflets cooperate to control the opening and closing of the blood flow channel within the stent.

[0101] Depending on the release mode, the stent is made of different materials, such as nickel-titanium alloy with shape memory that can expand in the body, or stainless steel material that can be released by ball expansion. The stent itself can be formed by cutting tubes or weaving wires, and the leaflets can be connected to the stent by stitching, bonding or integral molding.

[0102] To facilitate positioning within the body, positioning structures that can interact with surrounding native tissues can be provided on the periphery of the stent, such as anchors, arms, etc. To prevent peritoneal leakage, skirts or anti-leakage materials can be provided on the inner and / or outer sides of the stent. The leaflets, skirts, or anti-leakage materials can all be the bioprosthetic valve materials described in the above embodiments.

[0103] like Figure 12 When catheter intervention is used, the artificial heart valve 3 and the corresponding delivery system form a valve intervention system. The delivery system includes a catheter assembly 4 and a handle for controlling the catheter assembly. The artificial heart valve is in a radially compressed state during delivery in the body. The catheter assembly is released from its restraints or undergoes bulbodilation and radial expansion for release in the body.

[0104] The following specific embodiments provide further details: Compare with Example 1 During the treatment process, a simple glutaraldehyde cross-linking group was set as the control group. Glutaraldehyde cross-linked pig pericardium was prepared by immersing pig pericardium in 0.25% (w / w) glutaraldehyde for 72 hours at room temperature, and was recorded as control sample 1.

[0105] Example 1 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0106] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in a 5% (v / v) aqueous solution of glycidyl methacrylate in isopropanol for double bond modification at room temperature for 48 hours. The solvent used for the double bond modification solution was a 20% (v / v) aqueous solution of isopropanol.

[0107] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in a 3% (w / v) 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt solution for 2 hours. An initiator was added to the above solution, wherein the concentration of ammonium persulfate was 20 mM and the concentration of sodium bisulfite was 10 mM, to further initiate the polymerization between the double bonds on the double-bonded glutaraldehyde crosslinked biological valve material and the double bonds on the inner salt of 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid. After reacting at 37°C for 8 hours, a crosslinked porcine pericardium after double bond copolymerization was obtained, which was designated as sample 1.

[0108] Example 2 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0109] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in a 6% (v / v) propanol aqueous solution of glycidyl acrylate at room temperature for double bond modification. The reaction time was 72 hours, and the solvent used for the double bond modification solution was a 20% (v / v) propanol aqueous solution.

[0110] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in 5% (w / v) 2-methacryloyloxyethyl phosphocholine solution for 1 hour. An initiator was added to the above solution, wherein the concentration of potassium persulfate was 20 mM and the concentration of sodium sulfite was 10 mM, to further initiate the polymerization between the double bonds on the double bonds of the glutaraldehyde crosslinked biological valve material and the double bonds on 2-methacryloyloxyethyl phosphocholine. After reacting at 37°C for 8 hours, a crosslinked porcine pericardium after double bond copolymerization was obtained, which was designated as sample 2.

[0111] Example 3 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0112] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in an isopropanol aqueous solution containing 2% (v / v) glycidyl acrylate and 4% (v / v) allyl glycidyl ether at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 72 hours, and the solvent used for the double bond modification solution was a 30% (v / v) aqueous ethanol solution.

[0113] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in 5% (v / v) acrylamide solution for 3 hours. An initiator was added to the above solution, wherein the concentration of potassium persulfate was 20 mM and the concentration of sodium sulfite was 10 mM, to further initiate the polymerization between the double bonds on the double bonds of the glutaraldehyde crosslinked biological valve material and the double bonds on the acrylamide. After reacting at 37°C for 8 hours, a crosslinked porcine pericardium after double bond copolymerization was obtained, which was designated as sample 3.

[0114] Example 4 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in a 1.0% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde-crosslinked pig pericardium.

[0115] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in an aqueous solution of 3% (v / v) glycidyl methacrylate and 2% (v / v) glycidyl acrylate in isopropanol for double bond modification at room temperature for 48 hours. The solvent used for the double bond modification solution was 25% (v / v) isopropanol aqueous solution.

