Biological valve material based on aldehyde group crosslinking as well as preparation method and application of biological valve material

Abstract

The invention discloses a biological valve material based on aldehyde group crosslinking as well as a preparation method and application thereof. The preparation method comprises the following steps: step S110, contacting a biological material with an aldehyde group crosslinking agent solution for crosslinking; s120, soaking the biological material treated in the step S110 in a solution containing a first functional monomer, and reacting to access a first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and an oxirane group; step S130, soaking the biological material treated in the step S120 in a solution containing a second functional monomer, wherein the second functional monomer has a second carbon-carbon double bond; and step S200, carrying out polymerization reaction on the carbon-carbon double bonds under the action of an initiator to obtain the bioprosthetic valve material. According to the method, more and larger polymer cross-linked networks are formed through two times of cross-linking, the cross-linking degree of the biological material is improved, and the anti-calcification performance is improved.

Description

Aldehyde-crosslinked bioprosthetic valve material and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of interventional materials, and in particular to an aldehyde-crosslinked biological valve material, a preparation method thereof, and applications thereof. Background Art

[0002] Bioprosthetic heart valves are typically made from porcine or bovine pericardium, cross-linked with glutaraldehyde. They are used to replace damaged native heart valves in valvular heart disease. Compared to mechanical heart valves, bioprosthetic heart valves offer a number of advantages: They have superior fluid dynamics and are closer to native heart valves than mechanical valves. They are also less thrombogenic than mechanical valves, eliminating the need for lifelong anticoagulation therapy after implantation. Bioprosthetic heart valves are also compressible and can be implanted via minimally invasive procedures, avoiding open-chest surgery and minimizing the risk of valve replacement. These advantages have led to a year-on-year increase in the clinical application of bioprosthetic heart valves, making them the mainstream prosthetic valve.

[0003] Currently, nearly all bioprosthetic heart valves used clinically are cross-linked with glutaraldehyde. While glutaraldehyde cross-linking can improve the mechanical properties of the pericardium and reduce its immunogenicity to a certain extent, glutaraldehyde-cross-linked bioprosthetic valves still face challenges with stability and low cross-linking. This can lead to structural degradation and destruction after implantation, compromising their structural integrity and causing structural degradation and failure. Therefore, their stability and cross-linking still need to be further improved. Furthermore, degradation of bioprosthetic valve components can further induce mechanical damage to the valve leaflets and accelerate calcification and structural degradation, thereby impacting the normal blood flow properties of the bioprosthetic heart valve and reducing its service life.

[0004] At present, glutaraldehyde-crosslinked biological heart valves are still the mainstream biological heart valves used in clinical practice. In view of the fact that glutaraldehyde-crosslinked biological heart valves still have problems with stability and low crosslinking degree, as well as the risk of structural degradation and failure caused by structural degradation and damage, further research based on glutaraldehyde-crosslinked membranes not only meets actual production needs but also has great significance for scientific research.

[0005] The applicant of this application has long been committed to the research of biological heart valves. For example, in previous research, the Chinese invention patent application document with publication number CN 114748694A disclosed a co-cross-linked biological valve material and its preparation method and application, in which the biological valve material was functionally modified by introducing functional monomers for co-cross-linking during the cross-linking treatment; in the biological valve preparation methods disclosed in the Chinese invention patent application documents with publication numbers CN 114748693A, CN114748697A, CN 114748696A and CN 114748695A, while adding functional monomers for co-cross-linking, carbon-carbon double bonds were introduced from the functional monomers as a basis for further cross-linking, and the modification of the biological valve material was completed through two cross-linking processes.

[0006] In the studies mentioned above, whether it is the introduction of functional monomers for co-crosslinking modification during glutaraldehyde crosslinking, or the introduction of carbon-carbon double bonds as the basis for further crosslinking during the co-crosslinking process, new modified substances are introduced into the glutaraldehyde crosslinking process to participate in the crosslinking reaction.

[0007] Summary of the Invention

[0008] The present application provides a biological valve material based on aldehyde cross-linking, and its preparation method and application. Without changing the conventional glutaraldehyde cross-linking reaction, the carbon-carbon double bond is used as the basis for secondary cross-linking, providing a controllable cross-linking opportunity and range for the glutaraldehyde cross-linked membrane.

[0009] A method for preparing a biological valve material based on aldehyde cross-linking, comprising:

[0010] Step S110: contacting the biomaterial with an aldehyde cross-linking agent solution for cross-linking;

[0011] Step S120: Soaking the biomaterial treated in step S110 in a solution containing a first functional monomer to chemically connect the first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and an ethylene oxide group;

[0012] Step S130: soaking 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;

[0013] In step S200 , a carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.

[0014] Optionally, the aldehyde-based cross-linking agent is glutaraldehyde or formaldehyde.

[0015] Optionally, the biological material is animal tissue, and the animal tissue is selected from one or more of pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessel, skin or ligament.

[0016] Optionally, the animal tissue is fresh animal tissue or biological tissue that has been decellularized.

[0017] In step S200:

[0018] Optionally, an initiator is added to the system treated in the previous step; or the biological valve material treated in the previous step is taken out and immersed in a solution containing the initiator directly or after washing.

[0019] Optionally, the initiator is a single initiator or a mixed initiator.

[0020] Optionally, the mixed initiator is:

[0021] 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 to 100 mM.

[0022] Optionally, the single initiator is any component of the mixed initiators.

[0023] Optionally, the first functional monomer is selected from at least one of allyl glycidyl ether, glycidyl methacrylate and glycidyl acrylate.

[0024] 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-dimethylmethacrylamide, and double-bonded polylysine.

[0025] In step S110:

[0026] Optionally, the w / w concentration of the aldehyde cross-linking agent solution is 0.1% to 5%; and the cross-linking time is 0.5h-120h.

[0027] In step S120:

[0028] Optionally, the w / w concentration of the first functional monomer in the solution containing the first functional monomer is 1% to 10%; and the chemical reaction time is 2 to 120 hours.

[0029] Optionally, the solution containing the first functional monomer only contains the first functional monomer and a solvent that does not participate in the chemical reaction.

