WO2023088330A1 - 一种生物瓣膜材料及其制备方法和应用 - Google Patents

一种生物瓣膜材料及其制备方法和应用 Download PDF

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WO2023088330A1
WO2023088330A1 PCT/CN2022/132374 CN2022132374W WO2023088330A1 WO 2023088330 A1 WO2023088330 A1 WO 2023088330A1 CN 2022132374 W CN2022132374 W CN 2022132374W WO 2023088330 A1 WO2023088330 A1 WO 2023088330A1
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functional monomer
carbon
double bond
group
biological
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PCT/CN2022/132374
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English (en)
French (fr)
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王云兵
郑城
杨立
雷洋
李高参
罗日方
邝大军
麻彩丽
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四川大学
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Priority claimed from CN202210273134.5A external-priority patent/CN114748694A/zh
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Publication of WO2023088330A1 publication Critical patent/WO2023088330A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F299/00—Macromolecular compounds obtained by interreacting polymers involving only carbon-to-carbon unsaturated bond reactions, in the absence of non-macromolecular monomers
    • C08F299/02—Macromolecular compounds obtained by interreacting polymers involving only carbon-to-carbon unsaturated bond reactions, in the absence of non-macromolecular monomers from unsaturated polycondensates

Definitions

  • the invention relates to the technical field of interventional materials, in particular to a biological valve material and its preparation method and application.
  • Bio heart valves are usually prepared from pig or bovine pericardium, and are used to replace the human body's own heart valves with functional defects; biological heart valves have many advantages over mechanical heart valves: after implantation of biological heart valves, patients do not need Long-term use of anticoagulant drugs, bio-heart valves can use minimally invasive interventional surgery, these advantages make bio-heart valves gradually become the mainstream of the market in clinical applications.
  • Glutaraldehyde can cross-link the collagen in the pericardium and improve the mechanical properties of the diaphragm to meet the market's requirements for the mechanical properties of the diaphragm. requirements.
  • glutaraldehyde-crosslinked bioprosthetic valves have aldehyde groups, which are used to generate calcification sites, and thus have the disadvantage of poor blood compatibility, resulting in a limited lifespan in the body.
  • glutaraldehyde-crosslinked biofilm is still the mainstream bioprosthetic material, and research on improving the performance of glutaraldehyde-crosslinked membranes is still one of the current important research directions.
  • glutaraldehyde will be improved.
  • the crosslinking time and concentration of aldehyde but due to the self-polymerization reaction of glutaraldehyde itself, the degree of crosslinking is limited, and it is impossible to achieve all the crosslinking of all amino groups in the membrane, and blindly increasing the time and concentration will only increase the strength of the membrane.
  • the glutaraldehyde on the surface self-polymerizes, making the performance of the diaphragm hard.
  • This application provides a biological valve material and its preparation method and application, by introducing carbon-carbon double bonds on the basis of glutaraldehyde cross-linking as the basis of secondary cross-linking, and further realizing the secondary cross-linking by initiating the polymerization of carbon-carbon double bonds Secondary crosslinking to improve the performance of glutaraldehyde crosslinked membranes.
  • double bond monomers can be introduced into the glutaraldehyde crosslinking process for co-crosslinking, and the crosslinking method of introducing other crosslinking groups can be re-crosslinked.
  • the residual amino groups of the membrane after glutaraldehyde cross-linking can be introduced to introduce monomers with double bond cross-linking, and by introducing other cross-linking
  • the cross-linking method of the linking group provides a controllable cross-linking opportunity and range.
  • a method for preparing a biological valve material comprising:
  • Step S100 sequentially treating the biological material with the first treatment liquid and the second treatment liquid to obtain the pretreated biological material chemically grafted with the first carbon-carbon double bond;
  • the first treatment liquid and the second The treatment liquids are different from each other and contain one of reagent A and reagent B, wherein reagent A is the first functional monomer with the first carbon-carbon double bond, and reagent B is an aldehyde-based crosslinking agent;
  • step S200 the carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.
  • the first functional monomer also has an active group, and participates in a chemical reaction through the active group;
  • the first treatment liquid contains reagent A, and the active group can react with aldehyde groups;
  • the first treatment solution contains reagent B, and the active group can react with amino groups.
  • the biological material is animal tissue, including one or more of pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessel, skin or ligament.
  • the animal tissue is fresh animal tissue or biological tissue after decellularization.
  • the aldehyde-based crosslinking agent is glutaraldehyde or formaldehyde.
  • 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, directly or washed and then soaked in the solution containing the initiator;
  • the initiator is a single initiator or a mixed initiator
  • the mixed initiator is:
  • the single initiator is any component in each mixed initiator.
  • step S100 includes:
  • AS110 contacts the biological material with the first treatment liquid for physical penetration, and the first treatment liquid is a solution containing the first functional monomer;
  • AS120 contacts the biological material treated by AS110 with a second treatment solution to perform co-crosslinking and access to the first carbon-carbon double bond, and the second treatment solution is an aldehyde-based crosslinking agent solution.
  • the active group is amino or hydrazide.
  • step S100 includes:
  • the first treatment solution is an aldehyde-based crosslinking agent solution
  • step BS120 contacting the biological material treated in step BS110 with a second treatment solution to insert the first carbon-carbon double bond through a chemical reaction, and the second treatment solution is a solution containing the first functional monomer.
  • the active group is an oxirane group.
  • step S100 a non-condensation chemical reaction is used to insert the first carbon-carbon double bond.
  • the biological material has not undergone any chemical reaction involving other reagents before being treated with an aldehyde-based cross-linking agent.
  • the first carbon-carbon double bond is provided by the first functional monomer with an active group in the reaction system of step S100, and the reaction raw materials in step S100 only include the biological material, the first A functional monomer and the aldehyde-based crosslinking agent.
  • step S100 includes:
  • AS110 soaks the biological material in the first treatment liquid for physical penetration;
  • the first treatment liquid is a solution containing the first functional monomer;
  • the first functional monomer also has an active group;
  • the active The group is amino or hydrazide;
  • AS120 immersing the biological material treated in step AS110 in a second treatment solution for co-crosslinking and inserting the first carbon-carbon double bond, the second treatment solution being an aldehyde-based crosslinking agent solution.
  • the first functional monomer also has a functional group A.
  • the functional group A is selected from hydroxyl group, carboxyl group, amido group, sulfonic acid group, zwitterion, polyethylene glycol, urea group, carbamate group, carboxylate ion, sulfonate, At least one of at least one of sulfone and pyrrolidone.
  • step S100 also includes:
  • the second functional monomer also has a functional group B.
  • the functional group B is selected from the group consisting of hydroxyl, carboxyl, carboxylic acid choline, sulfonic acid choline, phosphorylcholine, pyrrolidone, sulfonic acid group, carboxylate ion, sulfonic acid ester, sulfoxide, amide At least one of group, methoxy group.
  • the second functional monomer is selected from polyethylene glycol diacrylate, 1,4-butanediol diacrylate, ethyl acrylate, N-methyl-2-acrylamide, N-2, 2-propenyl-2-acrylamide, N-ethylacrylamide, N,N'-vinylbisacrylamide, (ethane-1,2-diylbis(oxyl))bis(ethane-2 ,1-diyl)diacrylate, double bonded hyaluronic acid, acrylamide, 2-(prop-2-enamido)acetic acid, 2-acrylamido-2-methylpropanesulfonic acid, methacrylic acid Hydroxyethyl ester, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, N-isopropylacrylamide, N-(hydroxymethyl)acrylamide, N-( 2-Hydroxyethyl)methacrylamide, N,N-dimethylme
  • the second functional monomer is added to the system of the previous treatment; or the biological material after the previous treatment is washed and then soaked in the solution containing the second functional monomer;
  • the solution of the bifunctional monomer only includes the second functional monomer and a solvent that does not participate in the chemical reaction.
  • the solvent in the solution containing the second functional monomer is water, physiological saline, pH neutral buffer or an aqueous solution of ethanol; the mass of the second functional monomer in the solution containing the second functional monomer The percentage concentration is 1-10%; the soaking time is 2-20 hours.
  • the first functional monomer is selected from DL-2-amino-4-pentenoic acid, 2-methallylamine, 3-butene-1-amine, pent-4-en-1-amine , 2-aminoethyl methacrylate, methacrylohydrazide, acrylohydrazide, double bonded polylysine, 2-amino-7-ene-octanoic acid, 6-ene-heptanoic acid, 2-amino Pent-4-enoic acid, 4-(1-amino-2-methyl-propyl)-hepta-1,6-dien-4-ol, 4-(1-amino-ethyl)-hepta-1 , at least one of 6-dien-4-ols.
  • the solution containing the first functional monomer only includes the first functional monomer and a solvent that does not participate in chemical reactions.
  • the solvent in the solution containing the first functional monomer in step AS110 is water, physiological saline, isopropanol, pH neutral buffer or an aqueous solution of ethanol; in the solution containing the first functional monomer The concentration of the first functional monomer is 10-100mM; the soaking time is 2-20h.
  • the final concentration of the aldehyde-based cross-linking agent in the AS120 reaction system is 10-800 mM; the co-cross-linking time is 10-30 h.
  • step S100
  • step AS120(M) is also included: soak the biological material treated in step AS120 in a solution containing a third functional monomer to eliminate residual aldehyde groups; the third functional monomer has an amino group or an acyl group. Hydrazine.
  • step S100
  • Step AS120(M) is also included before step AS130: soak the biological material treated in step AS120 in a solution containing a third functional monomer to eliminate residual aldehyde groups; the third functional monomer has an amino group or an acyl group Hydrazine.
  • the solvent in the solution containing the third functional monomer is water, physiological saline, pH neutral buffer or an aqueous solution of ethanol; in the solution containing the third functional monomer
  • concentration of the third functional monomer is 10-100 mM; the soaking time is 2-48 hours.
  • the third functional group also has a functional group C.
  • the functional group C is selected from hydroxyl group, carboxyl group, amido group, sulfonic acid group, zwitterion, polyethylene glycol, urea group, carbamate group, carboxylate ion, sulfonate, At least one of at least one of sulfone and pyrrolidone.
  • the third functional monomer is selected from DL-2-amino-4-pentenoic acid, 2-methallylamine, 3-butene-1-amine, pent-4-en-1-amine , 2-aminoethyl methacrylate, methacrylohydrazide, acrylohydrazide, double bonded polylysine, 2-amino-7-ene-octanoic acid, 6-ene-heptanoic acid, 2-amino Pent-4-enoic acid, 4-(1-amino-2-methyl-propyl)-hepta-1,6-dien-4-ol, 4-(1-amino-ethyl)-hepta-1 , at least one of 6-dien-4-ols.
  • step S100 includes:
  • the first treatment solution is an aldehyde-based crosslinking agent solution
  • the second treatment solution is a solution containing the first functional monomer;
  • the first The functional monomer also has an active group; the active group is an oxirane group.
  • step S100 includes:
  • the first treatment solution is an aldehyde-based crosslinking agent solution
  • the second treatment solution is a solution containing the first functional monomer;
  • the first The functional monomer also has an active group; the active group is an oxirane group;
  • step BS130 soaking the biological material treated in step BS120 in a solution containing a second functional monomer, the second functional monomer has a second carbon-carbon double bond.
  • the second functional monomer is selected from 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-di Bis(oxyl))bis(ethane-2,1-diyl)diacrylate, N,N'-dimethylacrylamide, N,N-dimethylmethacrylamide, double bonded poly One or more of lysine.
  • the second functional monomer also has a functional group B.
  • the functional group B is selected from the group consisting of hydroxyl, carboxyl, carboxylic acid choline, sulfonic acid choline, phosphorylcholine, pyrrolidone, sulfonic acid group, carboxylate ion, sulfonic acid ester, sulfoxide, amide At least one of group, methoxy group.
  • the second functional monomer is selected from acrylamide, acrylic acid, sodium acrylate, methacrylic acid, sodium methacrylate, 2-(prop-2-enamido)acetic acid, 2-acrylamide-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-methyl Prop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, 2-methacryloyloxyethylphosphorylcholine, N-(2-hydroxyethyl)acrylamide, N- One or more of (methoxymethyl)methacrylamide, 2-acrylamide-2-methylpropanesulf
  • step BS130
  • the second functional monomer enters the biological material through physical infiltration.
  • the physical penetration can be understood as that when the biological material treated in step S120 is soaked in the solution containing the second functional monomer, the second functional monomer in the solution adheres to the surface of the biological material or embeds biological In the gaps in the material, no chemical reaction occurs between the second functional monomer and the biological material during this process.
  • the solution containing the second functional monomer only includes the second functional monomer and a solvent that does not participate in the reaction.
  • 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.
  • the v/v concentration of the second functional monomer in the solution containing the second functional monomer is 0.1%-6%.
  • the first functional monomer is selected from at least one of allyl glycidyl ether, glycidyl methacrylate and glycidyl acrylate.
  • step BS110
  • the w/w concentration of the aldehyde-based cross-linking agent solution is 0.1%-5%; the cross-linking time is 0.5h-120h.
  • step BS120
  • the solution containing the first functional monomer only includes the first functional monomer and a solvent that does not participate in chemical reactions.
  • 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.
  • the solvent in the solution containing the first functional monomer is methanol, ethanol, ethylene glycol, propanol, 1,2-propanediol, 1,3-propanediol, isopropanol, butanol, isobutanol , 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and any one of aqueous solution of glycerol, water, physiological saline, pH neutral buffer solution or one or more.
  • the application also provides a method for preparing a biological valve material, including:
  • Step BS110 contacting the biological material with an aldehyde-based cross-linking agent solution for cross-linking
  • Step BS120 immersing the biological material treated in step BS110 in a solution containing a first functional monomer to react with a first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and ethylene oxide alkyl;
  • step S200 the carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.
  • the application also provides a method for preparing a biological valve material, including:
  • Step BS110 contacting the biological material with an aldehyde-based cross-linking agent solution for cross-linking
  • Step BS120 immersing the biological material treated in step BS110 in a solution containing a first functional monomer to react with a first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and ethylene oxide alkyl;
  • Step BS130 soaking the biological material treated in step BS120 in a solution containing a second functional monomer, the second functional monomer has a second carbon-carbon double bond;
  • step S200 the carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.
  • the application also provides a method for preparing a biological valve material, including:
  • Step BS110 contacting the biological material with an aldehyde-based cross-linking agent solution for cross-linking
  • Step BS120 immersing the biological material treated in step BS110 in a solution containing a first functional monomer to react with a first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and ethylene oxide alkyl;
  • Step BS130 soaking the biological material treated in step BS120 in a solution containing a second functional monomer, the second functional monomer has a second carbon-carbon double bond and a functional group B;
  • step S200 the carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.
  • the present application also provides a biological valve material prepared by the preparation method.
  • the application also provides a biological valve material, including:
  • Step S100 sequentially treating the biological material with the first treatment liquid and the second treatment liquid to obtain the pretreated biological material chemically grafted with the first carbon-carbon double bond;
  • the first treatment liquid and the second The treatment liquids are different from each other and contain one of reagent A and reagent B, wherein reagent A is the first functional monomer with the first carbon-carbon double bond, and reagent B is an aldehyde-based crosslinking agent;
  • step S200 the carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.
  • the application also provides a biological valve material, including:
  • Step BS110 contacting the biological material with an aldehyde-based cross-linking agent solution for cross-linking
  • step BS120 the biological material treated in step BS110 is soaked in a solution containing a first functional monomer to react with a first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and ethylene oxide alkyl;
  • step S200 the carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.
  • the application also provides a biological valve material, including:
  • Step BS110 contacting the biological material with an aldehyde-based cross-linking agent solution for cross-linking
  • Step BS120 immersing the biological material treated in step BS110 in a solution containing a first functional monomer to react with a first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and ethylene oxide alkyl;
  • Step BS130 soaking the biological material treated in step BS120 in a solution containing a second functional monomer, the second functional monomer has a second carbon-carbon double bond;
  • step S200 the carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.
  • the application also provides a biological valve material, including:
  • Step BS110 contacting the biological material with an aldehyde-based cross-linking agent solution for cross-linking
  • step BS120 the biological material treated in step BS110 is soaked in a solution containing a first functional monomer to react with a first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and ethylene oxide alkyl;
  • Step BS130 soaking the biological material treated in step BS120 in a solution containing a second functional monomer, the second functional monomer has a second carbon-carbon double bond and a functional group B;
  • step S200 the carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.
  • the present application also provides a biological valve, including a stent and leaflets, the leaflets being the material of the biological valve.
  • the biological valve is a heart valve.
  • the present application also provides an interventional system, including a heart valve and a catheter assembly.
  • the heart valve is folded and delivered by the catheter assembly.
  • the heart valve includes a stent and leaflets, and the leaflets are the biological valve material.
  • the present application has at least one of the following beneficial effects:
  • the method of the present application introduces functional monomers for co-crosslinking during aldehyde group crosslinking, and introduces carbon-carbon double bonds at the same time as co-crosslinking, as the basis for secondary crosslinking, and prepares crosslinked biological compounds through two crosslinkings. Materials that can increase the degree of cross-linking of biomaterials and improve the mechanical properties of biomaterials.