[0116] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in a solution containing 1% (v / v) acrylamide and 1.5% (v / v) N-isopropylacrylamide for 1 hour. An initiator was added to the above solution, wherein the concentration of ammonium persulfate was 20 mM and the concentration of sodium bisulfite was 10 mM, to further initiate the polymerization between the double bonds on the double-bonded glutaraldehyde crosslinked biological valve material and the double bonds on acrylamide and N-isopropylacrylamide. After reacting at 37°C for 7 hours, a crosslinked porcine pericardium after double bond copolymerization was obtained, which was designated as sample 4.

[0117] Example 5 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0118] Glutaraldehyde-crosslinked porcine pericardium was washed with deionized water and then immersed in a 4% (v / v) aqueous solution of glycidyl methacrylate in ethanol at room temperature for double bond modification. The reaction time was 72 hours, and the solvent used for the double bond modification solution was a 20% (v / v) aqueous solution of ethanol.

[0119] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in 1.5% (v / v) N-isopropylacrylamide solution for 1 hour. An initiator was added to the above solution, wherein the concentration of sodium persulfate was 20 mM and the concentration of sodium bisulfite was 7 mM, to further initiate the polymerization between the double bonds on the double bonds of the glutaraldehyde crosslinked biological valve material and the double bonds on N-isopropylacrylamide. After reacting at 37°C for 7 hours, a crosslinked porcine pericardium after double bond copolymerization was obtained, which was designated as sample 5.

[0120] Example 6 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0121] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in a 4% (v / v) aqueous solution of glycidyl methacrylate in isobutanol for double bond modification at room temperature for 72 hours. The solvent used for the double bond modification solution was a 15% (v / v) aqueous solution of isobutanol.

[0122] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in a 2.0% (w / v) sodium acrylate solution for 5 hours. An initiator was added to the above solution, wherein the concentration of ammonium persulfate was 20 mM and the concentration of sodium bisulfite was 10 mM, to further initiate the polymerization between the double bonds on the double bonds of the glutaraldehyde crosslinked biological valve material and the double bonds on the sodium acrylate. After reacting at 37°C for 12 hours, a pig pericardium with double bond copolymerization and crosslinking was obtained, which was designated as sample 6.

[0123] Example 7 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0124] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in a 4% (v / v) aqueous solution of glycidyl acrylate in isopropanol for double bond modification at room temperature for 48 hours. The solvent used for the double bond modification solution was a 20% (v / v) aqueous solution of methanol.

[0125] After the double bond modification was completed, the double-bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double-bonded glutaraldehyde cross-linked porcine pericardium was immersed in a solution containing 1.0% (v / v) hydroxyethyl methacrylate and 0.5% (w / v) 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt for 1 hour. An initiator was added to the above solution, wherein the concentration of ammonium persulfate was 20 mM and the concentration of sodium bisulfite was 10 mM, to further initiate the polymerization between the double bonds on the double-bonded glutaraldehyde crosslinked biovalve material and the double bonds on hydroxyethyl methacrylate and 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt. After reacting at 37°C for 8 hours, a crosslinked porcine pericardium after double bond copolymerization was obtained, which was designated as sample 7.

[0126] Example 8 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0127] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in a 5% (v / v) aqueous solution of glycidyl methacrylate in ethylene glycol at room temperature for double bond modification. The reaction time was 72 hours, and the solvent used for the double bond modification solution was a 25% (v / v) aqueous solution of ethylene glycol.

[0128] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in 5% (v / v) N-(hydroxymethyl)acrylamide solution for 5 hours. An initiator was added to the above solution, wherein the concentration of ammonium persulfate was 20 mM and the concentration of sodium bisulfite was 10 mM, to further initiate the polymerization between the double bonds on the double bonds of the glutaraldehyde crosslinked bio-valve material and the double bonds on N-(hydroxymethyl)acrylamide. After reacting at 37°C for 12 hours, a pig pericardium with double bond copolymerization and crosslinking was obtained, which was designated as sample 8.

[0129] Example 9 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0130] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in a 7% (v / v) propanol aqueous solution of glycidyl acrylate at room temperature for double bond modification. The reaction time was 60 hours, and the solvent used for the double bond modification solution was a 30% (v / v) propanol aqueous solution.

[0131] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in 1.0% (v / v) N-(methoxymethyl)methacrylamide solution for 5 hours. An initiator was added to the above solution, wherein the concentration of ammonium persulfate was 20 mM and the concentration of tetramethylethylenediamine was 1 mM, to further initiate the polymerization between the double bonds on the double bonded glutaraldehyde crosslinked biological valve material and the double bonds on N-(methoxymethyl)methacrylamide. After reacting at 37°C for 12 hours, a pig pericardium with double bond copolymerization and crosslinking was obtained, which was designated as sample 9.