[0030] Optionally, the solvent in the solution containing the first functional monomer is one or more of an aqueous solution of any one of methanol, ethanol, ethylene glycol, propanol, 1,2-propylene glycol, 1,3-propylene glycol, isopropanol, butanol, isobutanol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and glycerol, water, physiological saline, and pH neutral buffer.

[0031] In step S130:

[0032] Optionally, the v / v concentration of the second functional monomer in the solution containing the second functional monomer is 0.1%-20%; and the soaking time is 0.5h-120h.

[0033] Furthermore, the v / v concentration of the second functional monomer in the solution containing the second functional monomer is 0.1%-6%.

[0034] Optionally, the second functional monomer enters the biomaterial by physical penetration.

[0035] The physical penetration can be understood as 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 embeds into the gaps within the biomaterial. During this process, no chemical reaction occurs between the second functional monomer and the biomaterial.

[0036] Optionally, the solution containing the second functional monomer only contains the second functional monomer and a solvent that does not participate in the reaction.

[0037] Optionally, the solvent in the solution containing the second functional monomer is one or a mixture of water, physiological saline, ethanol, isopropanol or a pH neutral buffer solution.

[0038] The present application also provides a biological valve material prepared by the above-mentioned preparation method.

[0039] The present application also provides a biological valve material, comprising:

[0040] Step S110: contacting the biomaterial with an aldehyde cross-linking agent solution for cross-linking;

[0041] Step S120: Soaking the biomaterial treated in step S110 in a solution containing a first functional monomer to chemically connect the first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and an ethylene oxide group;

[0042] Step S130: soaking 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;

[0043] In step S200 , a carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.

[0044] The present application also provides a biological valve, comprising a stent and leaflets, wherein the leaflets are made of the biological valve material.

[0045] Optionally, the biological valve is a heart valve.

[0046] The present application also provides an interventional system, comprising a heart valve and a catheter assembly, wherein the heart valve is folded and transported by the catheter assembly, and the heart valve comprises a stent and leaflets, wherein the leaflets are made of the biological valve material.

[0047] Compared with the prior art, this application has at least the following beneficial effects:

[0048] (1) The method of the present application introduces double bonds as the basis for secondary cross-linking on the biological valve material cross-linked with glutaraldehyde through double bond modification, and further introduces a functional monomer polymer cross-linking network to achieve secondary cross-linking by initiating polymerization between the double bonds on the biological valve material cross-linked with glutaraldehyde and the double bonds on the functional monomer, thereby further improving the cross-linking degree of the biological valve material.

[0049] (2) The present application introduces double bonds on the glutaraldehyde-crosslinked biological valve material, further triggers polymerization between the double bonds on the double-bonded biological valve material and the double bonds on the functional monomers, and introduces a functional monomer polymer crosslinking network. The crosslinking network can, to a certain extent, further reduce the binding of collagenase in the body to the collagen matrix on the biological valve material by physical blocking, protect the collagen matrix of the biological valve material, improve the stability of the glutaraldehyde-crosslinked biological valve material, and further reduce the risk of calcification caused by structural degradation of the biological valve material. Therefore, it also has certain anti-calcification properties.

[0050] (3) The present application introduces double bonds on the glutaraldehyde-crosslinked biological valve material, further triggers polymerization between the double bonds on the double-bonded glutaraldehyde-crosslinked biological valve material and the double bonds on the functional monomer, and introduces a functional monomer polymer cross-linking network. The cross-linking network can act as a polymer barrier to further reduce the binding of calcium ions in the environment with the mineralized areas on the biological valve material that are easily bound to calcium ions, thereby reducing the risk of calcification and playing an anti-calcification role.

[0051] (4) The present application introduces double bonds on the glutaraldehyde-crosslinked biological valve material, further triggers polymerization between the double bonds on the double-bonded glutaraldehyde-crosslinked biological valve material and the double bonds on the functional monomer, and increases the crosslinking degree on the biological valve material by introducing the functional monomer polymer crosslinking network, resulting in a more rigid structure of the biological valve material and increased elasticity; on the other hand, the functional monomer polymer crosslinking network fills the gaps between the collagen matrices on the biological valve material to inhibit the deformation of the collagen fibers, thereby making the texture of the biological valve material harder and improving its elasticity.

[0052] (5) Compared with the modification method of the applicant's previous research on the glutaraldehyde modification process in which carbon-carbon double bonds are introduced by adding functional monomers for co-crosslinking, in the modification process of the bioprosthetic valve material of the present application, glutaraldehyde crosslinking treatment is first performed, and then the residual amino groups and active groups such as hydroxyl and carboxyl groups on the glutaraldehyde crosslinking membrane are chemically connected to the functional monomers with carbon-carbon double bonds. The functional monomers with carbon-carbon double bonds are chemically connected with the amino groups, hydroxyl groups and carboxyl groups on the surface of the glutaraldehyde crosslinking membrane through the ethylene oxide group, and the carbon-carbon double bonds are mainly connected to the surface of the bioprosthetic valve material. In the process of glutaraldehyde crosslinking modification of the bioprosthetic valve material, no other substances that can participate in the crosslinking reaction are added, which can better protect the original fiber structure of the biomaterial, and can effectively ensure the mechanical properties of the membrane while ensuring the orientation direction of the original fibers of the biomaterial, avoiding the problem in the previous research that the direct addition of double-bond functional monomers during crosslinking may destroy the original fiber orientation of the biomaterial and increase the fiber disorder.