  • the functional monomer can also block some residual aldehyde groups on the biomaterial while introducing double bonds, so as to improve the anti-calcification and anticoagulant properties of the biomaterial, and can also further improve the cross-linking properties. connection efficiency.
  • the method of the present application can also introduce functional groups while introducing carbon-carbon double bonds, which can further improve the performance of biological materials, such as surface hydrophilicity, biocompatibility, etc.
  • This application introduces additional functional groups while introducing carbon-carbon double bonds for the second time, which can endow biomaterials with new characteristics and further improve the performance of biomaterials.
  • Biomaterials are co-crosslinked with functional monomers at the same time as glutaraldehyde crosslinking to improve the anti-calcification and anticoagulant properties of glutaraldehyde crosslinked biomaterials.
  • this application introduces double bonds into the glutaraldehyde-crosslinked biological valve material as the basis for secondary crosslinking through double bond modification, and further passes
  • the polymerization of double bonds on the biological valve material that triggers glutaraldehyde cross-linking can achieve secondary cross-linking.
  • the cross-linking degree of the biological valve material can be further improved, thereby improving the stability of the biological valve material.
  • It can further reduce the risk of calcification caused by structural degradation, so it also has certain anti-calcification properties.
  • Fig. 1 is the process flow chart of the application scheme one kind of preferred embodiment
  • Figure 2 is a schematic diagram of the chemical principle of the first embodiment of the present application scheme
  • Fig. 3 is the process flow diagram of a kind of preferred embodiment of scheme two of the present application.
  • Fig. 4 is the process flow sheet of a kind of more preferred embodiment of scheme three of the present application.
  • Fig. 5 is the process flow sheet of a kind of more preferred embodiment of scheme four of the present application.
  • Fig. 6 is the process flow diagram of a kind of preferred embodiment of scheme five of the present application.
  • Fig. 7 is the reaction schematic diagram of a kind of preferred embodiment of the present application.
  • Fig. 8 is the reaction schematic diagram of another kind of preferred embodiment of the present application.
  • Fig. 9 is the infrared spectrogram of sample 1 and control group 1 pericardium (GA) of embodiment 1;
  • Fig. 10 is the sample 1 of embodiment 1 and control group 1 pericardium (GA) lactate dehydrogenase relative activity result figure;
  • Fig. 11 is the sample 1 of embodiment 1 and control group 1 pericardial membrane (GA) rat subcutaneous implantation after hanging calcium amount detection result figure;
  • Fig. 12 is sample 2 of embodiment 2 and control group 2 pericardium (GA) water contact angle detection result figure;
  • Fig. 13 is sample 2 of embodiment 2 and control group 2 pericardium (GA) lactate dehydrogenase detection and hemolysis rate result figure;
  • Fig. 14 is sample 2 and control group 2 pericardium (GA) calcium ion concentration results figure of embodiment 2;
  • Fig. 15 is the basic schematic diagram of embodiment 3.
  • Fig. 16 is the result figure of Alizarin red staining of control group 3 slices
  • Figure 17 is a diagram of the Alizarin Red staining results of the slices after the subcutaneous implantation of sample 3 rats;
  • Figure 18 is a diagram of the Alizarin Red staining results of the slices after the subcutaneous implantation of sample 4 rats;
  • Figure 19 is a diagram of the Alizarin Red staining results of the slices after the subcutaneous implantation of sample 5 rats;
  • Figure 20 is a diagram of the Alizarin Red staining results of the slices after the subcutaneous implantation of sample 6 rats;
  • Figure 21 is a diagram of the Alizarin Red staining results of the slices after the subcutaneous implantation of sample 7 rats;
  • Figure 22 is a diagram of the Alizarin Red staining results of the slices after the subcutaneous implantation of sample 8 rats;
  • Fig. 23 is the basic schematic diagram of embodiment 10;
  • Figure 24 is an alizarin red-stained section view obtained after the control sample of the control group 4 was implanted for 30 days;
  • Figure 25 is an alizarin red-stained section view obtained after implantation of sample No. 10 for 30 days;
  • Figure 26 is an alizarin red-stained section view obtained after implantation of No. 11 sample for 30 days;
  • Figure 27 is an alizarin red-stained section view obtained after implantation of No. 12 sample for 30 days;
  • Figure 28 is a diagram of alizarin red-stained sections obtained after implantation of sample No. 13 for 30 days;
  • Figure 29 is a diagram of alizarin red-stained slices obtained after implantation of sample No. 14 for 30 days;
  • Figure 30 is an alizarin red-stained section view obtained after implantation of No. 15 sample for 30 days;
  • Fig. 31 is a scanning electron microscope image of control sample 4 blood contact experiment
  • Figure 32 is a scanning electron micrograph of sample No. 12 blood contact experiment
  • Figure 33 is the scanning electron microscope image of sample No. 13 blood contact experiment;
  • Figure 34 is the basic principle diagram of Example 17;
  • Figure 35 is an alizarin red-stained section diagram obtained after 30 days of implantation of the control sample of the control group 5;
  • Figure 36 is a diagram of alizarin red-stained sections of sample 17 after 30 days of implantation
  • Figure 37 is a diagram of alizarin red-stained sections of sample 18 after 30 days of implantation
  • Figure 38 is an alizarin red-stained section view obtained after implantation of sample 19 for 30 days;
  • Figure 39 is a diagram of alizarin red-stained sections obtained after implantation of sample 20 for 30 days;
  • Fig. 40 is a picture of alizarin red-stained section of sample 21 after 30 days of implantation.
  • Fig. 41 is the basic schematic diagram of embodiment 23;
  • Figure 42 is the blood scanning electron microscope picture of the control sample of control group 6;
  • Figure 43 is a scanning electron micrograph of the blood incubation experiment of sample 23;
  • Figure 44 is a scanning electron micrograph of the blood incubation experiment of sample 24;
  • Figure 45 is an alizarin red-stained section diagram obtained after the control sample of control group 6 was implanted for 60 days;
  • Figure 46 is a diagram of alizarin red-stained sections of sample 23 after 60 days of implantation
  • Figure 47 is a diagram of alizarin red-stained sections obtained after implantation of sample 24 for 60 days;
  • Figure 48 is a diagram of alizarin red-stained sections obtained after implantation of sample 25 for 60 days;
  • Figure 49 is an alizarin red-stained section view obtained after implantation of sample 26 for 60 days;
  • Figure 50 is a diagram of alizarin red-stained slices obtained after implantation of sample 27 for 60 days;
  • Fig. 51 is the reaction schematic diagram of embodiment 29 and 30;
  • Fig. 52 is the reaction schematic diagram of embodiment 31;
  • Fig. 53 is the reaction schematic diagram of embodiment 32;
  • Fig. 54 is the reaction schematic diagram of embodiment 33;
  • Figure 55 is a scanning electron micrograph of the blood contact experiment of control sample 7;
  • Figure 56 is a scanning electron micrograph of the blood contact experiment of sample 29;
  • Figure 57 is a scanning electron micrograph of sample 31 blood contact experiment
  • Figure 58 is a diagram of alizarin red-stained slices obtained after implantation in control sample 7 rats for 30 days;
  • Figure 59 is a diagram of alizarin red-stained sections obtained after implantation of sample 30 rats for 30 days;
  • Figure 60 is a diagram of alizarin red-stained sections obtained after implantation of sample 32 rats for 30 days;
  • Figure 61 is a diagram of alizarin red-stained sections obtained after implantation of sample 33 rats for 30 days;
  • Figure 62 is a diagram of alizarin red-stained sections obtained after implantation of sample 34 rats for 30 days;
  • Figure 63 is a process flow diagram of a more preferred embodiment of Scheme 6 of the present application.
  • Figure 64 is a reaction schematic diagram of a preferred embodiment of Scheme 6 of the present application.
  • Figure 65 is a picture of the Alizarin Red staining results of control group 8 (glutaraldehyde cross-linked pig pericardium) subcutaneously implanted in rats for 30 days;
  • Figure 66 is a diagram of the Alizarin Red staining results of sample 42 of Example 42 subcutaneously implanted in rats for 30 days;
  • Figure 67 is a diagram of the Alizarin Red staining results of sample 46 of Example 46 subcutaneously implanted in rats for 30 days;
  • Figure 68 is a diagram of the Alizarin Red staining results of sample 48 of Example 48 subcutaneously implanted in rats for 30 days;
  • Figure 69 is a process flow diagram of a double bond post-copolymerization and crosslinking embodiment of Scheme 7 of the present application.
  • Fig. 70 is the reaction schematic diagram of the embodiment of copolymerization and crosslinking after the seventh double bond of the application scheme
  • Figure 71 is a picture of the Alizarin Red staining results of control group 9 (glutaraldehyde cross-linked pig pericardium) subcutaneously implanted in rats for 30 days;
  • Figure 72 is a diagram of the Alizarin Red staining results of sample 52 of Example 52 subcutaneously implanted in rats for 30 days;
  • Figure 73 is a diagram of the Alizarin Red staining results of sample 53 of Example 53 subcutaneously implanted in rats for 30 days;
  • Figure 74 is a diagram of the Alizarin Red staining results of sample 56 of Example 56 after subcutaneous implantation in rats for 30 days;
  • Figure 75 is a process flow diagram of the embodiment of functionalized copolymerization and crosslinking after the eighth double bond of the application scheme
  • Fig. 76 is the reaction schematic diagram of the embodiment of functionalized copolymerization and crosslinking after eight double bonds in the scheme of the present application;
  • Figure 77 is a scanning electron microscope image of blood adhesion of control group 10 (glutaraldehyde cross-linked pig pericardium);
  • Fig. 78 is a scanning electron micrograph of blood adhesion of sample 62 in Example 62;
  • Figure 79 is a scanning electron micrograph of blood adhesion of sample 63 of Example 63;
  • Figure 80 is a scanning electron micrograph of blood adhesion of sample 68 in Example 68;
  • Figure 81 is a picture of the Alizarin Red staining results of the control group 10 (glutaraldehyde cross-linked pig pericardium) subcutaneously implanted in rats for 30 days;
  • Figure 82 is a diagram of the Alizarin Red staining results of Example 62 sample 62 after 30 days of subcutaneous implantation in rats;
  • Figure 83 is a diagram of the Alizarin Red staining results of Example 63 sample 63 after 30 days of subcutaneous implantation in rats;
  • Figure 84 is a diagram of the Alizarin Red staining results of Example 69 sample 69 after 30 days of subcutaneous implantation in rats;
  • Figure 85 is a schematic structural view of the heart valve of the present application.
  • Fig. 86 is a schematic structural diagram of the interventional system of the present application.
  • this application introduces carbon-carbon double bonds and then initiates secondary cross-linking of carbon-carbon double bonds to improve the biofilm based on glutaraldehyde cross-linking.
  • Anticoagulation, anticalcification, elasticity and other properties of the valve is provided, including:
  • Step S100 sequentially treating the biological material with the first treatment liquid and the second treatment liquid to obtain the pretreated biological material chemically grafted with the first carbon-carbon double bond;
  • the first treatment liquid and the second The treatment liquids are different from each other and contain one of reagent A and reagent B, wherein reagent A is the first functional monomer with the first carbon-carbon double bond, and reagent B is an aldehyde-based crosslinking agent;
  • step S200 the carbon-carbon double bond is polymerized under the action of an initiator to obtain a biological valve material.
  • the first carbon-carbon double bond introduced through a chemical reaction is then polymerized under the action of an initiator to further form a cross-linked network and improve the anticoagulation, anti-calcification, elasticity and other properties of the biovalve based on glutaraldehyde cross-linking .
  • the biological material used in this application is a conventional biological material in the existing glutaraldehyde crosslinking process, and the collagen content of the biological material is 60%-90%.
  • the biological material is animal tissue, and the animal source is pig, cow, horse or sheep, including one or more of pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessel, skin or ligament.
  • the animal tissue is fresh animal tissue or biological tissue after decellularization.
  • the biological tissue is treated with a surfactant as follows:
  • the ionic surfactant is mainly used to lyse cells, and the non-ionic surfactant is mainly used to remove lipids (such as phospholipids).
  • the ionic surfactant is sodium deoxycholate, potassium fatty acid soap, sodium lauryl sulfate, sodium cholate, cetyltrimethylammonium bromide, potassium salt of fatty acid, alkyl di At least one of methylsulfopropyl betaine.
  • the nonionic surfactant is at least one of Triton and Tween.
  • the ionic surfactant is sodium dodecylsulfonate, and the nonionic surfactant is triton.
  • the ionic surfactant is sodium dodecylsulfonate
  • the nonionic surfactant is Tween-20.
  • the cross-linking agent of the present application adopts the aldehyde-based cross-linking agent used in the current mainstream cross-linking method.
  • the aldehyde-based cross-linking agent can be one of glutaraldehyde and formaldehyde.
  • step S100 of the present application a non-condensation chemical reaction is used to insert the first carbon-carbon double bond.
  • step S100 the biological material is not subjected to any chemical reaction involving other reagents before being treated with an aldehyde-based cross-linking agent.
  • the first carbon-carbon double bond is provided by the first functional monomer with an active group in the reaction system of step S100, and the reaction raw materials in step S100 only include the biological material, the first A functional monomer and the aldehyde-based crosslinking agent.
  • the first treatment liquid contains reagent A (the first functional monomer with the first carbon-carbon double bond)
  • the first treatment liquid only contains the first functional monomer and does not participate in the chemical reaction solvent
  • the first treatment liquid contains reagent B (aldehyde-based cross-linking agent)
  • the first treatment liquid only contains aldehyde-based cross-linking agent and a solvent that does not participate in the chemical reaction.
  • the second treatment liquid is provided by the first functional monomer with an active group in the reaction system of step S100, and the reaction raw materials in step S100 only include the biological material, the first A functional monomer and the aldehyde-based crosslinking agent.
  • the biological material is sequentially treated with the first treatment liquid and the second treatment liquid
  • the order in which the reagents are in contact with the biological material, the way of feeding or contacting each other is not strictly limited,
  • the biological material can be taken out and contacted with the second treatment liquid, or the second treatment liquid can be directly added to the first treatment liquid immersed in the biological material.
  • the first treatment liquid and the second treatment liquid contain one of reagent A and reagent B which are different from each other. It can also be understood that: when the first treatment liquid contains reagent A, the second treatment liquid can only Containing reagent B, the second treatment solution mentioned here can only contain reagent B to be understood as emphasizing that it does not contain reagent A, rather than only reagent B in the second treatment solution; when the first treatment solution contains reagent B, the second treatment The second treatment solution can only contain reagent A. Similarly, the second treatment solution can only contain reagent A to be understood as emphasizing that it does not contain reagent B, rather than only reagent A in the second treatment solution.
  • the first functional monomer needs to participate in the chemical grafting reaction.
  • the first functional monomer also has an active group, and the active group participates in the chemical grafting reaction.
  • the first treatment liquid contains reagent A (the first functional monomer with the first carbon-carbon double bond), and the second treatment liquid contains reagent B (aldehyde-based cross-linking agent)
  • the first functional unit The active group of the body can react with the aldehyde group, and the first carbon-carbon double bond is indirectly connected to the biological material through a chemical reaction
  • the first treatment solution contains reagent B (aldehyde-based cross-linking agent)
  • the second treatment solution contains reagent B
  • the active group of the first functional monomer can react with the amino group to directly connect the first carbon-carbon double bond to the biological material.
  • the first carbon-carbon double bond can be indirectly connected to the biological material through a cross-linking agent, or can be directly connected to the active group on the biological material (including at least Amino) reactively incorporated into biomaterials.
  • the first treatment liquid contains reagent A (the first functional monomer with the first carbon-carbon double bond), and the second treatment liquid contains reagent B (aldehyde-based crosslinking agent ), under this alternative, the first functional monomer physically penetrates into the biological material; the amino group of the biological material and the active group of the first functional monomer are then combined with the aldehyde-based cross-linking agent Co-crosslinking is performed to access the first carbon-carbon double bond.
  • the first functional monomer first physically penetrates into the biological material, and then adds an aldehyde-based cross-linking agent to carry out co-cross-linking, that is, the chemical reaction, and the amino groups on the biological material are indirectly connected through the aldehyde-based cross-linking agent.
  • the active group of the first functional monomer is an amino group or a hydrazide.
  • the first treatment solution contains reagent B (aldehyde-based crosslinking agent), and the second treatment solution contains reagent A (the first functional unit with the first carbon-carbon double bond). body), under this alternative, the biomaterial first undergoes a cross-linking reaction with an aldehyde-based cross-linking agent, and then reacts with the active group of the first functional monomer to access the first carbon-carbon double bond.
  • step S100 an aldehyde-based cross-linking agent is first added, and the aldehyde-based cross-linking agent first reacts with some amino groups of the biological material, and then adds the first functional monomer to utilize the remaining amino groups and other groups on the biological material (such as Hydroxyl and carboxyl) 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, and in addition to the remaining amino groups on the biological material participating in the reaction, its hydroxyl and carboxyl groups can also react with the oxirane group, participate in the chemical reaction.
  • co-crosslinking is carried out by adding functional monomers during the glutaraldehyde modification process, and carbon-carbon double bonds are introduced at the same time as co-crosslinking, as the basis for secondary crosslinking.