[0132] Example 10 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0133] After washing with deionized water, glutaraldehyde-crosslinked porcine pericardium was immersed in an isopropanol aqueous solution containing 4% (v / v) glycidyl methacrylate and 2% (v / v) glycidyl acrylate at room temperature for double bond modification of glutaraldehyde-crosslinked porcine pericardium. The reaction time was 84 hours, and the solvent used for the double bond modification solution was 25% (v / v) aqueous ethanol solution.

[0134] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in a solution of 1% (v / v) acrylamide and 1.50% (w / v) 2-methacryloyloxyethyl phosphocholine for 3 hours. An initiator was added to the above solution, wherein the concentration of ammonium persulfate was 20 mM and the concentration of sodium bisulfite was 10 mM, to further initiate the polymerization between the double bonds on the double-bonded glutaraldehyde crosslinked biological valve material and the double bonds on acrylamide and 2-methacryloyloxyethyl phosphocholine. After reacting at 37°C for 7 hours, a crosslinked porcine pericardium after double bond copolymerization was obtained, which was designated as sample 10.

[0135] Performance characterization was performed on samples from Examples 1 to 10 and Control Example 1: To characterize the change in crosslinking degree of glutaraldehyde-crosslinked biovalve materials before and after double bond copolymerization crosslinking treatment, the thermal stability and crosslinking degree of the biovalve materials were characterized by measuring the heat shrinkage temperature; the stability of the biovalve materials was characterized by enzyme degradation experiments; the degree of calcification (anti-calcification performance) of the samples was characterized by subcutaneous implantation experiments in rats; the elasticity of the biovalve materials was characterized by testing the elastic angle; the hydrophilicity of the biovalve materials was characterized by water contact angle testing; and the antithrombotic performance of the materials was characterized by blood adhesion experiments.

[0136] Heat shrinkage temperature measurement Biological valve material was cut into circular sheets with a diameter of 0.6 cm, dried, and placed in a crucible. The material was then heated at a rate of 10 °C / min using a differential scanning calorimeter to measure its heat shrinkage temperature within the range of 40–120 °C. The heat shrinkage temperature was used to characterize the thermal stability and degree of cross-linking of the biological valve material; a higher heat shrinkage temperature corresponds to higher thermal stability and a higher degree of cross-linking.

[0137] Table 1 Thermal shrinkage temperature of each group of bioprosthetic valve materials

[0138] The heat shrinkage temperature of samples 1, 3, 6, 8, and control group 1 (glutaraldehyde-crosslinked porcine pericardium) was measured. As shown in Table 1, the heat shrinkage temperatures of samples 1, 3, 6, and 8 were all higher than those of control group 1 (glutaraldehyde-crosslinked porcine pericardium). This indicates that the thermal stability and crosslinking degree of samples 1, 3, 6, and 8 were all higher than those of the control group (glutaraldehyde-crosslinked porcine pericardium). The heat shrinkage temperature measurement results demonstrate that the method for preparing biological valve materials using post-functionalized copolymer crosslinking of double bonds described in this application can improve the thermal stability and crosslinking degree of biological valves.

[0139] Water contact angle test Cut the bioprosthetic valve material into 1 1cm 2 The sheet material was frozen at 80°C overnight and then transferred to a freeze dryer for 48 hours. The sheet material was then removed and placed on a water contact angle tester to measure the water contact angle of different materials to characterize the hydrophilicity of the materials. The smaller the water contact angle, the more hydrophilic the biological valve material.

[0140] Table 2 Water contact angles of bioprosthetic valve materials for each group

[0141] The results of the water contact angle test are shown in Table 2. Compared with control group 1 (glutaraldehyde crosslinked porcine pericardium), the water contact angles of samples 1, 2, 7, and 10 all decreased significantly. That is, samples 1, 2, 7, and 10 are more hydrophilic than control group 1 (glutaraldehyde crosslinked porcine pericardium). This indicates that the method of preparing biological valve materials by double bond post-functionalization copolymerization crosslinking can improve the hydrophilicity of biological valves.