[0053] (6) On the basis of chemical grafting of the first carbon-carbon double bond, a second carbon-carbon double bond is further introduced through physical penetration. More carbon-carbon double bonds provide more cross-linking basis for secondary cross-linking, which can further increase the cross-linking degree of the biological valve material and improve the mechanical properties of the biological valve material. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG1 is a process flow chart of an embodiment of the present invention for copolymerization and cross-linking after double bonds;

[0055] FIG2 is a reaction principle diagram of the embodiment of the present invention of copolymerization and cross-linking after double bonds;

[0056] FIG3 is an Alizarin red staining result of control group 1 (glutaraldehyde cross-linked porcine pericardium) 30 days after subcutaneous implantation in rats;

[0057] FIG4 is an Alizarin red staining result of Sample 1 of Example 1 after subcutaneous implantation in rats for 30 days;

[0058] FIG5 is an Alizarin red staining result of Sample 2 of Example 2 after subcutaneous implantation in rats for 30 days;

[0059] FIG6 is an Alizarin red staining result of Sample 5 of Example 5 after subcutaneous implantation in rats for 30 days;

[0060] FIG7 is a schematic structural diagram of the heart valve of the present application;

[0061] FIG8 is a schematic structural diagram of the intervention system of the present application. DETAILED DESCRIPTION

[0062] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0064] Currently, almost all bioprosthetic valves used in clinical implantation are made from glutaraldehyde-crosslinked bioprosthetic valve materials. The reaction of glutaraldehyde with the collagen matrix in the bioprosthetic valve material crosslinks the collagen in the bioprosthetic valve material, further reducing the immunogenicity of the bioprosthetic valve material itself and improving its mechanical strength. However, even after glutaraldehyde crosslinking, the bioprosthetic valve material still suffers from a low degree of crosslinking and faces the risk of structural degradation. This directly leads to the degradation of its components after implantation, compromising its structural integrity and causing structural degradation and decay. Furthermore, the degradation of bioprosthetic valve components further promotes mechanical damage to the valve leaflet structure and induces calcification, which affects the normal opening and closing of the valve and reduces the service life of the bioprosthetic valve as the structure degrades.

[0065] Currently, glutaraldehyde-crosslinked biological heart valves are still the mainstream biological heart valves used clinically. Given that glutaraldehyde-crosslinked biological heart valves still have problems with stability and low cross-linking degree, as well as the risk of structural degradation and failure caused by structural degradation and damage, a series of post-crosslinking and modifications based on glutaraldehyde cross-linking not only meet actual production needs but also have great significance for scientific research.

[0066] Therefore, the present application introduces carbon-carbon double bonds as a secondary cross-linking platform on the biological heart valve material by double-bonding the glutaraldehyde-cross-linked biological valve material under glutaraldehyde-cross-linking conditions, and introduces a polymer network of functional monomers on the glutaraldehyde-cross-linked biological valve material by inducing a copolymerization reaction between the double bonds in the double-bonded glutaraldehyde-cross-linked biological valve material and the double bonds of the functional monomer, thereby further expanding the cross-linking network. Furthermore, that is, on the basis of scheme one, a second carbon-carbon double bond is further introduced by physical penetration of a second functional monomer (containing a second carbon-carbon double bond), which will increase the cross-linking degree of the glutaraldehyde-cross-linked biological valve material membrane, enhance its structural stability, and further reduce the degree of calcification of the material to improve its anti-calcification performance.

[0067] Specifically, it includes (see Figure 1):

[0068] S110 immersing the biological valve material in an aldehyde-based crosslinking agent solution for crosslinking to prepare a glutaraldehyde-crosslinked biological valve material;

[0069] S120: Soaking the glutaraldehyde-crosslinked bioprosthetic valve material prepared in step S110 in a solution of a double-bonding reagent (first functional monomer) for double-bonding modification to prepare a double-bonded bioprosthetic valve material; the double-bonding reagent (first functional monomer) has at least one first carbon-carbon double bond and an ethylene oxide group.

[0070] S130 soaking the double-bonded bioprosthetic valve material obtained in step S120 with a functional monomer (second functional monomer) solution, wherein the second functional monomer has at least one second carbon-carbon double bond;

[0071] S200 adds an initiator to the solution obtained after the soaking in step S130, and brings the initiator into contact with the biological valve material and the functional monomer solution to initiate double bond polymerization.

[0072] In the present application, the biomaterial is first subjected to a cross-linking reaction with an aldehyde cross-linking agent (S110), and then the first carbon-carbon double bond is accessed by reacting with the active group of the first functional monomer (S120), and then the second carbon-carbon double bond is introduced by physical penetration of the second functional monomer (S130). During the preparation process, an aldehyde cross-linking agent is first added, and the aldehyde cross-linking agent first reacts with part of the amino group of the biomaterial, and then the first functional monomer is added, and the remaining amino group and other groups (such as hydroxyl and carboxyl) on the biomaterial are used to react with the active group on the first functional group to directly access the first carbon-carbon double bond. The active group of the first functional monomer is an oxirane group. In addition to the remaining amino group on the biomaterial participating in the reaction, its hydroxyl and carboxyl groups can also react with the oxirane group to participate in the chemical reaction. On the basis of accessing the first carbon-carbon double bond through chemical reaction, the second carbon-carbon double bond is introduced again through physical penetration by the second functional monomer. Finally, the first carbon-carbon double bond introduced by chemical reaction is polymerized under the action of an initiator to further form a cross-linked network, thereby improving the anti-coagulation, anti-calcification, elasticity and other properties of the bioprosthesis cross-linked based on glutaraldehyde.

[0073] The reaction principle of this application:

[0074] In this double bond cross-linking scheme, after the biological valve material is cross-linked with glutaraldehyde, a first carbon-carbon double bond is further introduced by using a double bond reagent (first functional monomer) solution. The double bond of the biological valve material cross-linked with glutaraldehyde is used as a platform for secondary cross-linking. The double bond reagent (first functional monomer) used has both a carbon-carbon double bond and an ethylene oxide group.

[0075] To facilitate understanding of the chemical principles involved in this scheme, further explanation is given by taking Figure 2 as an example: the double-bonding reagent (first functional monomer) is used to modify the glutaraldehyde-crosslinked biological valve material, and the ethylene oxide group in the double-bonding reagent (first functional monomer) reacts with the hydroxyl group, carboxyl group and a small amount of amino group remaining after glutaraldehyde cross-linking on the biological valve material to undergo a ring-opening reaction, thereby introducing a first carbon-carbon double bond into the glutaraldehyde-crosslinked biological valve material; further, the second functional monomer further introduces a second carbon-carbon double bond through physical penetration; further, by initiating a copolymerization reaction between the double bonds in the double-bonded glutaraldehyde-cross-linked biological valve material and the double bonds of the functional monomer, a functional monomer polymer is introduced as a cross-linking network to achieve further secondary cross-linking, thereby completing the double bond copolymerization and cross-linking treatment of the biological valve material.