  • the preparation of cross-linked biomaterials can increase the degree of cross-linking of biomaterials and improve the mechanical properties of biomaterials; in the second alternative, first carry out glutaraldehyde cross-linking treatment, and then use glutaraldehyde to cross-link the residue on the membrane Active groups such as amino groups, hydroxyl groups, and carboxyl groups are chemically connected to functional monomers with carbon-carbon double bonds, and the functional monomers with carbon-carbon double bonds cross-link the amino groups, hydroxyl groups, and carboxyl groups on the surface of the film through oxirane groups and glutaraldehyde Through chemical reaction connection, the carbon-carbon double bond is mainly connected to the surface of the biological valve material.
  • the fibrin such as amino groups, hydroxyl groups, and carboxyl groups
  • step S100 may include multiple sub-steps.
  • the starting materials involved are involved in at least one of the substeps, and are not strictly limited to reactions that participate in all substeps.
  • this application improves the existing glutaraldehyde crosslinking method, introduces functional monomers at the same time as glutaraldehyde crosslinking, performs co-crosslinking, improves the performance of glutaraldehyde crosslinking biomaterials, and is close to glutaraldehyde The problem of poor anti-calcification and anti-coagulant properties of aldehyde cross-linked biomaterials.
  • scheme 1 a functional monomer with a reactive group with an aldehyde group is introduced before glutaraldehyde cross-linking.
  • the crosslinking agent performs co-crosslinking.
  • AS110 contacts the biological material with a solution containing a first functional monomer for physical penetration;
  • the first functional monomer has at least one group that reacts with an aldehyde group;
  • the functional monomer first physically penetrates into the biological material.
  • the functional monomer has a group that reacts with the aldehyde group. Responses include at least:
  • the aldehyde groups at both ends of a part of the cross-linking agent react with the amino group of the biological material; 2) The aldehyde group at one end of the part of the cross-linking agent reacts with the amino group of the biological material, and the aldehyde group at the other end reacts with the first functional monomer; 3 ) One part of the cross-linking agent wherein the aldehyde group at one end reacts with the amino group of the biological material, and the aldehyde group at the other end forms a residual aldehyde group on the biological material; 4) Part of the residual aldehyde group reacts with the amino group of the first functional monomer.
  • the first functional monomer with a carbon-carbon double bond and a reactive group with an aldehyde group is introduced before glutaraldehyde cross-linking, and the first functional monomer is first physically Penetrate into the biomaterial, and then co-crosslink with the aldehyde-based crosslinking agent.
  • the first functional monomer reacts with the residual aldehyde group on the biomaterial to introduce the first carbon-carbon double bond into the biomaterial.
  • the double bond polymerization is initiated again, secondary cross-linking occurs, and the cross-linking treatment of the biological material is completed.
  • the biological material after the secondary cross-linking can also have functional groups to further improve the biocompatibility of the biomembrane.
  • AS110 immerses the biological material in a solution containing a first functional monomer for physical infiltration;
  • the first functional monomer has at least one first carbon-carbon double bond and at least one group reactive with an aldehyde group;
  • Step 1 The first functional monomer first physically penetrates into the biological material.
  • the introduced first functional monomer has the first carbon-carbon double bond and a group reactive with the aldehyde group (such as an amino group), and the functional monomer is fully penetrated.
  • an aldehyde-based crosslinking agent such as glutaraldehyde
  • the reactions that take place include at least:
  • the aldehyde groups at both ends of a part of the cross-linking agent react with the amino group of the biomaterial; 2) The aldehyde group at one end of the part of the cross-linking agent reacts with the amino group of the biomaterial, and the aldehyde group at the other end reacts with the amino group of the first functional monomer ; 3) the aldehyde group at one end of a part of the cross-linking agent reacts with the amino group of the biological material, and the aldehyde group at the other end forms a residual aldehyde group on the biological material; 4) part of the residual aldehyde group reacts with the amino group of the first functional monomer, and the A first carbon-carbon double bond is introduced into the biomaterial.
  • the second step the biomaterial that has completed cross-linking and introduced carbon-carbon double bonds is contacted with a solution containing an initiator to initiate double bond polymerization and secondary cross-linking occurs.
  • the present application further introduces a functional group on the basis of the functional monomer of Scheme 2, that is, the improved crosslinking scheme ( Denoted as Scheme 3), the first functional monomer with amino group, first carbon-carbon double bond and functional group A is introduced before glutaraldehyde crosslinking, the functional monomer first physically penetrates into the biological material, and then Co-crosslinking with an aldehyde-based crosslinking agent, the amino group of the first functional monomer reacts with the aldehyde group, the first carbon-carbon double bond and the functional group A are introduced into the biomaterial at the same time, and then the double bond polymerization is initiated, resulting in two Secondary cross-linking, the biomaterial after the secondary cross-linking has functional groups A, which can further improve the biocompatibility of the biomaterial.
  • the improved crosslinking scheme Denoted as Scheme 3
  • AS110 soaks the biological material in the solution containing the first functional monomer, and physically infiltrates; the first functional monomer has at least one amino group, at least one first carbon-carbon double bond and at least one functional group A;
  • Step 1 The first functional monomer first physically infiltrates into the biological material.
  • the introduced first functional monomer has an amino group, the first carbon-carbon double bond and a functional group A.
  • the first functional monomer is fully infiltrated and then added Aldehyde-based crosslinking agent (such as glutaraldehyde), for co-crosslinking, during the co-crosslinking process, the reactions that take place include at least:
  • the aldehyde groups at both ends of a part of the cross-linking agent react with the amino group of the biomaterial; 2) The aldehyde group at one end of the part of the cross-linking agent reacts with the amino group of the biomaterial, and the aldehyde group at the other end reacts with the amino group of the first functional monomer ; 3) the aldehyde group at one end of a part of the cross-linking agent reacts with the amino group of the biological material, and the aldehyde group at the other end forms a residual aldehyde group on the biological material; 4) part of the residual aldehyde group reacts with the amino group of the first functional monomer, and the A first carbon-carbon double bond is introduced into the biomaterial.
  • Step 2 The biological material after cross-linking and carbon-carbon double bond introduction is contacted with a solution containing an initiator to trigger the polymerization of the carbon-carbon double bond on the biological material and secondary cross-linking occurs.
  • the method of this scheme introduces the first functional monomer for co-crosslinking when the aldehyde group is crosslinked, and at the same time introduces the first carbon-carbon double bond as the basis for the secondary crosslinking, and prepares the crosslinked bio Materials that increase the degree of cross-linking of biomaterials.
  • the method of the present application introduces the functional group A while introducing the first carbon-carbon double bond, which can further improve the performance of the biological material, such as surface hydrophilicity, biocompatibility and the like.
  • the present application further improves the cross-linking means on the basis of the third scheme.
  • the first functional monomer with the first carbon-carbon double bond and the reactive group with the aldehyde group is introduced before glutaraldehyde cross-linking, the first functional monomer Physical penetration into the biological material first, and then co-crosslinking with an aldehyde-based cross-linking agent, the amino group of the first functional monomer reacts with the aldehyde group, and simultaneously introduces the first carbon-carbon double bond and functional group A into the biological material; Further, a part of the second carbon-carbon double bond is introduced through the physical infiltration of the second functional monomer, and finally the first carbon-carbon double bond on the biomaterial and the second carbon-carbon double bond of the second functional monomer are polymerized to form a cross-linked network , to further increase the degree of cross-linking of biomaterials.
  • AS110 physically permeates the biological material by immersing it in a solution containing the first functional monomer; the first functional monomer has at least one first carbon-carbon double bond and at least one group reactive with the residual aldehyde group on the biological material ;
  • reaction principle of the first step is the same as that of the first step of scheme three, and will not be repeated here.
  • Step 2 The second functional monomer first physically infiltrates into the biological material after co-crosslinking and carbon-carbon double bond is introduced at one time, and the second carbon-carbon double bond is further introduced. This step introduces the second carbon-carbon double bond as physical infiltration , after the second functional monomer is infiltrated, the second carbon-carbon double bond of the second functional monomer is initiated to copolymerize with the first carbon-carbon double bond on the surface of the biomaterial to perform secondary cross-linking to form a cross-linking network.
  • the method of this scheme crosslinks the biological material through aldehyde group co-crosslinking and double bond polymerization secondary crosslinking, and the biological material obtained by the two crosslinking treatments has a good crosslinking degree;
  • the second carbon-carbon double bond is further introduced through the second functional monomer, and the carbon-carbon double bond introduced through physical infiltration for the second time enables additional functional monomers to participate in the copolymerization during the double bond polymerization process, forming a more
  • the large polymer cross-linking network is beneficial to improve the degree of cross-linking and anti-calcification performance of biological valves.
  • the functional group B is further introduced through the second functional monomer.
  • the first functional monomer with the first carbon-carbon double bond and residual amino group is introduced before glutaraldehyde cross-linking, and the first functional monomer first physically penetrates into the
  • the biomaterial is then co-crosslinked with an aldehyde-based crosslinker, the amino group of the first functional monomer and the amino group on the biofilm introduce the first carbon-carbon double bond into the biomaterial by reacting with the aldehyde group of the glutaraldehyde crosslinker , and introduce functional group B at the same time; finally initiate the copolymerization of the second functional monomer and the carbon-carbon double bond on the biomaterial to form a cross-linked network while introducing functional functional group B to further improve the cross-linking degree and performance.
  • AS110 immerses the biological material in a solution containing a first functional monomer and reverse physical infiltration;
  • the first functional monomer has at least one amino group and at least one first carbon-carbon double bond;
  • biological materials are cross-linked through aldehyde group co-cross-linking and double-bond polymerization secondary cross-linking.
  • Part of the residual aldehyde group is removed; the first carbon-carbon double bond is introduced by co-crosslinking, and then the second carbon-carbon double bond is further introduced through the second functional monomer penetration.
  • the two-step introduction of carbon-carbon double bond can have additional
  • the functional monomers involved in the copolymerization form a larger polymer cross-linking network, which is conducive to improving the cross-linking degree and anti-calcification performance of biological valves; the second introduction of carbon-carbon double bonds also introduces functional groups B, which can endow The new characteristics of biomaterials further improve the performance of biomaterials.
  • step AS120(M) is also included after step AS120 of scheme 1 and scheme 2, scheme 3, and before step AS130 of scheme 4 and scheme 5: the organisms treated in step AS120 The material is soaked in the solution containing the third functional monomer to eliminate the remaining residual aldehyde group; the third functional monomer in this step has at least one group reactive with the aldehyde group.
  • both the first functional monomer and the third functional monomer have at least one group that reacts with the aldehyde group, and the first functional monomer reacts with the aldehyde group through the group during the co-crosslinking process, resulting in co-crosslinking Link; in the scheme comprising AS120(M), the third functional monomer reacts with the residual aldehyde group on the biomaterial through this group, eliminating the residual aldehyde group.
  • the groups reactive with aldehyde groups in the first functional monomer and the third functional monomer are each independently selected from amino groups or hydrazides.
  • the first functional monomer and the third functional monomer are each independently selected from at least one amino-substituted alkane, at least one amino-substituted cycloalkane, At least one amino-substituted olefin or amino-containing polymer.
  • the first functional monomer and the third functional monomer are each independently selected from ethylenediamine, 2-methylpropylamine, 1,4- Butylenediamine, n-hexylamine, oleylamine, 1,10-diaminodecane, octylamine, n-undecylamine, dodecylamine, tetradecylamine, hexadecanylamine, heptadecan-9-amine, cyclododecane Amine, Cycloheptanamine, Cyclooctylamine, 6-Methylheptane-1-amine, Nonadecan-10-amine, 3-Ethylpentan-1-amine, 2-Methylallylamine, 3-Butylamine One of en-1-amine, pent-4-en-1-amine, and 2-aminoethyl methacrylate.
  • the first functional monomer and the third functional monomer are each independently selected from methacrylohydrazide or acrylohydrazide.
  • the first functional monomer and the third functional monomer of Scheme 1 may have a functional group A in addition to an amino group; optionally, the functional group A is a hydrophilic group. Further optionally, the functional groups A of the first functional monomer and the third functional monomer are each independently selected from hydroxyl, carboxyl, amide, sulfonic acid, zwitterion, polyethylene glycol, ureido , carbamate group, carboxylate ion, sulfonate, sulfoxide, pyrrolidone at least one.
  • Hydroxyl As a hydrophilic group, it improves the surface hydrophilicity of co-crosslinked biomaterials to achieve anticoagulant effect;
  • Carboxyl group As a hydrophilic group, it improves the surface hydrophilicity of co-crosslinked biomaterials to achieve anticoagulant effect;
  • Amide group As a hydrophilic group, it improves the surface hydrophilicity of co-crosslinked biomaterials to achieve anticoagulant effect;
  • Carboxylate ions and sulfonic acid groups Improve the surface hydrophilicity of co-crosslinked biomaterials through ionic hydration to achieve anticoagulant effect;
  • Sulfoxide, pyrrolidone as a hydrophilic group, improve the surface hydrophilicity of co-crosslinked biomaterials to achieve anticoagulant effect;
  • Zwitterions Improve the surface hydrophilicity of co-crosslinked biomaterials through ionic hydration to achieve anticoagulant effect; it is beneficial to form the electrically neutral surface of co-crosslinked biological valves and reduce the adsorption of calcium ions to achieve anti-calcification Effect;
  • Polyethylene glycol As a hydrophilic group, it improves the surface hydrophilicity of co-crosslinked biomaterials; increases the steric hindrance between calcium ions and collagen, and improves the surface hydrophilicity of co-crosslinked biovalve materials;
  • Urethane group, urea group As a hydrophilic group, improve the surface hydrophilicity of co-crosslinked biomaterials to achieve anticoagulant effect
  • Urethane group As a hydrophilic group, it improves the surface hydrophilicity of the co-crosslinked biomaterial to achieve anticoagulant effect.
  • the first functional monomer and the second functional monomer are each independently selected from 2-amino-4-pentanoic acid, 2-amino-octanoic acid, 2-amino-5-hydroxypentanoic acid, 2-amino-2,3-dimethylbutyramide, 2-aminotetradecanoic acid, 2-amino-4-methylpentanoic acid, trishydroxymethylaminomethane, Amino-terminated polyethylene glycol and polyethylene glycol structural derivatives, amino oleic acid, natural amino acids, unnatural amino acids, polynatural amino acids (such as polylysine), DL-2-amino-4-pentenoic acid , 2-amino-7-ene-octanoic acid, 6-ene-heptanoic acid, 2-aminopent-4-enoic acid, 4-(1-amino-2-methyl-propyl)-hepta-1,6 One of -dien
  • first functional monomer and the second functional monomer of Scheme 1 it can be understood that the first functional monomer and the third functional monomer are each independently selected from the above-mentioned range, and may be the same or different.
  • the first functional monomer in Scheme 2 has at least one group that reacts with aldehyde groups. During the co-crosslinking process, the functional monomer reacts with the residual aldehyde groups on the biomaterial to introduce carbon-carbon double bonds.
  • the third functional monomer has at least one group reactive with aldehyde groups, and by reacting with the residual aldehyde groups on the biomaterial, the residual aldehyde groups are eliminated.
  • the aldehyde-reactive groups in the first functional monomer and the third functional monomer include but are not limited to amino groups and hydrazides.
  • the third functional monomer can also have at least one carbon-carbon double bond.
  • the amino group on the third functional monomer reacts with the residual aldehyde group on the biological material , while sealing the remaining residual aldehyde groups, carbon-carbon double bonds are reintroduced, increasing the number of carbon-carbon double bond groups used for subsequent double bond polymerization, which is conducive to improving the degree of crosslinking.
  • the groups reactive with aldehyde groups in the first functional monomer and the third functional monomer are each independently selected from one of amino groups and hydrazides, and may be the same or different. In a scheme that satisfies the functional monomer with at least one amino group and at least one carbon-carbon double bond, commercially available products can be directly used.
  • the first functional monomer and the third functional monomer described in scheme two Each independently selected from DL-2-amino-4-pentenoic acid, 2-methallylamine, 3-butene-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate At least one of ester, methacrylohydrazide and acrylohydrazide.
  • the first functional monomer of Scheme 3 has at least one active group.
  • the functional monomer reacts with the residual aldehyde group on the biomaterial through its active group, and introduces the first carbon-carbon double bond into the biomaterial.
  • the active group of the first functional monomer can be amino group or hydrazide.
  • the third functional monomer has at least one reactive group that reacts with the residual aldehyde group on the biomaterial to eliminate the residual aldehyde group.
  • the active group of the third functional monomer can be amino group or hydrazide.
  • the third functional monomer can also have at least one carbon-carbon double bond.
  • the first functional monomer may also have a functional group A
  • the third functional monomer may have a carbon-carbon double bond and an amino group.
  • it can also have a functional group C.
  • the functional group A and the functional group C are each independently selected from at least one of a hydroxyl group, a carboxyl group, an amide group and a sulfonic acid group.
  • the functional group A of the first functional monomer is at least one of hydroxyl, carboxyl, amido and sulfonic acid;
  • the functional group C of the third functional monomer is also hydroxyl, carboxyl, amido and At least one of the sulfonic acid groups; they may be the same or different.