[0142] Blood Adhesion Experiment The bioprosthetic valve material was cut into circular sheets with a diameter of 1 cm and transferred to a 48-well plate. 0.5 mL of fresh rabbit blood was then added to the surface of the material to ensure full contact with the blood for a blood adhesion test. After 1.5 hours of contact with the blood, the bioprosthetic valve material was removed from the blood and washed three times with physiological saline. The washed bioprosthetic valve material was then fixed in a 2.5% (w / v) glutaraldehyde solution for 2 hours. After fixation, the bioprosthetic valve material was dehydrated using gradient concentrations (50%, 75%, 90%, and 100%, v / v) of ethanol, followed by gold sputtering. Finally, the material was observed and photographed under a scanning electron microscope to characterize its antithrombotic properties.

[0143] Results analysis: such as Figures 3-6 As shown: After contact with blood, a large number of erythrocytes and platelets were observed to adhere to the control group 1 (glutaraldehyde-crosslinked porcine pericardium). Figure 3 ), while in sample 1 ( Figure 4 Sample 2 Figure 5 Sample 7 Figure 6 Only a small amount of erythrocyte adhesion was observed; the low blood cell adhesion on samples 1, 2, and 7 reduced the interaction between blood and bioprosthetic valve material, which further reduced the possibility of thrombosis on bioprosthetic valve material. That is, samples 1, 2, and 7 have better antithrombotic properties than control group 1 (glutaraldehyde crosslinked porcine pericardium); the blood adhesion experiment shows that the method of preparing bioprosthetic valve material by double bond post-functionalization copolymer crosslinking can improve the antithrombotic properties of bioprosthetic valve.

[0144] Elasticity test experiment The bioprosthetic valve material of uniform thickness was cut into 1... 4.6cm 2 A rectangular sample is held horizontally along the midline of its long side. The angle at which the sample hangs down relative to the horizontal plane of the midline is measured to characterize the sample's elasticity; the smaller the angle, the higher the elasticity.

[0145] Table 3 Elastic angles of bioprosthetic valve materials in each group

[0146] Elasticity tests were conducted on samples 1, 3, 6, 8, and control group 1 (glutaraldehyde-crosslinked porcine pericardium) to characterize their elasticity. The results are shown in Table 3. Compared to control group 1 (glutaraldehyde-crosslinked porcine pericardium), samples 1, 3, 6, and 8 exhibited lower elastic angles, indicating a significant improvement in elasticity. The method of preparing bioprosthetic valve materials through double-bond functionalization copolymerization and crosslinking can enhance the elasticity of bioprosthetic valves. Increased elasticity in bioprosthetic valve materials facilitates rapid morphological recovery after catheter implantation.

[0147] Enzyme degradation experiment The obtained bioprosthetic valve material was cut into circular sheets with a diameter of 1 cm, with 6-8 parallel samples per group. All circular sheet samples were placed in a 48-well plate and frozen overnight at -80°C, then transferred to a vacuum freeze dryer for 48 hours. The initial weight (W0) of each sample was recorded on a 0.0001 g balance and returned to the 48-well plate. 0.5 mL of collagenase I in PBS solution was added to each well of the 48-well plate, ensuring the bioprosthetic valve sample was completely immersed in the collagenase (100 U / mL) PBS solution. The 48-well plate was incubated at 37°C for 24 hours. After incubation, the bioprosthetic valve material samples were removed, rinsed three times, frozen overnight at -80°C, and then transferred to a vacuum freeze dryer for 48 hours. The final weight (Wt) of each sample after collagenase degradation was recorded on a 0.0001 g balance. The formula for calculating the enzyme degradation weight loss rate is as follows: Enzyme degradation weight loss rate =

[0148] The collagenase degradation weight loss rate of samples 1, 3, 6, 8 and control group 1 was determined, and the results are shown in Table 4.

[0149] Table 4. Enzymatic degradation weight loss rate of bioprosthetic valve materials in each group

[0150] As shown in Table 4, enzymatic degradation experiments were conducted on samples 1, 3, 6, 8, and control group 1 (glutaraldehyde-crosslinked porcine pericardium) to characterize the crosslinking efficiency of each group. The enzymatic degradation weight loss rate of each group was calculated after treating samples 1, 3, 6, 8, and control group 1 (glutaraldehyde-crosslinked porcine pericardium) with collagenase I, as shown in Table 4. The enzymatic degradation weight loss rates of samples 1, 3, 6, and 8 were all lower than those of control group 1 (glutaraldehyde-crosslinked porcine pericardium), indicating that the stability of samples 1, 3, 6, and 8 was higher than that of control group 1 (glutaraldehyde-crosslinked porcine pericardium), meaning that samples 1, 3, 6, and 8 had higher stability. The enzymatic degradation experiment results show that the method for preparing biological valve materials by double bond post-functionalization copolymer crosslinking of this application can improve the stability of biological valves.