[0076] Since more functional groups (hydroxyl and carboxyl groups other than amino groups) on the biological valve material are used for cross-linking, and the polymer of the functional monomer is further introduced as a cross-linking network through copolymerization with the functional monomer, the cross-linking network of the biological valve material is expanded. The cross-linking degree of the biological valve material after cross-linking by double bond copolymerization will be significantly improved, and its structural stability and anti-calcification performance will also be significantly improved with the introduction of the functional monomer polymer network.

[0077] After glutaraldehyde cross-linking, carbon-carbon double bonds are introduced. The carbon-carbon double bonds are mainly connected to the surface of the biological valve material. In the process of glutaraldehyde cross-linking modification of the biological valve material, no other substances that can participate in the cross-linking reaction are added, which can better protect the original fiber structure of the biomaterial. While effectively ensuring the mechanical properties of the membrane, the orientation direction of the original fibers of the biomaterial can be guaranteed, avoiding the problem in previous studies that the direct addition of double-bond functional monomers during cross-linking may destroy the original fiber orientation of the biomaterial and increase the fiber disorder.

[0078] On the basis of chemically grafting the first carbon-carbon double bond, the second carbon-carbon double bond is further introduced through physical penetration. More carbon-carbon double bonds provide more cross-linking basis for secondary cross-linking, which can further increase the cross-linking degree of the biological valve material and improve the mechanical properties of the biological valve material.

[0079] Optionally, in step S120 of the present application, non-condensing chemical bonding is used to connect the first carbon-carbon double bond.

[0080] Optionally, in step S110, the biomaterial is not subjected to any other chemical reaction involving any reagents before being treated with the aldehyde cross-linking agent.

[0081] Further optionally, in the reaction system of step S120, the first carbon-carbon double bond is provided by a first functional monomer having an active group, and the reaction raw materials in steps S110 and S120 only include the biomaterial, the first functional monomer and the aldehyde cross-linking agent.

[0082] In step S110:

[0083] The cross-linking agent of the present application adopts the aldehyde cross-linking agent used in the current mainstream cross-linking method. Optionally, the aldehyde cross-linking agent can be selected from glutaraldehyde and formaldehyde.

[0084] Optionally, the concentration of the glutaraldehyde solution is 0.1% to 5% (w / w); and the cross-linking time can be any time between 0.5 h and 120 h.

[0085] The biomaterial used in this application is a conventional biomaterial used in existing glutaraldehyde cross-linking processes, and the collagen content of the biomaterial is 60% to 90%. Furthermore, the biomaterial is animal tissue, and the animal source is pig, cow, horse, or sheep, including one or more of the pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessels, skin, or ligaments.

[0086] Optionally, the animal tissue is fresh animal tissue or biological tissue that has been decellularized.

[0087] Optionally, in the decellularization step, the biological tissue is treated with a surfactant as follows:

[0088] Decellularization of biological tissue using ionic surfactants; or

[0089] Non-ionic surfactants are used to decellularize biological tissues.

[0090] The ionic surfactant is mainly used for lysing cells, and the nonionic surfactant is mainly used for removing lipid substances (such as phospholipids).

[0091] Optionally, the ionic surfactant is at least one of sodium deoxycholate, fatty acid potassium soap, sodium lauryl sulfate, sodium cholate, hexadecyltrimethylammonium bromide, fatty acid potassium salt, and alkyldimethylsulfonpropyl betaine.

[0092] Optionally, the nonionic surfactant is at least one of Triton and Tween.

[0093] In step S120:

[0094] Optionally, the double-bonding agent, ie, the first functional monomer, is selected from at least one of allyl glycidyl ether, glycidyl methacrylate, and glycidyl acrylate.

[0095] Optionally, the concentration of the double-bonding agent in the solution containing the first functional monomer, ie, the double-bonding agent, is 1% to 10% (w / w); and the reaction time for the double-bonding modification is 2 to 120 hours.

[0096] Optionally, the solvent in the solution containing the first functional monomer, i.e., the double-bonding agent, is one or more of water, physiological saline, pH neutral buffer, or an aqueous solution of methanol, ethanol, ethylene glycol, propanol, 1,2-propylene glycol, 1,3-propylene glycol, isopropanol, butanol, isobutanol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, or glycerol.

[0097] Optionally, the biofilm material treated in S110 is taken out and washed or directly placed in a solution containing a double-bonding agent (first functional monomer).

[0098] In step S130:

[0099] The biological valve material processed in step S120 is immersed in the second functional monomer solution directly or after being washed.

[0100] Optionally, the second functional monomer has at least one second carbon-carbon double bond.

[0101] Further optionally, the second functional monomer is 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-dimethylmethacrylamide, and double-bonded polylysine.

[0102] Optionally, the concentration of the second functional monomer solution is 0.1% to 20% (v / v); further, the concentration of the second functional monomer solution is 0.1% to 6% (v / v).

[0103] Optionally, the solvent of the second functional monomer solution is one or a mixture of water, physiological saline, ethanol, isopropanol or a pH neutral buffer solution.

[0104] Optionally, the immersion time in the second functional monomer solution is 0.5 h to 120 h.

[0105] In step S200:

[0106] The bioprosthetic valve material treated in step S120 is washed with deionized water and then immersed in an initiator solution for treatment in step S200 or the initiator is directly added to the reaction system in step S120 to initiate polymerization, the latter being commonly known as a one-pot process.

[0107] Optionally, the solvent in the initiator-containing solution is water, physiological saline or pH neutral buffer.

[0108] As mentioned above, the concentration of the initiator can be understood as the concentration of the initiator in the solution contained in the reaction system in step S120 in the one-pot method, and can be understood as the concentration in the solution containing the initiator in the step-by-step method.

[0109] 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 to 100 mM.

[0110] The reaction time of step S200 is 3 to 24 hours.

[0111] In the present application, all reaction processes of S110, S120, S130 and S200 can be carried out at 0-50°C unless otherwise specified. Preferably, the temperature does not need to be specially controlled and can be carried out at room temperature, preferably not exceeding the temperature adapted to the human body, and preferably at 36-37°C.