  • the introduction of hydroxyl groups can improve the hydrophilicity of biological valves; the introduction of carboxyl groups can maintain the neutral pH of the AS110 reaction system; the introduction of amide groups can increase the hydrophilicity of biological valves through the hydrogen bond interaction between water molecules and amide groups; the introduction of sulfonic acid The group can increase the hydrophilicity of the biological valve through the ionic hydration between the water molecule and the sulfonic acid group.
  • the first functional monomer and the third functional monomer in one solution, commercially available products can be directly used.
  • the first functional monomer and the third functional monomer are independently selected from DL -2-amino-4-pentenoic acid, 2-amino-7-ene-octanoic acid, 6-ene-heptanoic acid, 2-aminopent-4-enoic acid, 4-(1-amino-2-methyl One of -propyl)-hepta-1,6-dien-4-ol and 4-(1-amino-ethyl)-hepta-1,6-dien-4-ol.
  • the first functional monomer of Scheme 4 has at least one group that reacts with the aldehyde group. During the co-crosslinking process, the first functional monomer reacts with some residual aldehyde groups on the biomaterial through the group, and the first carbon Carbon double bonds are incorporated into biomaterials.
  • groups reactive with aldehyde groups in the first functional monomer include but are not limited to amino groups and hydrazides.
  • the third functional monomer has at least one aldehyde-reactive group that reacts with remaining residual aldehyde groups on the biomaterial during soaking.
  • groups reactive with aldehyde groups in the third functional monomer include but are not limited to amino groups and hydrazides.
  • the first functional monomer of this scheme has at least one amino group and at least one first carbon-carbon double bond.
  • commercially available products can be directly used.
  • the first functional monomer is 2-methyl One of allylamine, 3-buten-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacrylohydrazide, and acrylohydrazide.
  • the third functional monomer of this scheme has at least one amino group.
  • the third functional monomer also has at least one carbon-carbon double bond.
  • the functional monomer The amino groups on the biomembrane react with the residual aldehyde groups on the biofilm, and at the same time, the carbon-carbon double bonds can be reintroduced while the remaining residual aldehyde groups are blocked, increasing the number of double bond bases for subsequent double bond polymerization.
  • the third functional monomer is 2-methallylamine, 3-buten-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacrylic acid One of hydrazide and acrylohydrazide.
  • the first functional monomer may also have a functional group A
  • the third functional monomer may have a carbon-carbon double bond and an amino group.
  • it can also have a functional group C.
  • the functional group A and the functional group C are each independently selected from at least one of a hydroxyl group, a carboxyl group, an amide group, and a sulfonic acid group.
  • the introduction of hydroxyl groups can improve the hydrophilicity of biological materials; the introduction of carboxyl groups can make biological materials appear electrically neutral; the introduction of hydroxyl groups can improve the hydrophilicity of biological valves; the introduction of carboxyl groups can maintain the pH neutrality of the reaction system of step AS110;
  • the hydrogen bond interaction between water molecules and amide groups increases the hydrophilicity of biological valves; the introduction of sulfonic acid groups can increase the hydrophilicity of biological valves through the ionic hydration between water molecules and sulfonic acid groups.
  • the functional monomers having at least one amino group, at least one carbon-carbon double bond and at least one functional group as described above in one solution, commercially available products can be directly used.
  • the first functional monomer and the third functional monomer are each independently selected from 2-amino-7-ene-octanoic acid, 6-ene-heptine, 2-aminopent-4-enoic acid, 4-(1-amino-2-methyl-propyl)-hepta-1,6-dien-4-ol, 4-(1-amino-ethyl)-hepta-1,6-dien-4 -A type of alcohol.
  • the second carbon-carbon double bond is further introduced through the second functional monomer.
  • the introduction process is physical penetration, and the second functional monomer does not interact with the biological material in this step.
  • the second functional monomer is polyethylene glycol diacrylate, 1,4-butanediol diacrylate, ethyl acrylate, N-methyl-2-propylene Amide, N-2,2-propenyl-2-acrylamide, N-ethylacrylamide, N,N'-vinylbisacrylamide, (ethane-1,2-diylbis(oxyl)) One of bis(ethane-2,1-diyl) diacrylate.
  • the first functional monomer has at least one amino group and at least one first carbon-carbon double bond.
  • commercially available products can be directly used.
  • the first functional monomer is 2-formazan One of aryl allylamine, 3-buten-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacrylohydrazide, and acrylohydrazide.
  • the third functional monomer of scheme five has at least one amino group.
  • the third functional monomer also has at least one carbon-carbon double bond.
  • the functional monomer The amino group on the biofilm reacts with the residual aldehyde group on the biofilm, and the carbon-carbon double bond can be reintroduced while the remaining residual aldehyde group is blocked.
  • the third functional monomer is 2-methallylamine, 3-buten-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacrylic acid One of hydrazide and acrylohydrazide.
  • the first functional monomer and the third functional monomer can also have functional groups in addition to carbon-carbon double bonds and amino groups.
  • the first functional monomer also has at least one functional Functional group A
  • the third functional monomer also has at least one functional group C';
  • the functional group A and functional group B described in this scheme are each independently selected from hydroxyl, carboxyl, amido, sulfonic One of the acid groups.
  • the introduction of hydroxyl groups can improve the hydrophilicity of biological materials; the introduction of carboxyl groups can make biological materials appear electrically neutral; the introduction of hydroxyl groups can improve the hydrophilicity of biological valves; the introduction of carboxyl groups can maintain the pH neutrality of the reaction system of step AS110;
  • the hydrogen bond interaction between water molecules and amide groups increases the hydrophilicity of biological valves; the introduction of sulfonic acid groups can increase the hydrophilicity of biological valves through the ionic hydration between water molecules and sulfonic acid groups.
  • the first functional monomer and the third functional monomer have functional groups
  • commercially available products can be used directly.
  • the first functional monomer and the third functional monomer are each independently selected from DL-2-amino-4-pentenoic acid, 2-amino-7-ene-octanoic acid, 6-ene-heptamine acid, 2-aminopent-4-enoic acid, 4-(1-amino-2-methyl-propyl)-hepta-1,6-dien-4-ol, 4-(1-amino-ethyl )-hepta-1,6-dien-4-ol in one.
  • the biological material is in contact with the solution containing the second functional monomer, and the second functional monomer physically penetrates into the biological material.
  • the functional group B is hydroxyl, carboxyl, carboxylic acid choline, sulfonic acid choline, phosphorylcholine, pyrrolidone, sulfonic acid One of group, carboxylate ion, sulfonate, sulfoxide, amide group, methoxy group.
  • the second functional monomer is polyethylene glycol diacrylate, acrylamide, 2-(prop-2-enamido)acetic acid, 2-acrylamide- 2-methylpropanesulfonic acid, hydroxyethyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, N-methyl-2-acrylamide, N-isopropylacrylamide, N-(hydroxymethyl)acrylamide, N-(2-hydroxyethyl)methacrylamide, N,N-dimethylmethacrylamide, 3-[N,N -Dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, 2-methacryloyloxyethyl phosphorylcholine A sort of. Commercially available.
  • the second functional monomer can also be prepared by modifying its own double bond.
  • the second functional monomer is double-bonded hyaluronic acid or double bonded hyaluronic acid. Bonded polylysine.
  • the first functional monomer and the third functional monomer can also be prepared by double bond modification, such as double bonded polylysine, in addition to the commercially available channels shown above.
  • the first functional monomer, the second functional monomer and the third functional monomer may independently select double-bonded hyaluronic acid or double-bonded polylysine.
  • the first functional monomer and the third functional monomer are independently selected from the above optional ranges (including commercially available and modified preparations), and may be the same or different.
  • An embodiment of the preparation of double-bonded polylysine comprising:
  • Polylysine was dissolved in deionized water, and then glycidyl methacrylate was added at a molar ratio of 1:1.5-1:5 (glycidyl methacrylate:amino). The mixture was placed on a shaker at 37°C for 5-10 days. Finally, use a dialysis bag with a molecular weight cut-off of 1000 to dialyze for 5-7 days, and freeze-dry to obtain partially double-bonded polylysine.
  • the biomaterials of the present application need to undergo conventional pretreatment.
  • the pretreatment includes conventional cleaning operations: obtain the biomaterials and store them in a low-temperature and humid state at 4°C; The biological material was washed with distilled water at 4°C and 100 RPM for 2 hours with gentle friction and fluid pressure until no adherent non-pericardial or non-collagenous tissue was visible.
  • the pretreated biological material is contacted with the solution containing the first functional monomer.
  • the contact process can be static contact or dynamic contact; when using static contact, the biological material is placed in the solution containing the first functional monomer.
  • the shaker can be shaken during the immersion process.
  • the temperature can be between 20°C and 50°C.
  • the final temperature of the contact process does not need to be specially controlled, and it can be at room temperature, preferably not exceeding the temperature adapted to the human body, preferably between 36°C and 37°C conduct.
  • the concentration of the first functional monomer in step AS110 and the contact time between the biological material and the solution containing the first functional monomer are appropriate to ensure that more of the first functional monomer penetrates into the biological material.
  • the first functional monomer The higher the concentration, the corresponding shorter contact time, the lower the concentration of the first functional monomer, the corresponding extended contact time.
  • the solvent of the solution described in step AS110 is water, physiological saline, pH neutral buffer or an aqueous solution of ethanol.
  • ethanol and water can be mixed in any proportion, usually about 50% ethanol;
  • the concentration of the functional monomer in the solution is 10-100 mM.
  • the contact time is 2-20 hours, so that the first functional monomer can fully penetrate into the biological material.
  • the concentration of the first functional monomer in the solution in step AS110 is 10-30 mM, and the soaking time is 2-5 hours.
  • a cross-linking agent is added to the reaction system.
  • the concentration of the cross-linking agent is 10-800 mM.
  • the temperature can be between 20°C and 50°C. Preferably, the temperature does not need to be specially controlled during the co-crosslinking process. ⁇ 37°C; the co-crosslinking reaction time should be as thorough as possible for the co-crosslinking reaction. Optionally, the co-crosslinking time is 10-30h under the condition that the cross-linking agent concentration is 10-800mM.
  • the concentration of the cross-linking agent in step AS120 is 50-500 mM; further, the concentration of the cross-linking agent in step AS120 is 50-150 mM, and the co-cross-linking time is 20-30 h.
  • the biological material and the crosslinking agent solution can be in static contact or dynamic contact, and during the dynamic contact process, the reaction system can be shaken while soaking to speed up the crosslinking process.
  • the concentration and soaking time of the third functional monomer in step AS120(M) are preferably more closed residual aldehyde groups.
  • the concentration of the third functional monomer in the solution is 10 ⁇ 100mM; soaking time is 2 ⁇ 48h.
  • the solvent in the solution described in step AS120(M) is water, physiological saline, pH neutral buffer or an aqueous solution of ethanol.
  • ethanol and water can be mixed in any proportion, usually 50 % of ethanol; the concentration of the third functional monomer in the solution containing the third functional monomer is 10-100mM; the soaking time is 2-48h.
  • the concentration of the third functional monomer is 30-50mM; the soaking time is 10-20h
  • the concentration of the third functional monomer is 10-30 mM; the soaking time is 3-8 hours.
  • the concentration of the third functional monomer in Scheme 3, Application 2 is 30-50 mM; the soaking time is 3-8 hours.
  • the concentration of the third functional monomer in the solution described in Scheme 5 is 20-40 mM; the soaking time is 2-4 hours.
  • the biological material treated in step AS120 is washed and then soaked in the third functional monomer solution; or the biological material treated in step AS120 is directly transferred to the third functional monomer solution .
  • the soaking temperature does not need to be specially controlled, and it can be at room temperature, preferably not exceeding the temperature adapted to the human body, preferably at 36-37°C.
  • a step of co-crosslinking or washing the biological material after completion of step AS120(M) is also included.
  • the biological material is taken out, the biological material is cleaned to remove residual functional monomers, crosslinking agents, etc., and then soaked in a solution containing the second functional key aggregation.
  • the biological material after co-crosslinking is contacted with the solution containing the second functional monomer, further introducing carbon-carbon double bonds, the final concentration of the second functional monomer and the contact time between the biological material and the solution containing the second functional monomer to ensure more It is advisable for the second functional monomer to penetrate into the biological material.
  • concentration of the second functional monomer is higher, and the corresponding contact time can be shorter, and the concentration of the second functional monomer is lower, and the corresponding contact time is adapted to extend.
  • the solvent in the solution containing the second functional monomer is water, physiological saline or pH neutral buffer or an aqueous solution of ethanol.
  • ethanol and water can be mixed in any proportion, usually 50% Left and right ethanol; the mass percent concentration of the second functional monomer is 1-10%.
  • the contact time is 2-20 hours. In order to fully penetrate the second functional monomer into the biological material.
  • the mass percent concentration of the second functional monomer in the solution containing the second functional monomer is 2-5%; the soaking time is 10-15 hours.
  • the contact process between the biological material and the solution containing the second functional monomer can be static contact or dynamic contact; the contact process can be at 20-50°C, preferably, the temperature does not need to be specially controlled, and room temperature is acceptable , it is advisable not to exceed the adaptation temperature of the human body, preferably at 36-37°C.
  • step AS120(M) the initiator is directly added to the system soaked in step AS120(M); or the biological material soaked in step AS120(M) is cleaned and then soaked in the solution containing the initiator middle.
  • an initiator is added to initiate free radical polymerization of the carbon-carbon double bond to perform secondary crosslinking (that is, step S200 in Scheme 4 and Scheme 5).
  • the initiator is a mixture of ammonium persulfate and sodium bisulfite; the concentrations of ammonium persulfate and sodium bisulfite in the solution are respectively 10 to 100 mM; further, persulfuric acid The concentrations of ammonium and sodium bisulfite were 20-40 mM, respectively.
  • the initiator is a mixture of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine; in the solution, ammonium persulfate and N,N, The mass percent concentrations of N', N'-tetramethylethylenediamine are 2%-5% and 0.2%-0.5% respectively.
  • the solvent in the solution containing the initiator is water, physiological saline or pH neutral buffer.
  • concentration of the initiator as mentioned above, in the one-pot method this concentration can be understood as the concentration of ammonium sulfate and sodium bisulfite in the solution contained in the reaction system of step AS120, in the step-by-step method, this concentration can be understood is the concentration in the solution containing the initiator.
  • the double-bond polymerization process can be carried out at 20-50°C.
  • the temperature in the double-bond polymerization process does not need to be specially controlled, and it can be at room temperature. °C.
  • the double bond polymerization time is preferably 2 to 48 hours, preferably 20 to 25 hours.
  • it also includes a post-treatment process after the double bond polymerization is completed, and the post-treatment includes conventional cleaning, softening, drying and other operations.
  • the biological material is dried after softening treatment: the drying process is one or more combinations of room temperature drying, blast drying, vacuum drying, and freeze drying.
  • the drying time is 1h to 10 days
  • the room temperature drying temperature is 10°C to 30°C
  • the blast drying or vacuum drying temperature is 15°C to 100°C
  • the freeze drying temperature is -20°C to -80°C.
  • step S2 Wash the biological material in step S1 with distilled water for 2 hours under gentle friction and fluid pressure at 4° C. and 100 RPM, until there is no visible adherent non-pericardium or non-collagen tissue;
  • step S3 soak the biological material cleaned in step S2 in an aqueous solution of arginine with a molar concentration of 10-100 mM, and soak for 12 hours at 37° C. to ensure sufficient physical penetration of arginine;
  • step S4 Add glutaraldehyde to the solution immersed in the biological material treated in step S3 for co-crosslinking.
  • the molar concentration of glutaraldehyde in the solution system is 10-800 mM, and react at 37° C. for 24 hours.
  • step S5 Soak the biological material treated in step S4 again in arginine solution (10-100 mM) for 2-48 hours.
  • step S6 Soak and wash the biological material treated in step S5 with distilled water to remove unreacted arginine and glutaraldehyde.
  • FIG. 2 A schematic diagram of the chemistry of this embodiment is shown in FIG. 2 .
  • Step 1 extracting the bioprosthetic valve material, and performing conventional pretreatment operations on the bioprosthetic valve material;
  • Step 2 soaking the biological material in the solution of the first functional monomer (amino-double bond compound);
  • Step 3 adding a crosslinking agent (glutaraldehyde) to the reaction system of step 2, co-crosslinking the first functional monomer (amino-double bond compound) and biological valve material, introducing free radicals (carbon-carbon double bond );
  • Step 4 soaking the biological material treated in Step 3 in the third functional monomer solution again.
  • Step five initiate the secondary crosslinking of free radical polymerization.
  • Step 6 After the secondary cross-linking, the biological material is cleaned and treated with glycerol, and the biological valve is preserved in a dry state or a wet state.