[0151] Anti-calcification test Cut the bioprosthetic valve material into 1 1cm 2 Sterilized tissue samples were implanted subcutaneously into rats and removed 30 days later. Each sample was divided into two parts: one part was decapsulated, freeze-dried, weighed, digested with 6M hydrochloric acid, and the calcium content per gram was determined; the other part was fixed with paraformaldehyde tissue fixative. After fixation, the samples were removed, trimmed and smoothed with a scalpel, and transferred to a dehydration box. The samples were dehydrated using graded ethanol. After dehydration, the samples were transferred to an embedding machine and embedded in molten paraffin, then transferred to a -20°C freezer for cooling and shaping. 5μm thick sections were cut from the trimmed paraffin blocks using a microtome, transferred from a slide to a glass slide, and dewaxed and rehydrated. The sections were stained with alizarin red for 3 minutes, washed with water, dried, and cleared with xylene for 5 minutes. The sections were mounted with neutral resin, and the staining results were imaged using a pathological slide scanner.

[0152] The bioprosthetic valve material was cut into 1 piece for samples 1, 2, 8 and control group 1 (glutaraldehyde cross-linked porcine pericardium). 1cm 2 The sheet material was subjected to an anti-calcification test.

[0153] Table 5. Calcium content of bioprosthetic valve materials in each group 30 days after subcutaneous implantation in rats.

[0154] The degree of calcification in each group of samples (samples 1, 2, and 8) and control group 1 (glutaraldehyde-crosslinked porcine pericardium) was characterized by detecting calcium content 30 days after subcutaneous implantation in rats. As shown in Table 5, the calcium content of samples 1, 2, and 8 was lower than that of the control group (glutaraldehyde-crosslinked porcine pericardium) 30 days after subcutaneous implantation in rats. This result indicates the anti-calcification performance of the method for preparing functionalized bio-valve materials by double bond post-functionalization copolymer crosslinking proposed in this application.

[0155] Alizarin red staining was used to directly observe the degree of calcification in control group 1 (glutaraldehyde-crosslinked porcine pericardium), sample 1, sample 2, and sample 8 after subcutaneous implantation in rats for 30 days. Images of alizarin red staining results for sample sections implanted subcutaneously in rats 30 days later are shown below. Figure 7-10 As shown, the darker the color of the sample after alizarin red staining, the higher the degree of calcification. This is in contrast to the alizarin red staining results of control group 1 (glutaraldehyde-crosslinked porcine pericardium). Figure 7 Example 1 Figure 8 Example 2 Figure 9 Example 8 Figure 10The alizarin red staining images of the sections showed a significant decrease in color, directly indicating that the degree of calcification in samples 1, 2, and 8 was lower than that in control group 1. In other words, samples 1, 2, and 8 exhibited a stronger anti-calcification effect compared to the control group. Alizarin red staining results of bioprosthetic valve materials implanted subcutaneously in rats 30 days later showed that the method for preparing functionalized bioprosthetic valve materials using double-bond post-functionalization copolymer crosslinking proposed in this application can improve the anti-calcification performance of bioprosthetic valves.

[0156] Example 11 Freshly harvested pig pericardium was soaked in physiological saline and shaken for 2 hours. Then, the pig pericardium was soaked in 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde cross-linked pig pericardium.

[0157] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in a 5% (v / v) aqueous solution of glycidyl methacrylate in ethylene glycol at room temperature for double bond modification. The reaction time was 72 hours, and the solvent used for the double bond modification solution was a 25% (v / v) aqueous solution of ethylene glycol.