[0112] In the present application, all reactions of S110, S120, S130 and S200 can be either static reactions or dynamic reactions unless otherwise specified. The dynamic reactions can be carried out under the action of a peristaltic pump or other equipment that can circulate the solution, or can be carried out by shaking at a speed of 10 rpm-150 rpm. The peristaltic cycle or shaking time can be continuous or intermittent.

[0113] The present application optionally further includes dehydration and drying treatment after the double bond polymerization is completed to produce a dry film. After the double bond polymerization is completed, the biological valve material is routinely cleaned and softened, and then dehydrated and dried.

[0114] The cleaning solution can be one or a mixture of water, physiological saline, ethanol, isopropanol or 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.

[0115] Optionally, the dehydration treatment is to expose the membrane sheet after double bond polymerization or the valve sewn from the membrane sheet to a dehydration solution.

[0116] Optionally, the dehydration solution is a mixed solution of an alcohol solution and water, the alcohol solution accounts for 20-90% (v / v), and the alcohol reagent can be one of ethanol and isopropanol or a mixture of the two.

[0117] Optionally, the drying treatment is to expose the dehydrated membrane or valve to a softener solution for a treatment time of 20 minutes to 10 hours.

[0118] Optionally, the main component of the softener solution is a mixed solution of one or two of glycerol and polyethylene glycol, the glycerol concentration is 10-100% (v / v), and the other components are one or more of water, ethanol, and isopropanol, accounting for 0-90% (v / v).

[0119] Optionally, the valve after drying can be sterilized by ethylene oxide sterilization or electron beam sterilization.

[0120] 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.

[0121] As shown in Figure 7, in one embodiment, an artificial heart valve is provided, including a stent 1 and leaflets 2 connected to the stent 1. The stent is cylindrical as a whole, and the side walls are a hollow grid structure. The interior of the stent is a blood flow channel, and the multiple leaflets cooperate with each other to control the degree of opening and closing of the blood flow channel in the stent.

[0122] Depending on the release mode, the stent is processed using corresponding materials, such as nickel-titanium alloy with shape memory that can self-expand in the body, or stainless steel that is released by balloon expansion, etc. The stent itself can be formed by cutting tubes or weaving wires, and the leaflets can be connected to the stent by sewing, bonding or integral mold molding.

[0123] To ensure positioning within the body, positioning structures that can interact with surrounding native tissues, such as anchors or arms, can be provided on the periphery of the stent. To prevent 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 made of the bioprosthetic valve materials described in the above embodiments.

[0124] As shown in Figure 8, when catheter intervention is used, the artificial heart valve 3 and the corresponding delivery system constitute 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 when delivered in the body. The catheter assembly is released from its restraints or undergoes balloon expansion and radial expansion and release in the body.

[0125] The following is further described with specific examples:

[0126] Comparative Example 1

[0127] During the treatment process, a simple glutaraldehyde cross-linking group was set as a control group, and the porcine pericardium was immersed in 0.625% (w / w) glutaraldehyde at room temperature for 72 hours to prepare glutaraldehyde cross-linked porcine pericardium, which was recorded as control sample 1.

[0128] Example 11

[0129] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0130] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in a 5% (v / v) isopropanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 72 hours, and the solvent of the double bond modification solution used was 18% (v / v) isopropanol aqueous solution.

[0131] After the double bond modification was completed, the double bond glutaraldehyde cross-linked pig pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium was immersed in a 2% (v / v) polyethylene glycol diacrylate solution for 2 hours;

[0132] 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 polymerization between the double bonds on the double-bonded glutaraldehyde cross-linked biological valve material and the double bonds on polyethylene glycol diacrylate. After reacting at 37°C for 8 hours, a pig pericardium cross-linked after double bond copolymerization was obtained, which was recorded as sample 1.

[0133] Example 2

[0134] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0135] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in a 6% (v / v) propanol aqueous solution of glycidyl acrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 72 hours, and the solvent of the double bond modification solution used was a 20% (v / v) propanol aqueous solution.

[0136] After the double bond modification was completed, the double bond glutaraldehyde cross-linked pig pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium was immersed in 2.5% (v / v) N-methyl-2-acrylamide solution for 1 hour;

[0137] 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 polymerization between the double bonds on the double-bonded glutaraldehyde-cross-linked biological valve material and the double bonds on N-methyl-2-acrylamide. After reacting at 37°C for 8 hours, a pig pericardium cross-linked after double bond copolymerization was obtained, which was recorded as Sample 2.

[0138] Example 3

[0139] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0140] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and 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 of the double bond modification solution used was 30% (v / v) ethanol aqueous solution.

[0141] After the double bond modification was completed, the double bond glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked porcine pericardium was immersed in a 2.5% (v / v) (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) diacrylate solution for 1 hour;

[0142] 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-bonded glutaraldehyde cross-linked biological valve material and the double bonds on (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) diacrylate. After reacting at 37°C for 8 hours, a pig pericardium cross-linked after double bond copolymerization was obtained, which was recorded as sample 3.

[0143] Example 4

[0144] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0145] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in an isopropanol aqueous solution of 3% (v / v) glycidyl methacrylate and 2% (v / v) glycidyl acrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 48 hours, and the solvent of the double bond modification solution used was a 25% (v / v) isopropanol aqueous solution.

[0146] After the double bond modification was completed, the double bond glutaraldehyde cross-linked pig pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium was immersed in a 1.5% (v / v) ethane-1,2-diyl diacrylate solution for 1 hour;

[0147] 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 polymerization between the double bonds on the double-bonded glutaraldehyde cross-linked biological valve material and the double bonds on ethane-1,2-diyl diacrylate. After reacting at 37°C for 7 hours, a pig pericardium cross-linked after double bond copolymerization was obtained, which was recorded as Sample 4.

[0148] Example 5

[0149] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0150] The glutaraldehyde-crosslinked porcine pericardium was washed with deionized water and immersed in a 4% (v / v) ethanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 72 hours, and the solvent of the double bond modification solution was 20% (v / v) ethanol aqueous solution.

[0151] After the double bond modification was completed, the double bond glutaraldehyde cross-linked pig pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium was immersed in 1.4% (v / v) N,N'-dimethylacrylamide solution for 1 hour;

[0152] 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 polymerization between the double bonds on the double-bonded glutaraldehyde cross-linked biological valve material and the double bonds on N,N'-dimethylacrylamide. After reacting at 37°C for 7 hours, a pig pericardium cross-linked after double bond copolymerization was obtained, which was recorded as Sample 5.