  • Step 1 extracting the bioprosthetic valve material, and performing conventional pretreatment operations on the bioprosthetic valve material;
  • Step 2 the amino-double bond compound solution (that is, the functional monomer, and also has a functional group) solution soaks the biological valve material;
  • Step 3 add glutaraldehyde (crosslinking agent) to the reaction system of step 2, carry out co-crosslinking to amino-double bond compound (functional monomer) and biological valve material, introduce carbon-carbon double bond (free radical) and functional groups;
  • Step 4 soaking the biological material treated in Step 3 in the amino-double bond compound (functional monomer) solution again.
  • Step five initiate the secondary crosslinking of free radical polymerization.
  • Step 6 After the secondary cross-linking, the biological material is cleaned and treated with glycerol, and the biological valve is preserved in a dry state or a wet state.
  • Step 1 extracting the bioprosthetic valve material, and performing conventional pretreatment operations on the bioprosthetic valve material;
  • Step 2 soaking the biological material in the solution of the first functional monomer (amino-double bond compound);
  • Step 3 adding a crosslinking agent (glutaraldehyde) to the reaction system in step 2, co-crosslinking the functional monomer (amino-double bond compound) and biological valve material, introducing free radicals (carbon-carbon double bond), Functional groups can also be further introduced;
  • a crosslinking agent glucosealdehyde
  • Step 4 soaking the biological material treated in Step 3 in the solution of amino-double bond compound (third functional monomer) again.
  • Step 5 soaking treatment of free radical polymerization monomer (second functional monomer);
  • Step six initiate the secondary crosslinking of free radical polymerization.
  • Step 7 after the secondary cross-linking, the biological material is cleaned and treated with glycerin, and the biological valve is preserved in a dry state or a wet state.
  • Step 1 extracting the bioprosthetic valve material, and performing conventional pretreatment operations on the bioprosthetic valve material;
  • Step 2 the amino-double bond compound (first functional monomer) solution soaks the biological material
  • Step 3 add glutaraldehyde (crosslinking agent) to the reaction system of step 2, carry out co-crosslinking to amino-double bond compound (first functional monomer) and biological valve material, introduce carbon-carbon double bond (free radical ), functional groups can also be further introduced;
  • Step 4 soaking the biological material treated in Step 3 in the solution of amino-double bond compound (third functional monomer) again.
  • Step 5 soaking treatment of free radical polymerization monomer (second functional monomer);
  • Step six initiate the secondary crosslinking of free radical polymerization.
  • Step 7 after the secondary cross-linking, the biological material is cleaned and treated with glycerin, and the biological valve is preserved in a dry state or a wet state.
  • step S2 Wash the biological material in step S1 with distilled water for 2 hours under gentle friction and fluid pressure at 4° C. and 100 RPM, until there is no visible adherent non-pericardium or non-collagen tissue;
  • step S3 soak the biological material cleaned in step S2 in an aqueous solution of DL-2-amino-4-pentenoic acid with a molar concentration of 10-100 mM, and soak for 12 hours at 37° C. to ensure that DL-2-amino-4-pentenoic acid Sufficient physical penetration of 4-pentenoic acid;
  • step S4 Add glutaraldehyde to the solution immersed in the biological material treated in step S3 to undergo copolymerization.
  • the molar concentration of glutaraldehyde in the solution system is 10-500 mM, and react at 37° C. for 24 hours.
  • step S5 Soak and wash the biological material treated in step S4 with distilled water to remove unreacted DL-2-amino-4-pentenoic acid and glutaraldehyde.
  • step S6 Soak the biological material treated in step S5 in a 5% aqueous solution of polyethylene glycol diacrylate for 12 hours at 37° C. to ensure sufficient physical penetration of polyethylene glycol diacrylate.
  • step S7 Add ammonium persulfate and sodium bisulfite initiators to the biological material treated in step S6 for initiation, and the molar concentration of ammonium persulfate and sodium bisulfite is 10-100 mM.
  • FIG. 7 A schematic diagram of the chemistry of this embodiment is shown in FIG. 7 .
  • step S3 using DL-2-amino-4-pentenoic acid/glutaraldehyde/pericardium co-crosslinking to introduce free radically polymerizable allyl groups is compared to the method reported in the literature Similar studies have a higher efficiency of introducing free radical polymerizable groups, and this scheme can further increase the degree of crosslinking of the pericardium while introducing allyl groups.
  • a biological valve material with excellent anti-calcification and anti-coagulation properties can be prepared, and the relative activity of lactate dehydrogenase is 0.1-0.25, and the amount of calcium attached is 30-50 ⁇ g/mg.
  • the copolymerization cross-linking method adopted is to use DL-2-amino-4-pentenoic acid and glutaraldehyde as the copolymerization cross-linking agent to introduce carbon-carbon double bonds on the biomaterial, and then add polyethylene glycol diacrylate , under the triggering of ammonium persulfate and sodium bisulfite initiators, polyethylene glycol-modified materials covalently bonded to the pericardium are obtained through copolymerization and cross-linking on the surface of the pericardium, which can improve the structural stability of biological valve materials anti-calcification and anticoagulant properties, potentially extending its lifespan.
  • step S1 The biological material in step S1 is washed with distilled water at 4°C and 100 RPM for 2 hours with gentle friction and fluid pressure, until there is no visible adherent non-pericardium or non-collagen tissue, and at the same time, it is achieved by osmotic shock Effective decellularization of pericardial tissue;
  • FIG. 8 the schematic diagram of the modification of hyaluronic acid and poly-lysine and the schematic diagram of the principle of partially double-bonded poly-lysine modified pericardium and double-bonded hyaluronic acid radical polymerization are shown in FIG. 8 .
  • This research strategy is to use double cross-linking, including glutaraldehyde cross-linking and free radical polymerization cross-linking, and the material has a high degree of cross-linking;
  • the binding mode of hyaluronic acid and pericardial material in this method is chemical covalent binding, which has higher stability.
  • glycidyl methacrylate was used to modify polylysine and hyaluronic acid respectively to obtain partially double-bonded polylysine and double-bonded hyaluronic acid.
  • glutaraldehyde Under the action of glutaraldehyde, the pericardium and partially double-bonded polylysine (having both amino groups and double bonds) were copolymerized and cross-linked to simultaneously realize the cross-linking and double-bond modification of the pericardium.
  • the hyaluronic acid-modified glutaraldehyde pericardial material was obtained by copolymerizing the double-bonded glutaraldehyde valve with the double-bonded hyaluronic acid free radical.
  • the present application further introduces double bonds and initiates post-crosslinking, that is, on the basis of glutaraldehyde crosslinking film, the first functional monomer (containing the first carbon-carbon double bond and ethylene oxide Alkyl) and glutaraldehyde cross-linked biofilm chemical reaction to introduce the first carbon-carbon double bond, this scheme is recorded as scheme six, which will improve the cross-linking degree, stability and mechanical properties of glutaraldehyde cross-linked biological valve material film and anti-calcification.
  • BS110 Soak the bio-valve material in an aldehyde-based cross-linking agent solution to cross-link; prepare glutaraldehyde-cross-linked bio-valve material;
  • BS120 Soak the glutaraldehyde-crosslinked biological valve material prepared in step BS110 in a solution containing a double bond reagent (the first functional monomer) to modify the double bond to prepare a double bonded biological valve material;
  • the bonding agent (first functional monomer) has at least one first carbon-carbon double bond and an oxiranyl group.
  • the first functional monomer after glutaraldehyde cross-links the bio-valve material, the first functional monomer, the double-bonding reagent solution, is further used to introduce the first carbon-carbon double bond to realize the glutaraldehyde-cross-linked bio-valve material
  • the double bond, the first functional monomer used, that is, the double bond reagent has both the first carbon-carbon double bond and the oxirane group.
  • the first functional monomer that is, the double bond agent to modify the glutaraldehyde cross-linked biological valve material
  • the monomer namely, the oxirane group in the double bond reagent, and the hydroxyl group, carboxyl group on the glutaraldehyde crosslinked biological valve material, and a small amount of amino groups remaining after glutaraldehyde crosslinking undergo a ring-opening reaction, and then undergo a ring-opening reaction in the glutaraldehyde crosslinking
  • the first carbon-carbon double bond is directly introduced into the biological valve material; further, the polymerization of these double bonds on the glutaraldehyde-crosslinked biological valve material is induced to realize the secondary cross-linking, and the post-crosslinking treatment of the biological valve material is completed.
  • the degree of cross-linking of the biological valve material after the secondary cross-linking will be
  • Step BS120 of the program
  • the double-bonding agent that is, the first functional monomer is selected from at least one of allyl glycidyl ether, glycidyl methacrylate and glycidyl acrylate.
  • the concentration of the double-bonding reagent in the solution containing the first functional monomer, that is, the double-bonding reagent is 1%-10% (w/w); the reaction time for the double-bonding modification is 2-120 hours.
  • the solvent in the solution containing the first functional monomer that is the double bond reagent is water, physiological saline, pH neutral buffer or methanol, ethanol, ethylene glycol, propanol, 1,2-propylene glycol, One or more of 1,3-propanediol, isopropanol, butanol, isobutanol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, glycerin aqueous solution .
  • the biofilm material treated in S110 is taken out, washed or directly placed in a solution containing a double-bonding reagent (the first functional monomer).
  • step S200 of the scheme
  • step BS120 The biological valve material treated in step BS120 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 in step BS120 to initiate the polymerization reaction, which is commonly known as a one-pot method.
  • the solvent in the solution containing the initiator is water, physiological saline or pH neutral buffer.
  • concentration of the initiator as mentioned above, in the one-pot method this concentration can be understood as the concentration of the initiator in the solution contained in the reaction system of step BS120, and in the step-by-step method, this concentration can be understood as the concentration of the solution containing the initiator. concentration in solution.
  • 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 persulfate
  • the suitable double bond polymerization time is 3-24 hours.
  • this solution has at least one of the following beneficial effects:
  • this scheme By introducing double bonds into the glutaraldehyde-crosslinked biological valve material, this scheme further triggers the polymerization of the double bonds, improves the stability of the glutaraldehyde-crosslinked material, and further reduces the calcification caused by structural degradation Therefore, it also has certain anti-calcification properties.
  • the biological valve products currently used for clinical implantation are almost all made of glutaraldehyde-crosslinked biological valve materials.
  • the collagen in the biological valve material can be cross-linked by the reaction of glutaraldehyde with the collagen matrix in the biological valve material And further reduce the immunogenicity of the biological valve material itself, and improve the mechanical strength of the biological valve material; however, the biological valve material still has the problem of low cross-linking degree after glutaraldehyde cross-linking and faces the risk of structural degradation and degradation , which will directly lead to the degradation of its components after implantation, causing its structural integrity to be damaged and structural degradation and decay to occur. Furthermore, the degradation of biological valve components will further promote the mechanical damage of its valve leaflet structure and induce calcification, which will affect the normal opening and closing movement of the valve and reduce the service life of biological valves with structural degradation.
  • glutaraldehyde-crosslinked biological heart valves are still the mainstream biological heart valves used clinically.
  • a series of post-crosslinking and modification based on glutaraldehyde crosslinking not only meet the actual production needs but also have great significance for scientific research.
  • the present application treats the glutaraldehyde cross-linked bio-valve material under conditions based on glutaraldehyde cross-linking, and further introduces carbon into the glutaraldehyde-cross-linked bio-valve material.
  • the double bond is used as a platform for secondary cross-linking.
  • the glutaraldehyde-cross-linked bio-valve material By initiating a copolymerization reaction between the double bond in the double-bonded glutaraldehyde-cross-linked bio-valve material and the double bond of the functional monomer, the glutaraldehyde-cross-linked bio-valve material is introduced
  • the polymer network of functional monomers further expands the cross-linked network, that is, on the basis of scheme six, further introduces the second carbon-carbon double bond through physical penetration of the second functional monomer (containing the second carbon-carbon double bond), and records
  • this will increase the degree of cross-linking of the glutaraldehyde cross-linked biological valve material membrane, improve its structural stability, and further reduce the degree of calcification of the material to improve its anti-calcification performance.
  • BS110 Soak the bio-valve material in an aldehyde-based cross-linking agent solution to cross-link; prepare glutaraldehyde-cross-linked bio-valve material;
  • BS120 Soak the glutaraldehyde-crosslinked biological valve material prepared in step BS110 in a solution of a double bond reagent (the first functional monomer) to modify the double bond to prepare a double bonded biological valve material; the double bond
  • the chemical reagent (the first functional monomer) has at least one first carbon-carbon double bond and an oxirane group.
  • step BS130 soaking the double-bonded biological valve material obtained in step BS120 with a solution of a functional monomer (second functional monomer), the second functional monomer having at least one second carbon-carbon double bond;
  • step BS130 Adding an initiator to the solution after soaking in step BS130, making it contact with the biological valve material and the functional monomer solution to initiate double bond polymerization.
  • the biological valve material is further introduced into the first carbon-carbon double bond by using a double bond reagent (first functional monomer) solution to achieve glutaraldehyde cross-linking.
  • a double bond reagent (first functional monomer) solution to achieve glutaraldehyde cross-linking.
  • the double bond of the biological valve material is used as a platform for secondary cross-linking, and the double bond reagent (first functional monomer) used has both carbon-carbon double bonds and oxirane groups.
  • Figure 70 In order to facilitate the understanding of the chemical principles involved in this scheme, take the example shown in Figure 70 as an example to further illustrate: use the double bond reagent (the first functional monomer) to modify the glutaraldehyde cross-linked biological valve material, and through the double bond
  • the oxirane group in the chemical reagent (the first functional monomer) undergoes a ring-opening reaction with the hydroxyl group, carboxyl group on the glutaraldehyde cross-linked biological valve material, and a small amount of amino groups remaining after glutaraldehyde cross-linking, and then in the glutaraldehyde Introduce the first carbon-carbon double bond into the cross-linked biological valve material; further, the second functional monomer further introduces the second carbon-carbon double bond through physical penetration;
  • the double bond in the valve material and the double bond of the functional monomer undergo a copolymerization reaction, and the functional monomer polymer is introduced as a cross-linked network to realize further secondary cross-linking and complete the post-
  • the biological valve material 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 into the polymer of the functional monomer as a cross-linking network through the copolymerization of the functional monomer, the biological valve material is expanded.
  • the cross-linking network, the cross-linking degree of the cross-linked biological valve material after the 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.
  • step BS120 of the scheme selection of double-bonding reagent (first functional monomer), concentration of double-bonding reagent (first functional monomer), solvent selection of double-bonding reagent (first functional monomer), double-bonding reagent (first functional monomer) solution solvent selection,
  • the reaction time and operation process of the bonding modification are the same as those in Scheme 6. I won't repeat them here.
  • Step BS130 of the program
  • the second functional monomer has at least one second carbon-carbon double bond.
  • the second functional monomer is 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-di Bis(oxyl))bis(ethane-2,1-diyl)diacrylate, N,N'-dimethylacrylamide, N,N-dimethylmethacrylamide, double bonded poly One or more of lysine.
  • 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) .
  • the solvent of the second functional monomer solution is one or a mixture of water, physiological saline, ethanol, isopropanol or pH neutral buffer solution.
  • the soaking time in the second functional monomer solution is 0.5h-120h.
  • step S200 of the scheme
  • step BS120 The biological valve material treated in step BS120 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 in step BS120 to initiate the polymerization reaction, which is commonly known as a one-pot method.
  • the solvent in the solution containing the initiator is water, physiological saline or pH neutral buffer.
  • concentration of the initiator as mentioned above, in the one-pot method this concentration can be understood as the concentration of the initiator in the solution contained in the reaction system of step BS120, and in the step-by-step method, this concentration can be understood as the concentration of the solution containing the initiator. concentration in solution.
  • 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 persulfate
  • the reaction time of step S200 is 3-24 hours.
  • the solution has at least one of the following beneficial effects:
  • this scheme introduces carbon-carbon double bonds into the glutaraldehyde-crosslinked biological valve material through double bond modification as the basis for secondary crosslinking, further By initiating the polymerization between the carbon-carbon double bonds on the glutaraldehyde-crosslinked biological valve material and the carbon-carbon double bonds on the functional monomers, the functional monomer polymer crosslinking network is introduced to achieve secondary crosslinking, which can be further Improve the degree of cross-linking of biological valve materials.
  • the scheme further triggers the relationship between the carbon-carbon double bond on the double-bonded biological valve material and the carbon-carbon double bond on the functional monomer.
  • Polymerization by introducing functional monomer polymer cross-linked network, the cross-linked network can further reduce the combination of collagenase in the body and the collagen matrix on the biological valve material through physical blocking to a certain extent, and protect the biological valve material collagen
  • the matrix improves the stability of the glutaraldehyde-crosslinked biological valve material, further reduces the risk of calcification caused by the structural degradation of the biological valve material, and therefore also has certain anti-calcification properties.
  • the scheme After introducing carbon-carbon double bonds on the glutaraldehyde cross-linked bio-valve material, the scheme further triggers the carbon-carbon double bond on the glutaraldehyde-cross-linked bio-valve material and the carbon carbon on the functional monomer.
  • the degradation of biological valve components will further promote the mechanical damage of its valve leaflet structure and induce calcification, which will affect the normal opening and closing movement of the valve and reduce the service life of biological valves with structural degradation. Although it is less thrombogenic than mechanical valves, thrombus still exists in biological valves, which will destroy the normal function of biological valves and bring the risk of secondary valve replacement. On the other hand, the occurrence of calcification will directly lead to the failure of biological valves.