[0158] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in 5% (v / v) N-(hydroxymethyl)acrylamide solution for 5 hours. An initiator was added to the above solution, wherein the concentration of ammonium persulfate was 20 mM and the concentration of sodium bisulfite was 10 mM, to further initiate the polymerization between the double bonds on the double bonds of the glutaraldehyde crosslinked bioprosthetic valve material and the double bonds on N-(hydroxymethyl)acrylamide. After reacting at 37°C for 12 hours, a crosslinked porcine pericardium was obtained after double bond copolymerization. The crosslinked porcine pericardium material was immersed in a 60% isopropanol aqueous solution for 45 min, and then immersed in a solution of 10% glycerol, 3% polyethylene glycol (Mn=200), and 87% ethanol at room temperature for 3 h. Excess glycerol was removed from the surface of the porcine pericardium material, and it was sterilized with ethylene oxide and designated as sample 11.

[0159] Example 12 Fresh porcine pericardium was placed in a PS solution containing 0.5% sodium deoxycholate (surfactant) and shaken at room temperature for 4 hours. Then it was washed three times with a 0.9% sodium chloride aqueous solution (i.e., physiological saline).

[0160] Subsequently, the pig pericardium was immersed in a 0.25% (w / w) glutaraldehyde solution at room temperature for 72 hours to prepare glutaraldehyde-crosslinked pig pericardium.

[0161] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and then immersed in a 5% (v / v) aqueous solution of glycidyl methacrylate in isopropanol for double bond modification at room temperature for 48 hours. The solvent used for the double bond modification solution was a 20% (v / v) aqueous solution of isopropanol.

[0162] After the double bond modification was completed, the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked porcine pericardium was immersed in a 3% (w / v) 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt solution for 2 hours. An initiator was added to the above solution, wherein the concentration of ammonium persulfate was 20 mM and the concentration of sodium bisulfite was 10 mM, to further initiate the polymerization between the double bonds on the double-bonded glutaraldehyde crosslinked biological valve material and the double bonds on the inner salt of 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid. After reacting at 37°C for 8 hours, a pig pericardium with crosslinked double bond copolymerization was obtained, which was designated as sample 12.

[0163] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for preparing biological valve materials through copolymer crosslinking, characterized in that, include: Step S110: The biomaterial is cross-linked by contacting the aldehyde cross-linking agent solution; Step S120: Immerse the biomaterial treated in step S110 in a solution containing a first functional monomer, wherein the first functional monomer has a first carbon-carbon double bond and an ethylene oxide group, and the biomaterial chemically reacts with the ethylene oxide group to attach the first carbon-carbon double bond. Step S130: Immerse the biomaterial treated in step S120 in a solution containing a second functional monomer, wherein the second functional monomer has a second carbon-carbon double bond and a functional group B. In step S200, carbon-carbon double bonds undergo a polymerization reaction under the action of an initiator to obtain a biological valve material.

2. The method according to claim 1, characterized in that, The aldehyde crosslinking agent is glutaraldehyde or formaldehyde.

3. The method according to claim 1, characterized in that, The biological material is animal tissue, which is selected from one or more of the following: pericardium, valves, intestinal membrane, meninges, pulmonary membrane, blood vessels, skin, and ligaments.

4. The method according to claim 3, characterized in that, The animal tissue is either fresh animal tissue or biological tissue that has undergone decellularization.

5. The method according to claim 1, characterized in that, In step S200: the initiator is added to the system treated in the previous step; or the biological material treated in the previous step is taken out and directly or after washing is immersed in a solution containing the initiator.

6. The method according to claim 1, characterized in that, The initiator can be a single initiator or a mixture of initiators.

7. The method according to claim 6, characterized in that, The mixed initiator is: The initiator is a mixture of ammonium persulfate and sodium bisulfite, or a mixture of ammonium persulfate and sodium bisulfite, or a mixture of sodium persulfate and sodium bisulfite, or a mixture of potassium persulfate and sodium bisulfite, or a mixture of potassium persulfate and sodium bisulfite, or a mixture of potassium persulfate and tetramethylethylenediamine, or a mixture of ammonium persulfate and tetramethylethylenediamine, or a mixture of sodium persulfate and tetramethylethylenediamine; the concentration of each component in the mixture is 1~100 mM.

8. The method according to claim 7, characterized in that, The single initiator can be any component of the mixed initiators.

9. The method according to claim 1, characterized in that, In step S200, the polymerization reaction takes 3 to 24 hours.

10. The method according to claim 1, characterized in that, The first functional monomer is selected from at least one of allyl glycidyl ether, glycidyl methacrylate, and glycidyl acrylate.