[0153] Example 6

[0154] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0155] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in a 4% (v / v) isobutanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 72 hours, and the solvent of the double bond modification solution used was a 15% (v / v) isobutanol aqueous solution.

[0156] After the double bond modification was completed, the double bond glutaraldehyde cross-linked pig pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium was immersed in 1.4% (v / v) N,N'-dimethylmethacrylamide solution for 5 hours;

[0157] 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 polymerization between the double bonds on the double-bonded glutaraldehyde-cross-linked biological valve material and the double bonds on N,N'-dimethylmethacrylamide. After reacting at 37°C for 12 hours, a double-bond copolymerized and cross-linked porcine pericardium was obtained, which was recorded as Sample 6.

[0158] Example 7

[0159] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0160] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in a 4% (v / v) isopropanol aqueous solution of glycidyl acrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 48 hours, and the solvent of the double bond modification solution used was a 20% (v / v) methanol aqueous solution.

[0161] After the double bond modification was completed, the double bond glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked porcine pericardium was immersed in a solution containing 1.0% (v / v) N,N'-dimethylacrylamide and 0.5% (v / v) N,N'-dimethylmethacrylamide for 1 hour;

[0162] 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 polymerization between the double bonds on the double-bonded glutaraldehyde-cross-linked biological valve material and the double bonds on N,N'-dimethylacrylamide and N,N'-dimethylmethacrylamide. After reacting at 37°C for 8 hours, a pig pericardium cross-linked after double bond copolymerization was obtained, which was recorded as Sample 7.

[0163] Example 8

[0164] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0165] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in a 5% (v / v) aqueous solution of glycidyl methacrylate in ethylene glycol at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 72 hours, and the solvent of the double bond modification solution used was a 25% (v / v) aqueous solution of ethylene glycol.

[0166] After the double bond modification was completed, the double bond glutaraldehyde cross-linked pig pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium was immersed in 1.25% (v / v) N,N'-dimethylmethacrylamide solution for 5 hours;

[0167] 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 polymerization between the double bonds on the double-bonded glutaraldehyde-cross-linked biological valve material and the double bonds on N,N'-dimethylmethacrylamide. After reacting at 37°C for 12 hours, a double-bond copolymerized and cross-linked porcine pericardium was obtained, which was recorded as Sample 8.

[0168] Example 9

[0169] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0170] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in a 7% (v / v) propanol aqueous solution of glycidyl acrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 60 hours, and the solvent of the double bond modification solution used was 30% (v / v) propanol aqueous solution.

[0171] After the double bond modification is completed, the double bond glutaraldehyde cross-linked pig pericardium is washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium is immersed in a 1.0% (v / v) N-ethylacrylamide solution for 5 hours;

[0172] Initiators were added to the above solution, with ammonium persulfate concentrations of 20 mM and tetramethylethylenediamine concentrations of 1 mM to further initiate polymerization between the double bonds on the double-bonded glutaraldehyde-cross-linked biological valve material and the double bonds on N,N'-dimethylmethacrylamide. After reacting at 37°C for 12 hours, a double-bond copolymerized and cross-linked porcine pericardium was obtained, which was recorded as Sample 9.

[0173] Example 10

[0174] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0175] After washing with deionized water, the 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 the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 84 hours, and the solvent of the double bond modification solution used was 25% (v / v) ethanol aqueous solution.

[0176] After the double bond modification was completed, the double bond glutaraldehyde cross-linked pig pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium was immersed in 1.50% (v / v) N,N'-dimethylmethacrylamide solution for 3 hours;

[0177] 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 polymerization between the double bonds on the double-bonded glutaraldehyde-cross-linked biological valve material and the double bonds on N,N'-dimethylmethacrylamide. After reacting at 37°C for 7 hours, a pig pericardium cross-linked after double bond copolymerization was obtained, which was recorded as sample 10.

[0178] The performance of the samples of Examples 1 to 10 and the sample of Comparative Example 1 was characterized:

[0179] In order to characterize the change in the cross-linking degree of glutaraldehyde-cross-linked biological valve materials before and after double bond copolymerization cross-linking treatment, the thermal stability and cross-linking degree of the biological valve materials were characterized by measuring the thermal shrinkage temperature of the biological valve materials; the stability of the biological valve materials was characterized by an enzyme degradation experiment; the calcification degree (anti-calcification performance) of the samples was characterized by a rat subcutaneous implantation experiment; and the elastic angle of the biological valve materials was tested to characterize their elasticity.

[0180] Thermal shrinkage temperature determination:

[0181] The bioprosthetic valve material was cut into circular sheets with a diameter of 0.6 cm, dried, and placed in a crucible. The thermal shrinkage temperature of the bioprosthetic valve material was measured on a differential scanning calorimeter at a heating rate of 10°C / min in the range of 40-120°C. This thermal shrinkage temperature was used to characterize the thermal stability and degree of cross-linking of the bioprosthetic valve material; higher thermal shrinkage temperatures corresponded to greater thermal stability and cross-linking.

[0182] Table 1 Thermal shrinkage temperature of each group of bioprosthetic valve materials

[0183] Sample name Thermal shrinkage temperature (℃) Control group 1 (glutaraldehyde cross-linked pig pericardium) 85.2 Sample 1 91.3 Sample 2 92.8 Sample 3 90.7 Sample 5 91.4 Sample 10 90.1

[0184] Thermal shrinkage temperature measurements were performed on control group 1 (glutaraldehyde-crosslinked porcine pericardium), sample 1, sample 2, sample 3, sample 5, and sample 10. As shown in Table 1, the thermal shrinkage temperatures of sample 1, sample 2, sample 3, sample 5, and sample 10 were all higher than those of control group 1 (glutaraldehyde-crosslinked porcine pericardium). This means that the thermal stability and degree of crosslinking of sample 1, sample 2, sample 3, sample 5, and sample 10 were all higher than those of the control group (glutaraldehyde-crosslinked porcine pericardium). The results of the thermal shrinkage temperature measurement experiments indicate that the method of preparing a bioprosthetic valve material by double-bond post-copolymerization and crosslinking of the present application can improve the thermal stability and degree of crosslinking of bioprosthetic valves.