  • the biological heart valve material is treated with double bonds under the condition of glutaraldehyde cross-linked bio-valve material, and carbon-carbon double bonds are introduced into the glutaraldehyde-cross-linked bio-valve material as functionalization Copolymerization and cross-linking platform, by initiating the double-bond glutaraldehyde cross-linked bio-valve material copolymerization reaction with the double bond of the functional monomer, introducing a functional polymer network on the glutaraldehyde-cross-linked bio-valve material, Further expand the cross-linking network to realize the functional copolymerization cross-linking after the double bond of the biological valve material, that is, on the basis of the seventh scheme, the second functional monomer also has a functional group B, which is recorded as the eighth scheme.
  • BS110 Soak the bio-valve material in an aldehyde-based cross-linking agent solution to cross-link; prepare glutaraldehyde-cross-linked bio-valve material;
  • BS120 Soak the glutaraldehyde-crosslinked biological valve material prepared in step BS110 in a solution of a double bond reagent (the first functional monomer) to modify the double bond to prepare a double bonded biological valve material; the double bond
  • the chemical reagent (the first functional monomer) has at least one first carbon-carbon double bond and an oxirane group.
  • step BS130 soaking the double-bonded biological valve material obtained in step BS120 with a second functional monomer solution, the second functional monomer has at least one second carbon-carbon double bond and at least one functional group B;
  • step BS130 Adding an initiator to the solution after soaking in step BS130, making it contact with the biological valve material and the functional monomer solution to initiate double bond polymerization.
  • the biological valve material is further introduced into the first carbon-carbon double bond by using a double bond reagent (first functional monomer) solution to achieve glutaraldehyde cross-linking.
  • the double bond of biological valve material is used as a platform for secondary cross-linking.
  • the double bond agent (first functional monomer) used has both carbon-carbon double bonds and oxirane groups, and the second functional monomer has a second Carbon-carbon double bond and functional group B.
  • the biological valve material 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 into the polymer of the functional monomer as a cross-linking network through the copolymerization of the functional monomer, the biological valve material is expanded.
  • the cross-linking network, the cross-linking degree of the biological valve material treated with functionalized copolymerization cross-linking after the double bond will be significantly improved, and its structural stability and anti-calcification performance will also be significantly improved with the introduction of the functional polymer network.
  • the functional polymer network is introduced into the biological valve treatment through double bond post-functionalization copolymerization, so that the biological valve material is rich in functional groups, thus endowing the biological valve material with the corresponding performance of the functional group; functionality
  • the group B may be selected from hydroxyl, carboxyl, carboxylic acid choline, sulfonic acid choline, phosphoryl choline, pyrrolidone, sulfonic acid group, carboxylate ion, sulfonate, sulfoxide, amide group, methoxyl group, These groups can combine water molecules with water molecules through hydrogen bonds and ionic hydration, which further enhances the hydrophilicity of the surface of the biological valve material, and forms a certain hydration layer on the biological valve to resist excessive adhesion of proteins and cells in the body. Improve antithrombotic properties and biocompatibility.
  • Hydroxyl As a hydrophilic group, it improves the surface hydrophilicity of biological materials to achieve anticoagulant effect;
  • Carboxyl group As a hydrophilic group, it improves the surface hydrophilicity of biological materials to achieve anticoagulant effect;
  • Carboxylate ions and sulfonic acid groups through ionic hydration, the surface hydrophilicity of biological materials can be improved to achieve anticoagulant effect;
  • Sulfoxide, pyrrolidone as a hydrophilic group, improve the surface hydrophilicity of biological materials to achieve anticoagulant effect;
  • Zwitterions Improve the surface hydrophilicity of biological materials through ionic hydration to achieve anticoagulant effects; it is beneficial to form an electrically neutral surface of biological valves and reduce the adsorption of calcium ions to achieve anti-calcification effects;
  • Polyethylene glycol As a hydrophilic group, it improves the surface hydrophilicity of biological materials; increases the steric hindrance between calcium ions and collagen, and improves the surface hydrophilicity of biological valve materials;
  • Urethane group, urea group as a hydrophilic group, improve the surface hydrophilicity of biological materials to achieve anticoagulant effect;
  • Urethane group As a hydrophilic group, it improves the surface hydrophilicity of biological materials to achieve anticoagulant effect.
  • Amide As a hydrophilic group, it improves the surface hydrophilicity of biomaterials to achieve anticoagulant effects; as a toughening group, it can dynamically adjust the elasticity of biomaterials to improve the utilization of biomaterials.
  • the prepared Valve hydrodynamic performance is excellent.
  • step BS120 of the scheme selection of double-bonding reagent (first functional monomer), concentration of double-bonding reagent (first functional monomer), solvent selection of double-bonding reagent (first functional monomer), double-bonding reagent (first functional monomer) solution solvent selection,
  • the reaction time and operation process of the bonding modification are the same as those in Scheme 6. I won't repeat them here.
  • Step BS130 of the program
  • the second functional monomer has at least one second carbon-carbon double bond and at least one functional group B.
  • the second functional monomer is acrylamide, acrylic acid, sodium acrylate, methacrylic acid, sodium methacrylate, 2-(prop-2-enamido)acetic acid, 2-acrylamide-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-methylpropane -2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, 2-methacryloyloxyethylphosphorylcholine, N-(2-hydroxyethyl)acrylamide, N-( Methoxymethyl) methacrylamide, 2-acrylamide-2-methylpropanesulfonic acid, 2-acrylamide,
  • the concentration of the second functional monomer solution is 0.1%-6% (v/v).
  • the solvent of the second functional monomer solution is one or a mixture of water, physiological saline, ethanol, isopropanol or pH neutral buffer solution.
  • the soaking time in the second functional monomer solution is 0.5h-120h.
  • step S200 of the scheme
  • step S200 of this solution is the same as the processing of step S200 of solution 7, and will not be repeated here.
  • the solution has at least one of the following beneficial effects:
  • the scheme is modified on the basis of glutaraldehyde cross-linked bio-valve material, and carbon carbon is introduced into the glutaraldehyde-cross-linked bio-valve material through the reaction of double bond reagents with glutaraldehyde-cross-linked bio-valve material Double bond, the obtained double bonded glutaraldehyde cross-linked bio-valve material is used as a platform for functional copolymerization and cross-linking, further by triggering the carbon-carbon double bond on the glutaraldehyde cross-linked bio-valve material and the carbon-carbon double bond on the functional monomer Polymerization between bonds to introduce functional monomer polymers as a functionalized crosslinking network to achieve functionalized copolymerization and crosslinking can further improve the degree of crosslinking of biological valve materials and introduce functional groups. By increasing the degree of cross-linking, the stability of biological valve materials will be improved.
  • the solution is to introduce carbon-carbon double bonds into the glutaraldehyde-crosslinked bioprosthetic material, and further trigger the polymerization between the carbon-carbon double bonds on the double-bonded bioprosthetic material and the carbon-carbon double bonds on the functional monomers to
  • a functional polymer cross-linked network is introduced on the biological valve material, which can act as a polymer barrier to a certain extent to reduce the contact and interaction between the collagenase in the body and the collagen matrix on the biological valve material, significantly reducing the Collagenase degrades the collagen matrix on the biological valve material, improves the stability of the glutaraldehyde-crosslinked biological valve material, and further reduces the risk of structural degradation of the biological valve caused by the structural degradation of the biological valve material.
  • this scheme further triggers the double-bonded carbon-carbon double bond on the glutaraldehyde-crosslinked bioprosthetic material and the carbon-carbon double bond on the functional monomer.
  • Polymerization between polymers introduces a functional polymer cross-linked network, which can serve as a polymer barrier to further reduce the binding of calcium ions to the mineralized areas on the bioprosthetic valve material that are easy to combine with calcium ions, reducing the risk of calcification. And then play the role of anti-calcification.
  • the functional group hydroxyl, carboxyl, carboxylic acid choline, sulfonic acid choline, phosphorylcholine, pyrrolidone, sulfonic acid group, carboxylate ion, sulfonic acid ester, sulfoxide, amide group, methoxyl group, It can bind water molecules with water molecules through hydrogen bonds and ion hydration, which further enhances the hydrophilicity of the surface of biological valve materials, forms a certain hydration layer in the body to resist excessive adhesion of proteins and cells, and improves antithrombotic performance and biocompatibility.
  • the concentration of the glutaraldehyde solution is 0.1%-5% (w/w), and the cross-linking time can be any time between 0.5h-120h.
  • dehydration and drying treatment after the double bond polymerization is completed to form a dry film.
  • the biological valve material is routinely cleaned, softened, and then dehydrated and dried.
  • the cleaning solution can be one or a mixture of water, normal saline, ethanol, isopropanol or pH neutral buffer solution, and the pH can be adjusted to 5.0-9.5 before use and during use, or not adjusted .
  • the dehydration treatment is to expose the membrane with double bonds polymerized or the valve sewn from the membrane to a dehydration solution.
  • the dehydration solution is a mixed solution of alcohol solution and water, the alcohol solution accounts for 20-90% (v/v), and the alcohol reagent can be one or both of ethanol and isopropanol. kind of mixture.
  • the drying treatment is to expose the dehydrated membrane or valve to softener solution, and the treatment time is 20min-10h.
  • the main component of the softener solution is a mixed solution of one or two of glycerin and polyethylene glycol, the concentration of glycerin is 10-100% (v/v), and other components are water, ethanol, iso One or several kinds of propanol, accounting for 0-90% (v/v).
  • the way of sterilizing the valve after the drying treatment may be one of ethylene oxide sterilization or electron beam sterilization.
  • the bio-valve material prepared by the above method can be used for intervening bio-valve, such as through minimally invasive intervention; it can also be used for surgical bio-valve, such as implanting through surgery.
  • an artificial heart valve in one embodiment, including a stent 1 and a leaflet 2 connected in the stent 1.
  • the stent is generally cylindrical in shape, and the side wall is a hollow grid structure.
  • the stent The inside is a blood flow channel, and the multiple leaflets cooperate with each other to control the opening and closing of the blood flow channel in the stent.
  • the corresponding materials are selected during processing, such as nickel-titanium alloy with shape memory and self-expanding in vivo, or stainless steel released by ball expansion, etc.
  • the stent itself can be formed by cutting tubes or braiding wires , the leaflets can be connected to the bracket by stitching, bonding or integral molding.
  • positioning structures that can interact with surrounding native tissues can also be provided on the periphery of the stent, such as anchors, arms, etc.
  • skirts can also be provided on the inside and/or outside of the stent. Weekly leakage materials, etc.
  • the valve leaflets, the skirt or the anti-peripheral leakage material can all use the biological valve material of the above embodiments.
  • the artificial heart valve 3 and the corresponding delivery system form a valve intervention system.
  • the delivery system includes the catheter assembly 4 and the handle for controlling the catheter assembly.
  • the artificial heart valve is in a radially compressed state when it is delivered in the body. In vivo release of the catheter assembly or balloon dilation and radial expansion.
  • porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, then soaked in 30mM DL-2-amino-4-pentenoic acid aqueous solution at 37°C for 12 hours, and then Glutaraldehyde was added to make the concentration 100 mM, soaked for 24 hours at 37° C. and 100 RPM, and then washed with distilled water.
  • the glutaraldehyde treatment group was set as the control group 1, that is, the pericardium was soaked in 0.625% glutaraldehyde for 24 hours.
  • Control Example 1 Wash the freshly collected porcine pericardium in distilled water at 4°C and 100 RPM for 2 hours until there is no visible adherent non-pericardium or non-collagenous tissue, and then soak the cleaned biological material in 0.625% glutaraldehyde , soaked at 37° C. for 24 hours to prepare a biological valve material, which is referred to as Control Example 1.
  • the biological valve material of the above-mentioned Example 1 and Comparative Example 1 was selected to measure the relative activity of lactate dehydrogenase.
  • the measurement method is as follows: wash the sample to be tested (the original film with a diameter of 6 mm) with 0.9% normal saline for 5 minutes, incubate with 100 ⁇ L of platelet-rich plasma in a 96-well plate at 37°C for 1 hour, suck out the serum, and use The surface of the sample was washed three times with PBS, five minutes each time. The positive control was 100 ⁇ L of platelet-rich plasma.
  • the lactate dehydrogenase release detection kit (Beyotime Biotechnology, Shanghai, China) was used to detect the relative content of adhered platelets on the sample surface according to the instructions. The absorbance value at 490nm was determined using a microplate reader (BioTek Synergy H1, USA).
  • the biovalve materials of the above-mentioned Example 1 and Comparative Example 1 were arbitrarily selected to measure the amount of calcium attached.
  • the method for determining the amount of attached calcium is as follows: the sample to be tested (1cm ⁇ 1cm in size) is rinsed in 0.9% physiological saline for 5 minutes. Samples were surgically implanted in two subcutaneous pockets in the central dorsal wall region of 45-50 g male Sprague-Dawley rats (one sample per rat per group). After 30 days, the implanted samples were removed from the dorsal wall of the rats. After removing the fibrous capsule around the sample, it was freeze-dried and weighed dry.
  • Lactate dehydrogenase relative activity Glutaraldehyde control group 1 0.410 ⁇ 0.072
  • Example 1 0.100 ⁇ 0.019
  • 100U/mL type I collagenase Invitrogen, NY, USA
  • Example 1 The weight loss rate (%) of Example 1: 3.234 ⁇ 0.125; the weight loss rate (%) of Comparative Example 1: 8.036 ⁇ 0.760.
  • modified hyaluronic acid Preparation of modified hyaluronic acid: Weigh 2 g of sodium hyaluronate with a molecular weight of 10,000 and dissolve it in 20 ml of PBS, then add 6.5 ml of glycidyl methacrylate and 4.5 ml of triethylamine in sequence. Place on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 5000 for 7 days, freeze-dry to obtain double-bonded hyaluronic acid;
  • modified polylysine Polylysine was dissolved in deionized water, and then glycidyl methacrylate (glycidyl methacrylate: amino group) was added in a molar ratio of (1:1.5). The mixture was placed on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 1000 to dialyze for 7 days, and freeze-dry to obtain partially double-bonded poly-lysine;
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 180 mM modified polylysine aqueous solution at room temperature for 12 hours, and then added glutaraldehyde solution To a mass concentration of 2.5%, react on a shaker at 37°C for 24 hours, take out the pericardial material and wash it, soak it in a 50mg/ml modified hyaluronic acid aqueous solution at room temperature for 12 hours, then use 2.5% ammonium persulfate and 0.25% N, N,N',N'-tetramethylethylenediamine was soaked at 37°C for 12 hours, and finally washed with distilled water, which was designated as sample 2.
  • the water contact angle test, the lactate dehydrogenase activity test, the hemolysis rate test and the calcification test were respectively performed on the sample 2 prepared in Example 2 and the control group 2 samples.
  • Control group 2 Wash the freshly collected porcine pericardium with distilled water for 2 hours at 4°C and 100 RPM, and then soak it in a glutaraldehyde solution with a mass concentration of 0.625% for 24 hours. After the reaction is completed, take it out and soak it in 0.2 % glutaraldehyde solution, recorded as control sample 2.
  • the materials of the control group and Example 1 were cut into square pieces of 1*1cm, placed between two glass pieces, flattened, vacuum freeze-dried, and tested for water contact angle.
  • Lactate dehydrogenase activity test Fresh rabbit blood was collected and centrifuged at 1500 rpm for 15 minutes to obtain platelet-rich plasma. The materials of the control group and Example 1 were cut into discs with a diameter of 10 mm and washed three times with PBS, put into a 48-well plate, added with 100 ⁇ L of platelet-rich plasma and soaked at 37° C. for 1 h. 100 ⁇ L of platelet-rich plasma was selected as a positive control for quantitative detection. After incubation, wash 3 times with PBS. The relative amount of platelet adhesion was determined with a lactate dehydrogenase assay kit. The absorbance at 490nm of each group was recorded with a microplate reader, and the relative activity of lactate dehydrogenase in each group was calculated, and the relative number of platelets was expressed by the relative activity of lactate dehydrogenase.
  • the test results of the water contact angle are shown in a to c in Figure 12.
  • the control group is the glutaraldehyde treatment group, i.e. Control Example 2
  • the experimental group is the hydrophilic treatment group, i.e. Example 2.
  • the water contact angle of the experimental group is reduced.
  • the final water contact angle results of Example 2 and glutaraldehyde control group 2 are shown in Table 3.
  • the detection results of lactate dehydrogenase activity and hemolysis rate are shown in Figure 13, where a is the comparison result of lactate dehydrogenase activity, b is the comparison result of hemolysis rate, c is the comparison result of calcium ion concentration, and the control group is the glutaraldehyde treatment group That is, the control example 2, the experimental group is the hydrophilic treatment group, that is, the embodiment 2, and the lactate dehydrogenase activity and hemolysis rate of the experimental group are both reduced. Further, the results of the final lactate dehydrogenase activity and hemolysis rate of Example 2 and the glutaraldehyde control group 2 are shown in Table 4.
  • the results of the calcium ion concentration detection are shown in Figure 14.