11. The method according to claim 1, characterized in that, In step S110: the w / w concentration of the aldehyde crosslinking agent solution is 0.1%~5%; the crosslinking time is 0.5h-120h.

12. The method according to claim 1, characterized in that, In step S120: the w / w concentration of the first functional monomer in the solution containing the first functional monomer is 1%~10%; the reaction time is 2~120 hours.

13. The method according to claim 1, characterized in that, The solution containing the first functional monomer contains only the first functional monomer and a solvent that does not participate in the chemical reaction.

14. The method according to claim 1, characterized in that, The solvent in the solution containing the first functional monomer is one or more of the following: an aqueous solution of methanol, ethanol, ethylene glycol, propanol, 1,2-propanediol, 1,3-propanediol, isopropanol, butanol, isobutanol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, glycerol, water, physiological saline, and pH neutral buffer.

15. The method according to claim 1, characterized in that, The second functional monomer is selected from one or more of the following: polyethylene glycol diacrylate, 1,4-butanediol diacrylate, ethane-1,2-diyl diacrylate, ethyl acrylate, N-methyl-2-acrylamide, N-2,2-propenyl-2-acrylamide, N-ethylacrylamide, N,N'-vinylbisacrylamide, (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)diacrylate, N,N'-dimethylacrylamide, N,N-dimethylmethylacrylamide, and double-bonded polylysine.

16. The method according to claim 1, characterized in that, In step S130: the v / v concentration of the second functional monomer in the solution containing the second functional monomer is 0.1%-20%; the soaking time is 0.5h-120h.

17. The method according to claim 1, characterized in that, The v / v concentration of the second functional monomer in the solution containing the second functional monomer is 0.1%-6%.

18. The method according to claim 1, characterized in that, The second functional monomer enters the biomaterial through physical permeation.

19. The method according to claim 1, characterized in that, The solution containing the second functional monomer contains only the second functional monomer and a solvent that does not participate in the reaction.

20. The method according to claim 1, characterized in that, The solvent in the solution containing the second functional monomer is one or a mixture of several of the following: water, physiological saline, ethanol, isopropanol, and pH neutral buffer solution.

21. The method according to claim 1, characterized in that, The functional group B is selected from at least one of hydroxyl, carboxyl, carboxycholine, sulfonic acid choline, phosphoric acid choline, pyrrolidone, sulfonic acid group, carboxylate ion, sulfonate, sulfoxide, amide group, and methoxy group.

22. The method according to claim 1, characterized in that, The second functional monomer is selected from one or more of acrylamide, acrylic acid, sodium acrylate, methacrylic acid, sodium methacrylate, 2-(prop-2-enoylamino)acetic acid, 2-acrylamido-2-methylpropanesulfonic acid, hydroxyethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, N-methyl-2-acrylamide, N-isopropylacrylamide, N-(hydroxymethyl)acrylamide, N-(2-hydroxyethyl)methacrylamide, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, 2-methacryloyloxyethyl phosphocholine, N-(2-hydroxyethyl)acrylamide, N-(methoxymethyl)methacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and double-bonded hyaluronic acid.

23. A biological valve material, characterized in that, It is prepared by the method described in any one of claims 1 to 22.

24. A biological valve material, characterized in that, include: Step S110: The biomaterial is cross-linked by contacting the aldehyde cross-linking agent solution; Step S120: Immerse the biomaterial treated in step S110 in a solution containing a first functional monomer, wherein the first functional monomer has a first carbon-carbon double bond and an ethylene oxide group, and the biomaterial chemically reacts with the ethylene oxide group to attach the first carbon-carbon double bond. Step S130: Immerse the biomaterial treated in step S120 in a solution containing a second functional monomer, wherein the second functional monomer has a second carbon-carbon double bond and a functional group B. In step S200, carbon-carbon double bonds undergo a polymerization reaction under the action of an initiator to obtain a biological valve material.

25. A biological valve, comprising a scaffold and leaflets, characterized in that, The leaflet is the biological valve material as described in claim 23 or 24.

26. The biological valve according to claim 25, characterized in that, The bioprosthetic valve is a heart valve.

27. An interventional system comprising a heart valve and a catheter assembly, wherein the heart valve is delivered by the catheter assembly after folding, characterized in that, The heart valve includes a stent and leaflets, wherein the leaflets are the bioprosthetic valve material as described in claim 23 or 24.

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