[0185] Elasticity test experiment

[0186] Cut the bioprosthetic valve material into pieces with uniform thickness of 1◇4.6cm 2 A rectangular sample is clamped horizontally along the midline of the long side of the rectangular sample. The angle at which the sample sags relative to the horizontal plane of the midline is tested to characterize the elasticity of the sample. The smaller the angle, the higher the elasticity.

[0187] Table 2 Elastic angle of each group of bioprosthetic valve materials

[0188] Sample name Elastic angle (°)

[0189] Control group 1 (glutaraldehyde cross-linked porcine pericardium) 65 Sample 1 35 Sample 2 45 Sample 3 56 Sample 5 47 Sample 10 50

[0190] Elasticity tests were performed on Samples 1, 2, 3, 5, 10, and Control Group 1 (glutaraldehyde-crosslinked porcine pericardium) to characterize their elasticity. The results of the elasticity tests are shown in Table 2. Compared with Control Group 1 (glutaraldehyde-crosslinked porcine pericardium), Samples 1, 2, 3, 5, and 10 had lower elastic angles, indicating that their elasticity was significantly improved compared with that of the Control Group (glutaraldehyde-crosslinked porcine pericardium). The method of preparing bioprosthetic valve materials by double bond post-copolymerization and cross-linking can improve the elasticity of bioprosthetic valves, and the enhanced elasticity of bioprosthetic valve materials is conducive to rapid recovery of their shape after transcatheter implantation.

[0191] Enzyme degradation experiment

[0192] The bioprosthetic valve material was cut into circular sheets with a diameter of 1 cm, with 6-8 parallel test samples per group. All circular sheet samples were placed in a 48-well plate, frozen at -80°C overnight, and then transferred to a vacuum freeze dryer for 48 hours. The weight of each sample was measured on a 1 / 100,000 balance and recorded as the initial weight (W0) before being returned to the 48-well plate. 0.5 mL of collagenase I in PBS was added to each well of the 48-well plate, ensuring that the bioprosthetic valve sample was completely immersed in the collagenase (100 U / mL) PBS solution. The 48-well plate was placed in a 37°C constant temperature incubator for 24 hours. After incubation, the bioprosthetic valve material sample was removed, rinsed three times, frozen at -80°C overnight, and then transferred to a vacuum freeze dryer for 48 hours. The weight of each sample after degradation with the collagenase solution was measured on a 1 / 100,000 balance and recorded as the final weight (Wt). The weight loss rate due to enzymatic degradation was calculated as follows:

[0193]

[0194] The collagenase degradation weight loss rate of sample 1, sample 2, sample 5, sample 10 and control group 1 was measured, and the results are shown in Table 3.

[0195] Table 3 Weight loss rate of enzymatic degradation of bioprosthetic valve materials in each group

[0196] Sample name Enzyme degradation weight loss rate (%) Control group (glutaraldehyde cross-linked pig pericardium) 7.27±1.18 Example 11 1.12±0.40 Example 12 3.61±0.22 Example 15 2.90±0.45 Example 20 3.27±0.55

[0197] Enzyme degradation experiments were performed on sample 1, sample 2, sample 5, sample 10 and control group 1 (glutaraldehyde cross-linked porcine pericardium) to characterize the cross-linking efficiency of each group of samples. After treating sample 1, sample 2, sample 5, sample 10 and control group 1 (glutaraldehyde cross-linked porcine pericardium) with collagenase I, the enzymatic degradation weight loss rate of each group of samples was calculated as shown in Table 3. The enzymatic degradation weight loss rates of sample 1, sample 2, sample 5, and sample 10 were all lower than that of control group 1 (glutaraldehyde cross-linked porcine pericardium), indicating that the stability of sample 1, sample 2, sample 5, and sample 10 was higher than that of control group 1 (glutaraldehyde cross-linked porcine pericardium), that is, sample 1, sample 2, sample 5, and sample 10 were higher. The results of the enzymatic degradation experiment show that the method of preparing biological valve materials by double bond post-copolymerization and cross-linking of the present application can improve the stability of biological valves.

[0198] Anti-calcification test

[0199] Cut the bioprosthetic valve material into 1◇1cm 2 After sterilization, the samples were implanted subcutaneously in rats and removed 30 days later. Each sample was divided into two parts. One part was decapsulated, freeze-dried, weighed, and digested with 6M hydrochloric acid for determination of calcium content per gram. The other part was fixed with paraformaldehyde tissue fixative. After fixation, the samples were removed, trimmed with a scalpel, and transferred to a dehydration box. The samples were dehydrated using a gradient of ethanol. After dehydration, the samples were transferred to an embedding machine and embedded in melted paraffin wax. They were then cooled in a -20°C refrigerator and trimmed. 5μm-thick sections were cut from the trimmed wax blocks on a microtome, transferred from the slide spreader to glass slides, dewaxed, and rehydrated. The sections were stained with alizarin red for 3 minutes, washed with water, dried, and permeabilized with xylene for 5 minutes. The sections were mounted with neutral gum, and images of the staining results were captured on a pathology slide scanner.

[0200] For samples 1, 2, 5 and control group 1 (glutaraldehyde cross-linked pig pericardium), the bioprosthetic valve material was cut into 1◇1cm 2 The sheet was tested for anti-calcification.

[0201] Table 4 Calcium content of bioprosthetic valve materials in each group 30 days after subcutaneous implantation in rats

[0202] Sample name Calcium content (mg / g) Control group 1 (glutaraldehyde cross-linked pig pericardium) 67.3±10.5 Sample 15.4±2.7 Sample 28.1±3.6 Sample 5 13.9±4.7

[0203] Calcium content was measured in samples 1, 2, and 5, as well as in control group 1 (glutaraldehyde-crosslinked porcine pericardium), 30 days after subcutaneous implantation in rats. As shown in Table 4, the calcium content in samples 1, 2, and 5 was lower than that in control group 1 (glutaraldehyde-crosslinked porcine pericardium) 30 days after subcutaneous implantation in rats. This result demonstrates that the double-bond post-copolymerization and cross-linking method for preparing bioprosthetic valve materials can improve the anti-calcification properties of bioprosthetic valves.