  • the control group is the glutaraldehyde experimental group, i.e. the control example 2, and the experimental group is the hydrophilic treatment group, i.e. the embodiment 2, and the calcium ion content in the experimental group is all reduced. Further, the final calcium ion concentration results of Example 2 and glutaraldehyde control group 2 are shown in Table 5.
  • the method provided in this embodiment can improve the hydrophilic performance, blood compatibility and anti-calcification ability of the biological material, potentially prolonging its service life.
  • the freshly collected porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 100 mM glutaraldehyde solution, and cross-linked at room temperature for 24 hours at 100 RPM to obtain control sample 3.
  • the preparation process is shown in FIG. 3 .
  • a method for preparing a biological valve material by co-crosslinking combined with double bond crosslinking is shown in FIG. 15 for the basic principle.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, then soaked in 20mM 2-methallylamine aqueous solution at 37°C for 2 hours, and then added glutaraldehyde to make the final concentration 100mM , soaked for 24 hours at 37°C, 100RPM rotating speed shaking condition. Take out the pig pericardium and wash it with distilled water. After cleaning, soak in 20 mM 2-methylallylamine aqueous solution at 37° C.
  • sample 3 For the convenience of distinguishing the samples prepared in each embodiment, the sample obtained in this embodiment is designated as sample 3.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, then soaked in 20mM 3-buten-1-amine aqueous solution at 37°C for 2 hours, and then added glutaraldehyde to make the final concentration 100mM, soak for 24 hours at 37°C, shaking at 100RPM. Take out the pig pericardium and wash it with distilled water. After cleaning, soak in deionized water, add ammonium persulfate and sodium bisulfite initiators to initiate, the molar concentrations of ammonium persulfate and sodium bisulfite are both 30mM, and react at 37°C for 24 hours.
  • the sample obtained in this embodiment is designated as sample 4.
  • the freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, then soaked in 20mM 2-aminoethyl methacrylate aqueous solution at 37°C for 2 hours, and then added glutaraldehyde to make it final.
  • the concentration is 100mM, and soaked for 24 hours at 37°C and shaking at 100RPM.
  • the sample obtained in this embodiment is designated as sample 5.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, and then soaked in an aqueous solution containing 10mM 2-aminoethyl methacrylate and 10mM 2-methallylamine at 37°C for 2 hours , and then add glutaraldehyde to make the final concentration 100mM, and soak for 24 hours at 37°C and 100RPM rotating speed shaking condition. Take out the pig pericardium and wash it with distilled water.
  • the freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, then soaked in 20mM 2-aminoethyl methacrylate aqueous solution at 37°C for 2 hours, and then added glutaraldehyde to make it final.
  • the concentration is 100mM, and soaked for 24 hours at 37°C and shaking at 100RPM.
  • sample 7 After cleaning, soak in distilled water, add ammonium persulfate and sodium bisulfite initiators to initiate, the molar concentration of ammonium persulfate and sodium bisulfite are both 30mM, and react at 37°C for 24 hours.
  • the sample obtained in this embodiment is designated as sample 7.
  • the freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, then soaked in 20mM 2-aminoethyl methacrylate aqueous solution at 37°C for 2 hours, and then added glutaraldehyde to make it final.
  • the concentration is 100mM, and soaked for 24 hours at 37°C and shaking at 100RPM.
  • ammonium persulfate and sodium bisulfite initiators were added directly to the solution for initiation, the molar concentrations of ammonium persulfate and sodium bisulfite were both 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • the sample obtained in this embodiment is designated as sample 8.
  • modified hyaluronic acid Preparation of modified hyaluronic acid: Weigh 2 g of sodium hyaluronate with a molecular weight of 10,000 and dissolve it in 20 ml of PBS, then add 6.5 ml of glycidyl methacrylate and 4.5 ml of triethylamine in sequence. Place on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 5000 for 7 days, freeze-dry to obtain double-bonded hyaluronic acid;
  • modified polylysine Dissolve polylysine in deionized water, and then add glycidyl methacrylate (glycidyl methacrylate: amino group) at a molar ratio of (1:1.5). The mixture was placed on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 1000 to dialyze for 7 days, and freeze-dry to obtain partially double-bonded poly-lysine;
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, and then soaked in 60 mM (calculated as lysine) modified poly-lysine aqueous solution at room temperature for 12 hours.
  • Sample 5 sample 6, sample 7, sample 8 and control group 3 were cut into circular sheets with a diameter of 1 cm, and 6 parallel samples were set for each group. All circular sheet samples were placed in a 48-well plate, frozen overnight at minus 80°C and then transferred to a vacuum freeze dryer for 48 hours to freeze dry. The weight of each sample was weighed on a one-hundred-thousandth balance and recorded as the initial weight (W0) and returned to the 48-well plate.
  • Enzyme degradation experiments were carried out on sample 5, sample 6, sample 7, sample 8 and control group 3 to characterize the cross-linking efficiency of each group of samples, and collagenase I was used to treat sample 5, sample 6, sample 7, sample 8 and control group 3
  • the enzymatic degradation weight loss rate of each group of samples was calculated as shown in Table 6.
  • the enzymatic degradation weight loss rate of sample 5, sample 6, sample 7, and sample 8 was lower than that of control group 3, which indicated that the enzymatic degradation stability of sample 5, sample 6, sample 7, and sample 8 was higher than that of control group 3, that is, sample 5.
  • the cross-linking efficiency of sample 6, sample 7 and sample 8 is higher.
  • the results of enzyme degradation experiments show that the method of the present application for preparing biological valve materials by co-crosslinking combined with double bond crosslinking can improve the crosslinking degree of biological valve materials.
  • Alizarin red staining experiment is carried out to control group 3 and the sample gained in embodiment 3 ⁇ 8, alizarin red staining experiment:
  • Sample 3 sample 4, sample 5, sample 6, sample 7, sample 8 and control group 3 were implanted subcutaneously in rats and taken out after 30 days, and fixed with paraformaldehyde tissue fixative. After the fixation, take it out and repair it with a scalpel, then transfer it to the dehydration box.
  • Material samples were subjected to gradient dehydration with 50%, 75%, 85%, 95% (v/v) and absolute ethanol. After dehydration, the material samples were transferred to an embedding machine for embedding with melted paraffin, and then transferred to a -20°C refrigerator to cool and trim the shape. Sections 3-5 ⁇ m thick were cut from trimmed wax blocks on a microtome, transferred from the spreader to glass slides and dewaxed and rehydrated. The sections were stained with Alizarin Red staining solution for 3 minutes, washed with water, dried and then permeabilized with xylene for 5 minutes. After the sections were sealed with neutral gum, images of staining results were collected on a pathological slide scanner
  • Sample 3, sample 4, sample 5, sample 6, sample 7, sample 8 and control group 3 after being implanted subcutaneously in rats for 30 days were stained by alizarin red to characterize the degree of calcification of samples in each group.
  • Figure 16-22 shows the images of the Alizarin Red staining results of the sample slices 30 days after being implanted subcutaneously in rats, where the darker the color of the sample after Alizarin Red staining, the higher the degree of calcification.
  • Freshly collected porcine pericardium was washed in distilled water at 4°C and 100 RPM for 2 hours, then soaked in 100 mM glutaraldehyde solution, and cross-linked at room temperature for 24 hours at 100 RPM to obtain control sample 4.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking, and then soaked in 20mM 2-aminopent-4-enoic acid aqueous solution for 4 hours at 37°C;
  • Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the sample obtained after reacting at 37° C. for 24 hours was recorded as sample No. 13.
  • Enzyme degradation experiments were carried out on sample No. 12, sample No. 13 and control group 4 to characterize the degree of cross-linking of samples in each group, and the enzymatic degradation of samples in each group was calculated after treating sample No. 12, sample 13 and control group 4 with collagenase I
  • the weight loss rate is shown in Table 7.
  • the enzymatic degradation weight loss rate of sample No. 12 and sample No. 13 was lower than that of control group 4, which indicated that the enzymatic degradation stability of sample No. 12 and sample No. 13 was higher than that of control group 4.
  • the connection is higher.
  • the results of the enzyme degradation experiment show that the method for preparing the bio-valve material by double bond post-crosslinking in this embodiment can improve the cross-linking degree of the bio-valve material.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking, and then soaked in 20mM 2-aminopent-4-enoic acid aqueous solution for 4 hours at 37°C;
  • the pig pericardium was transferred to 50mM 2-aminopent-4-enoic acid aqueous solution and soaked for 4 hours;
  • Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of ammonium persulfate and sodium bisulfite were both 30 mM, and the sample obtained after reacting at 37° C. for 24 hours was recorded as sample No. 14.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking, and then soaked in 20mM 2-aminopent-4-enoic acid aqueous solution for 4 hours at 37°C;
  • the pericardium was immersed in distilled water, and ammonium persulfate and sodium bisulfite initiators were added to initiate.
  • the molar concentrations of ammonium persulfate and sodium bisulfite were both 30 mM, and reacted at 37 ° C for 24 hours, followed by washing with distilled water and using
  • the dry film sample obtained by dehydration of glycerin is recorded as No. 15 sample.
  • modified hyaluronic acid Preparation of modified hyaluronic acid: Weigh 2 g of sodium hyaluronate with a molecular weight of 10,000 and dissolve it in 20 ml of PBS, then add 6.5 ml of glycidyl methacrylate and 4.5 ml of triethylamine in sequence. Place on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 5000 for 7 days, freeze-dry to obtain double-bonded hyaluronic acid;
  • modified polylysine Dissolve polylysine in deionized water, and then add glycidyl methacrylate (glycidyl methacrylate: amino group) at a molar ratio of (1:1.5). The mixture was placed on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 1000 to dialyze for 7 days, and freeze-dry to obtain partially double-bonded poly-lysine;
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, and then soaked in 60 mM (calculated as lysine) modified poly-lysine aqueous solution at room temperature for 12 hours.
  • the samples (No. 10 sample, No. 11 sample, No. 12 sample, No. 13 sample, No. 14 sample, No. 15 sample and control sample 4) of control example 4 and embodiment 10 ⁇ 15 gained samples carry out subcutaneous implantation experiment in rats. After 30 days of implantation, the samples of each group were taken out for Alizarin red staining experiment to characterize the degree of calcification of each group of samples after 30 days of subcutaneous implantation in rats.
  • Alizarin red staining experiment was carried out on samples No. 10, No. 11, No. 12, No. 13, No. 14, No. 15 and control sample 4 by the same method as the alizarin red staining experiment described above.
  • sample No. 15 is obvious It is lighter, which shows that the calcification degree of sample No. 10, sample No. 11, sample No. 12, sample No. 13, sample No. 14, and sample No. 15 is lower than that of control sample 3, that is, sample No. 10, sample No. 11, sample No. 12, Compared with control group 4, sample No. 13, sample No. 14, and sample No. 15 have a certain anti-calcification effect.
  • the Alizarin Red staining results of samples No. 10, No. 11, No. 12, No. 13, No. 14, No. 15 and control group 4 after 30 days of implantation into the subcutaneous skin of rats showed that the double bond post-transplantation of this application
  • the method of joint preparation of biological valve materials can improve the anti-calcification performance of biological valves.
  • Control samples 4, 12, and 13 with uniform surface and thickness were cut into sheets with a diameter of 1 cm, rinsed with normal saline, drained and placed in a 24-well plate, and 300 ⁇ L of rabbit blood was added to each well and incubated at 37 Incubate with shaking at 70 bpm for 1 hour at °C. After the incubation, the rabbit blood was discarded, and 500 ⁇ L of normal saline was added to each well, and the unadhered blood was washed away under slight shaking of the shaker. After washing, the samples were transferred to 2.5% (w/w) glutaraldehyde solution for fixation for 4 hours.
  • the fixed samples were dehydrated with graded ethanol (25%, 50%, 75% and 100%, v/v), 20 minutes per grade.
  • the dried samples were fixed on the test bench with conductive glue and sprayed with gold.
  • the images of blood adhesion on each group of samples were observed and taken on a field emission scanning electron microscope.
  • control sample 4 sample No. 12, and sample No. 13 in the blood contact experiment are shown in Figures 31 to 33.
  • FIGs 31 to 33 After contacting and incubating with rabbit blood, more blood cell adhesion and aggregation were observed in the scanning electron microscope image of control sample 4, while less blood cells adhered to samples No. 12 and No. 13, and only a few blood cells adhered scatteredly to the surface.
  • the results show that samples No. 12 and No. 13 can inhibit the adhesion of blood cells to a certain extent, thereby reducing the risk of coagulation, and have an anticoagulant effect.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 100 mM glutaraldehyde solution, and cross-linked for 24 hours at room temperature with 100 RPM.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • sample 17 Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • sample 17 the sample obtained in this embodiment is designated as sample 17.
  • reaction schematic diagram of this embodiment is shown in FIG. 34 .
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • sample 18 Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • sample 18 the sample obtained in this embodiment is designated as sample 18.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • sample 19 the sample obtained in this embodiment is designated as sample 19.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM with shaking.
  • N,N'-ethylene bisacrylamide to make the final concentration 5%, and soak at 37°C for 12 hours to ensure sufficient physical penetration of N,N'-ethylene bisacrylamide.
  • sample 20 Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • sample 20 the sample obtained in this embodiment is designated as sample 20.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • sample 21 Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • sample 21 the sample obtained in this embodiment is designated as sample 21.
  • modified hyaluronic acid Preparation of modified hyaluronic acid: Weigh 2 g of sodium hyaluronate with a molecular weight of 10,000 and dissolve it in 20 ml of PBS, then add 6.5 ml of glycidyl methacrylate and 4.5 ml of triethylamine in sequence. Place on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 5000 for 7 days, freeze-dry to obtain double-bonded hyaluronic acid;
  • modified polylysine Dissolve polylysine in deionized water, and then add glycidyl methacrylate (glycidyl methacrylate: amino group) at a molar ratio of (1:1.5). The mixture was placed on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 1000 to dialyze for 7 days, and freeze-dry to obtain partially double-bonded poly-lysine;
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, and then soaked in 60 mM (calculated as lysine) modified poly-lysine aqueous solution at room temperature for 12 hours.
  • Sample 17, sample 18, sample 19, sample 20, sample 21 and control group 5 were subjected to enzymatic degradation experiments to characterize the degree of cross-linking of each group of samples, and collagenase I was used to treat sample 17, sample 18, sample 19, sample 20, After the sample 21 and the control group 5, the enzymatic degradation weight loss rate of each group of samples was calculated, and the results are shown in Table 8.
  • the enzymatic degradation weight loss rate of sample 17, sample 18, sample 19, sample 20, and sample 21 was lower than that of control group 5, indicating that the enzymatic degradation stability of sample 17, sample 18, sample 19, and sample 20 was higher than that of control group 5. , that is, the cross-linking degree of sample 17, sample 18, sample 19, and sample 20 is higher.
  • the results of enzyme degradation experiments show that the preparation method of the double-bond cross-linked bio-valve material after co-cross-linking can improve the cross-linking degree of the bio-valve material.
  • Alizarin red staining experiment was carried out on sample 17, sample 18, sample 19, sample 20, sample 21 and control group 5 using the same test method as the aforementioned alizarin red staining experiment.
  • sample 19, sample 20, and sample 21 The calcification degree of sample 19, sample 20, and sample 21 is lower than that of control sample 5, that is, sample 17, sample 18, sample 19, sample 20, and sample 21 have certain anti-calcification effects compared with control sample 5.
  • Alizarin red staining results of samples 17, 18, 19, 20, 21, and control group 5 after being implanted subcutaneously in rats for 30 days showed that the preparation of double-bond cross-linked biological valve materials after co-crosslinking in this application
  • the method can improve the anti-calcification performance of the biological valve.
  • Freshly collected porcine pericardium was washed in distilled water at 4°C and 100 RPM for 2 hours, then soaked in 100 mM glutaraldehyde solution, and cross-linked at room temperature for 24 hours at 100 RPM to obtain control sample 6.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM with shaking.
  • sample 23 Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • sample 23 the sample obtained in this embodiment is designated as sample 23.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM with shaking.
  • sample 24 Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • sample 24 the sample obtained in this embodiment is designated as sample 24.
  • the blood contact test was carried out on the control sample 6, sample 23 and sample 24 using the same test method as the blood contact test described above.
  • control sample 6 sample 23 and sample 24 in the blood contact experiment are shown in Fig. 42, Fig. 43 and Fig. 44.
  • Fig. 42, Fig. 43 and Fig. 44 After contacting and incubating with rabbit blood, more blood cell adhesion and aggregation were observed in the scanning electron micrograph of control sample 6, while less blood cells adhered to sample 23 and sample 24, and only a few blood cells adhered scatteredly to the surface.
  • the results show that samples 23 and 24 can inhibit the adhesion of blood cells to a certain extent, thereby reducing the risk of coagulation, and have anticoagulant effects.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM with shaking.
  • Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • the sample obtained in this embodiment is designated as sample 25.
  • Sample 23, sample 24, sample 25 and control group 6 were used to determine the weight loss rate of collagenase degradation by the same method as the enzymatic degradation experiment described above.