[0204] The control group 1 (glutaraldehyde cross-linked pig pericardium), sample 1, sample 2, and sample 5, which were implanted into the subcutaneous tissue of rats for 30 days, were stained with alizarin red to directly observe the degree of calcification of each group of samples. The alizarin red staining result images of the sample slices implanted into the subcutaneous tissue of rats for 30 days are shown in Figures 3-12, wherein the darker the color of the sample after alizarin red staining, the higher the degree of calcification. Compared with the alizarin red staining results of the slices of the control group 1 (glutaraldehyde cross-linked pig pericardium) (Figure 3), the alizarin red staining colors of the slices of sample 1 (Figure 4), sample 2 (Figure 5), and sample 5 (Figure 6) are obviously lighter and paler, which directly indicates that the degree of calcification of sample 1, sample 2, and sample 5 is lower than that of the control group 1, that is, sample 1, sample 2, and sample 5 have a stronger anti-calcification effect than that of the control group 1. The alizarin red staining results of the biological valve material implanted into the subcutaneous tissue of rats for 30 days show that the method for preparing biological valve material by double bond copolymerization and cross-linking of the present application can improve the anti-calcification performance of biological valve.

[0205] Example 11

[0206] Freshly harvested porcine pericardium was immersed in physiological saline and shaken for 2 hours, and then immersed in 0.625% (w / w) glutaraldehyde solution at room temperature and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the porcine pericardium to prepare glutaraldehyde cross-linked porcine pericardium.

[0207] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in a 5% (v / v) isopropanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 72 hours, and the solvent of the double bond modification solution used was 18% (v / v) isopropanol aqueous solution.

[0208] After the double bond modification was completed, the double bond glutaraldehyde cross-linked pig pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium was immersed in a 2% (v / v) polyethylene glycol diacrylate solution for 2 hours;

[0209] 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 polymerization between the double bonds on the double-bonded glutaraldehyde cross-linked biological valve material and the double bonds on polyethylene glycol diacrylate. After reacting at 37°C for 8 hours, a double-bond copolymerized cross-linked porcine pericardium was obtained.

[0210] The pig pericardium material cross-linked after double bond copolymerization was immersed in a 60% isopropyl alcohol solution for 45 minutes, and then immersed in a 10% glycerol, 3% polyethylene glycol (Mn=200), and 87% ethanol solution at room temperature for 3 hours. Excess glycerol was removed from the surface of the pig pericardium material, and the material was sterilized with ethylene oxide. This was designated as Sample 12.

[0211] Example 12

[0212] Fresh porcine pericardium was placed in a PS solution containing 0.5% sodium deoxycholate (surfactant) by mass, shaken at room temperature for 4 hours, and then washed three times with a 0.9% sodium chloride aqueous solution (ie, normal saline).

[0213] Subsequently, the pig pericardium was immersed in a 0.625% (w / w) glutaraldehyde solution at room temperature, and the solution was immersed and shaken for 72 hours to perform glutaraldehyde cross-linking treatment on the pig pericardium to prepare glutaraldehyde cross-linked pig pericardium.

[0214] The glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in a 5% (v / v) isopropanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium. The reaction time was 72 hours, and the solvent of the double bond modification solution used was 18% (v / v) isopropanol aqueous solution.

[0215] After the double bond modification was completed, the double bond glutaraldehyde cross-linked pig pericardium was washed with deionized water; then, the double bond glutaraldehyde cross-linked pig pericardium was immersed in a 2% (v / v) polyethylene glycol diacrylate solution for 2 hours;

[0216] 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 polymerization between the double bonds on the double-bonded glutaraldehyde cross-linked biological valve material and the double bonds on polyethylene glycol diacrylate. After reacting at 37°C for 8 hours, a pig pericardium cross-linked after double bond copolymerization was obtained, which was recorded as sample 12.

[0217] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A method for preparing a biological valve material based on aldehyde cross-linking, characterized in that: include: Step S110: contacting the biomaterial with an aldehyde cross-linking agent solution for cross-linking; Step S120: Soaking the biomaterial treated in step S110 in a solution containing a first functional monomer to chemically connect the first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and an ethylene oxide group; Step S130: soaking 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; In step S200 , a carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.

2. The preparation method according to claim 1, characterized in that The aldehyde cross-linking agent is glutaraldehyde or formaldehyde.

3. The preparation method according to claim 1, characterized in that The biological material is animal tissue, and the animal tissue is selected from one or more of pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessel, skin or ligament.

4. The preparation method according to claim 3, characterized in that The animal tissue is fresh animal tissue or biological tissue that has been decellularized.

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

6. The preparation method according to claim 1, characterized in that The initiator is a single initiator or a mixed initiator.

7. The preparation method according to claim 6, characterized in that The mixed 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 to 100 mM.

8. The preparation method according to claim 7, characterized in that The single initiator is any component in each mixed initiator.

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

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

12. The preparation 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% to 10%; and the reaction time is 2 to 120 hours.

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

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

15. The preparation method according to claim 1, characterized in that 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-dimethylmethacrylamide, and double-bonded polylysine.

16. The preparation 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%; and the soaking time is 0.5h-120h.

17. The preparation 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 preparation method according to claim 1, characterized in that The second functional monomer enters the biomaterial by physical penetration.

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

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

21. A biological valve material, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 20.

22. A biological valve material, characterized in that: include: Step S110: contacting the biomaterial with an aldehyde cross-linking agent solution for cross-linking; Step S120: Soaking the biomaterial treated in step S110 in a solution containing a first functional monomer to chemically connect the first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and an ethylene oxide group; Step S130: soaking 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; In step S200 , a carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.

23. A bioprosthetic valve comprising a stent and a valve leaflet, characterized in that: The leaflet is the biological valve material according to claim 21 or 22.

24. The bioprosthesis according to claim 23, wherein: The biological valve is a heart valve.

25. An interventional system comprising a heart valve and a catheter assembly, wherein the heart valve is folded and transported by the catheter assembly, wherein: The heart valve comprises a stent and leaflets, and the leaflets are made of the biological valve material according to claim 21 or 22.

Images