  • Enzyme degradation experiments were carried out on sample 23, sample 24, sample 25 and control group 6 to characterize the degree of cross-linking of samples in each group, and the cross-linking degree of samples in each group was calculated after treating sample 23, sample 24, sample 25 and control group 6 with collagenase I.
  • the enzymatic degradation weight loss rate is shown in the table above.
  • the enzymatic degradation weight loss rate of sample 23, sample 24, sample 25 is all lower than control group 6, this shows that the enzymatic degradation stability of sample sample 23, sample 24, sample 25 is all higher than control group 6, namely sample 23, sample 24, Sample 25 has a higher degree of crosslinking.
  • the results of the enzyme degradation experiment show that the method for preparing the bio-valve material of the present application can increase the cross-linking degree of the bio-valve material.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM with shaking.
  • N-isopropylacrylamide was added to make the final concentration 5wt%, and soaked at 37°C for 12 hours to ensure sufficient physical penetration of N-isopropylacrylamide.
  • Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • sample 26 the sample obtained in this embodiment is designated as sample 26.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM with shaking.
  • Ammonium persulfate and sodium bisulfite initiators were added for initiation, the molar concentrations of both ammonium persulfate and sodium bisulfite were 30 mM, and the reaction was carried out at 37° C. for 24 hours.
  • sample 27 The sample obtained in this example was designated as sample 27.
  • modified hyaluronic acid Preparation of modified hyaluronic acid: Weigh 2 g of sodium hyaluronate with a molecular weight of 10,000 and dissolve it in 20 ml of PBS, then add 6.5 ml of glycidyl methacrylate and 4.5 ml of triethylamine in sequence. Place on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 5000 for 7 days, freeze-dry to obtain double-bonded hyaluronic acid;
  • modified polylysine Dissolve polylysine in deionized water, and then add glycidyl methacrylate (glycidyl methacrylate: amino group) at a molar ratio of (1:1.5). The mixture was placed on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 1000 to dialyze for 7 days, and freeze-dry to obtain partially double-bonded poly-lysine;
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, and then soaked in 60 mM (calculated as lysine) modified poly-lysine aqueous solution at room temperature for 12 hours.
  • Alizarin red staining experiment was carried out on sample 23, sample 24, sample 25, sample 26, sample 27 and control sample 6 using the same test method as the alizarin red staining experiment described above.
  • sample 25, sample 26 and sample 27 The degree of calcification of sample 25, sample 26 and sample 27 is lower than that of control sample 6, that is, sample 23, sample 24, sample 25, sample 26 and sample 27 have a certain anti-calcification effect compared with control sample 6.
  • Alizarin red staining results of samples 23, 24, 25, 26, 27 and control group 6 after being implanted subcutaneously in rats for 30 days showed that the method for preparing biological valve materials by the method of the present application can improve the resistance of biological valves. Calcification properties.
  • the freshly collected porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 100 mM glutaraldehyde solution, and then cross-linked at room temperature and 100 RPM for 24 hours to obtain control sample 7.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the pig pericardium was soaked in 50 mM arginine aqueous solution for 12 hours, and then washed with distilled water to obtain a sample which was designated as sample 29.
  • Freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM with shaking
  • sample 30 The dry film obtained by immersing in glycerin is designated as sample 30.
  • Example 29 The reaction principles of Example 29 and Example 30 are shown in Figure 51.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • sample 31 pig pericardium was soaked in 50 mM tris aqueous solution for 12 hours, and then washed with distilled water to obtain a sample which was designated as sample 31.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the pig pericardium was soaked in 20mM oleylamine in ethanol aqueous solution (50% ethanol, v/v) for 12 hours; subsequently washed with ethanol aqueous solution (50% ethanol, v/v) to get the sample and record it as sample 32.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the pig pericardium was soaked in 20mM dodecylamine in ethanol aqueous solution (50% ethanol, v/v) for 12 hours; followed by washing with ethanol aqueous solution (50% ethanol, v/v) to obtain the sample as Sample 33.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • sample 34 the cleaned sample is designated as sample 34.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • sample 35 the sample obtained after washing with aqueous solution was designated as sample 35.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the pig pericardium was soaked in 30 mM 3-buten-1-amine aqueous solution for 12 hours; the sample was then washed with distilled water and recorded as sample 36.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the porcine pericardium was soaked in oleylamine ethanol solution (50% ethanol, v/v) for 12 hours; the sample was then washed with distilled water and recorded as sample 37.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the pig pericardium was soaked in oleylamine ethanol water solution (50% ethanol, v/v) for 12 hours; then washed with ethanol water solution (50% ethanol, v/v) to obtain a sample designated as sample 38.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the porcine pericardium was soaked in 30 mM 2-aminopent-4-enoic acid aqueous solution for 12 hours; the sample was then washed with distilled water and recorded as sample 39.
  • the freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C and 100 RPM shaking;
  • the pig pericardium was soaked in 50 mM tris aqueous solution for 12 hours; the sample was then washed with distilled water and recorded as sample 40.
  • modified hyaluronic acid Preparation of modified hyaluronic acid: Weigh 2 g of sodium hyaluronate with a molecular weight of 10,000 and dissolve it in 20 ml of PBS, then add 6.5 ml of glycidyl methacrylate and 4.5 ml of triethylamine in sequence. Place on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 5000 for 7 days, freeze-dry to obtain double-bonded hyaluronic acid;
  • modified polylysine Dissolve polylysine in deionized water, and then add glycidyl methacrylate (glycidyl methacrylate: amino group) at a molar ratio of (1:1.5). The mixture was placed on a shaker at 37°C for 7 days. Finally, use a dialysis bag with a molecular weight cut-off of 1000 to dialyze for 7 days, and freeze-dry to obtain partially double-bonded poly-lysine;
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, and then soaked in 60 mM (calculated as lysine) modified poly-lysine aqueous solution at room temperature for 12 hours.
  • the blood contact test was carried out on the control sample 7, sample 29 and sample 31 using the same test method as the blood contact test described above.
  • samples 35-40 show that they also have similar properties to samples 29 and 31, and can improve the anticoagulant performance of biological valve materials.
  • the alizarin red staining experiment was carried out using the same test method as the alizarin red staining experiment described above for sample 30, sample 32, sample 33, sample 34 and control sample 7.
  • control sample 7, sample 30, sample 32, sample 33, and sample 34 implanted subcutaneously in rats for 30 days were stained by alizarin red staining experiment to characterize the degree of calcification of samples in each group.
  • the images of the Alizarin Red staining results of the sample sections after the control sample 7, Sample 30, Sample 32, Sample 33, and Sample 34 were implanted subcutaneously in rats for 30 days are shown in Figure 58-62, wherein the color of the samples after Alizarin Red staining is more Darker indicates higher degree of calcification.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution, and cross-linked at room temperature for 48 hours at 100 RPM Get control sample 8.
  • the freshly collected porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution at room temperature for 48 hours.
  • the glutaraldehyde cross-linking treatment was performed on the biological valve to obtain the glutaraldehyde cross-linked porcine pericardium.
  • Glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water, and double-bonded by glutaraldehyde-crosslinked porcine pericardium by soaking in 5% (v/v) glycidyl methacrylate in propanol aqueous solution at room temperature Modification, the reaction time is 72 hours, and the solvent of the double bond solution used is 20% (v/v) propanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium was soaked in a mixture of 20mM potassium persulfate and 10mM sodium bisulfite The double bonded glutaraldehyde cross-linked porcine pericardium was further induced in the solution to polymerize the double bonds, and after reacting at 37° C. for 8 hours, a post-double bond cross-linked porcine pericardium was obtained, which was designated as sample 42.
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution at room temperature for 48 hours to treat the biological valve.
  • Glutaraldehyde cross-linked porcine pericardium was obtained by dialdehyde cross-linking treatment.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water, and then soaked in 6% (v/v) glycidyl acrylate in isopropanol aqueous solution at room temperature for double bond modification of glutaraldehyde cross-linked porcine pericardium , the reaction time was 72 hours, and the solvent of the double bond solution used was 20% (v/v) isopropanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium was soaked in a mixture of 20mM ammonium persulfate and 5mM sodium bisulfite The double bonded glutaraldehyde cross-linked porcine pericardium was further induced in the liquid to polymerize the double bonds, and after reacting at 37° C. for 8 hours, the double bond post-cross-linked porcine pericardium was obtained, which was designated as sample 43.
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution at room temperature for 48 hours to treat the biological valve.
  • Glutaraldehyde cross-linked porcine pericardium was obtained by dialdehyde cross-linking treatment.
  • the glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and soaked in isopropyl acrylate containing 4% (v/v) glycidyl acrylate and 4% (v/v) allyl glycidyl ether at room temperature.
  • the double bond modification of the glutaraldehyde cross-linked porcine pericardium was carried out in the alcohol aqueous solution, and the reaction time was 72 hours, and the solvent of the double bond solution used was 30% (v/v) ethanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium was soaked in a mixture of 20mM ammonium persulfate and 10mM sodium bisulfite The double-bond glutaraldehyde cross-linked pig pericardium was further induced in the liquid to polymerize the double bond, and after 7 hours of reaction at 37°C, the pig pericardium cross-linked after the double bond was obtained, which was recorded as sample 44, and the code was GAGA-PP -3.
  • porcine pericardium was washed with distilled water at 100 RPM at 4°C for 2 hours, soaked in 0.30% (w/w) glutaraldehyde solution at room temperature, and soaked at room temperature for 48 hours.
  • Glutaraldehyde cross-linked porcine pericardium was obtained by dialdehyde cross-linking treatment.
  • the glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and soaked in an aqueous solution of 5% (v/v) glycidyl methacrylate and 2% (v/v) glycidyl acrylate in isopropanol at room temperature
  • the double bond modification of glutaraldehyde cross-linked porcine pericardium was carried out in , the reaction time was 72 hours, and the solvent of the double bond solution used was 35% (v/v) isopropanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium was soaked in a mixture of 20mM sodium persulfate and 5mM sodium bisulfite The double bonded glutaraldehyde cross-linked porcine pericardium was further induced in the solution to polymerize the double bonds, and after reacting at 37° C. for 8 hours, the double bond post-cross-linked porcine pericardium was obtained, which was designated as sample 45.
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution at room temperature for 48 hours to treat the biological valve.
  • Glutaraldehyde cross-linked porcine pericardium was obtained by dialdehyde cross-linking treatment.
  • Glutaraldehyde-crosslinked porcine pericardium was washed with deionized water, and glutaraldehyde-crosslinked porcine pericardium was soaked in 4% (v/v) glycidyl methacrylate ethanol aqueous solution at room temperature for glutaraldehyde-crosslinked pig pericardium.
  • the reaction time is 72 hours, and the solvent of the double bond solution used is 20% (v/v) ethanol aqueous solution.
  • ammonium persulfate and sodium bisulfite are added to initiate the polymerization of double bonds on the double bonded glutaraldehyde cross-linked porcine pericardium, wherein the concentration of ammonium persulfate is 20 mM, and the concentration of sodium bisulfite is 5 mM
  • sample 46 a double bond post-crosslinked porcine pericardium was obtained, which was designated as sample 46.
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution at room temperature for 48 hours to treat the biological valve.
  • Glutaraldehyde cross-linked porcine pericardium was obtained by dialdehyde cross-linking treatment.
  • Glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water, and the double bonds of glutaraldehyde-crosslinked porcine pericardium were soaked in 4% (v/v) glycidyl methacrylate in isobutanol aqueous solution at room temperature. modification, the reaction time was 72 hours, and the solvent of the double bond solution used was 15% (v/v) isobutanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium was soaked in a mixture of 20mM ammonium persulfate and 5mM sodium bisulfite The double bonded glutaraldehyde cross-linked porcine pericardium was further induced in the liquid to polymerize the double bonds, and after reacting at 37° C. for 8 hours, the double bond post-cross-linked porcine pericardium was obtained, which was designated as sample 47.
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution at room temperature for 48 hours to treat the biological valve.
  • Glutaraldehyde cross-linked porcine pericardium was obtained by dialdehyde cross-linking treatment.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water, and the glutaraldehyde cross-linked porcine pericardium was modified by immersing in 4% (v/v) glycidyl acrylate in isopropanol aqueous solution at room temperature , the reaction time was 48 hours, and the solvent of the double bond solution used was 20% (v/v) aqueous methanol.
  • the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium was soaked in a mixture of 20mM ammonium persulfate and 6.5mM sodium sulfite The polymerization reaction of the double bonds on the cross-linked porcine pericardium was further induced by double-bonded glutaraldehyde, and after 10 hours of reaction at 37° C., a post-double bond cross-linked porcine pericardium was obtained, which was designated as sample 48.
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution at room temperature for 48 hours to treat the biological valve.
  • Glutaraldehyde cross-linked porcine pericardium was obtained by dialdehyde cross-linking treatment.
  • the glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water, and the double bonds of the glutaraldehyde-crosslinked porcine pericardium were soaked in 4% (v/v) glycidyl methacrylate in ethylene glycol aqueous solution at room temperature. modification, the reaction time is 72 hours, and the solvent of the double bond solution used is 20% (v/v) ethylene glycol aqueous solution.
  • the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium was soaked in a mixture of 40mM ammonium persulfate and 15mM sodium bisulfite The double bonded glutaraldehyde cross-linked porcine pericardium was further induced in the solution to polymerize the double bonds, and after reacting at 37° C. for 7 hours, the double bond cross-linked porcine pericardium was obtained, which was designated as sample 49.
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution at room temperature for 48 hours to treat the biological valve.
  • Glutaraldehyde cross-linked porcine pericardium was obtained by dialdehyde cross-linking treatment.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water, and then soaked in 7% (v/v) glycidyl acrylate aqueous propanol at room temperature to modify the double bond of the glutaraldehyde cross-linked porcine pericardium,
  • the reaction time was 60 hours, and the solvent of the double bond solution used was 40% (v/v) propanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked porcine pericardium was washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium was soaked in a mixture of 30mM sodium persulfate and 10mM sodium bisulfite The double bonded glutaraldehyde cross-linked porcine pericardium was further induced in the solution to polymerize the double bonds, and after reacting at 37° C. for 8 hours, a post-double bond cross-linked porcine pericardium was obtained, which was designated as sample 50.
  • porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then soaked in 0.30% (w/w) glutaraldehyde solution at room temperature for 48 hours to treat the biological valve.
  • Glutaraldehyde cross-linked porcine pericardium was obtained by dialdehyde cross-linking treatment.
  • glutaraldehyde-crosslinked porcine pericardium was soaked in isopropanol containing 6% (v/v) glycidyl methacrylate and 3% (v/v) glycidyl acrylate at room temperature.
  • the double bond modification of the glutaraldehyde cross-linked porcine pericardium is carried out in an aqueous solution, and the reaction time is 84 hours, and the solvent of the double bond solution used is 50% (v/v) ethanol aqueous solution.
  • the double bonded glutaraldehyde crosslinked pig pericardium was washed with deionized water; then the double bonded glutaraldehyde crosslinked pig pericardium was soaked in a mixture of 40mM ammonium persulfate and 10mM sodium sulfite to further induce Double bonded glutaraldehyde cross-linked double bond polymerization reaction of pig pericardium, after reaction at 37°C for 12 hours, a double bond cross-linked pig pericardium was obtained, which was designated as sample 51.
  • the thermal stability and cross-linking degree of the bio-valve material were characterized by measuring the heat shrinkage temperature of the bio-valve material;
  • the stability of the biovalve material was characterized by degradation experiments;
  • the degree of calcification (anti-calcification performance) of the samples was characterized by subcutaneous implantation experiments in rats.
  • the thermal stability and cross-linking degree of biological valve materials are characterized by measuring the heat shrinkage temperature; the higher the heat shrinkage temperature, the higher the corresponding thermal stability and cross-linking degree.
  • Embodiment 42, embodiment 43, embodiment 50, embodiment 51 and control group 8 are carried out heat shrinkage temperature measurement and find: as shown in table 10, embodiment 42, embodiment 43, The thermal shrinkage temperature of embodiment 50, embodiment 51 is all higher than control group 8 (glutaraldehyde cross-linked porcine pericardium), namely the thermal stability and cross-linking of embodiment 42, embodiment 43, embodiment 50, embodiment 51 The degrees were higher than those of the control group 8 (glutaraldehyde cross-linked porcine pericardium).
  • the experimental results of heat shrinkage temperature measurement show that the double bond post-crosslinking method of the present application for preparing biological valve materials can improve the thermal stability and crosslinking degree of biological valves.
  • Sample 44, sample 47, sample 45, sample 51 and control group 8 were used to determine the weight loss rate of collagenase degradation by the same method as the enzymatic degradation experiment described above.
  • Control group 8 (glutaraldehyde cross-linked pig pericardium) 7.45 ⁇ 1.33 Sample 44 5.31 ⁇ 0.30 Sample 45 4.47 ⁇ 1.05 Sample 47 5.12 ⁇ 0.97 Sample 51 3.06 ⁇ 0.59
  • Enzyme degradation experiments were carried out on control group 8 (glutaraldehyde cross-linked porcine pericardium), sample 44, sample 45, sample 47, and sample 51 to characterize the cross-linking efficiency of each group of samples, and collagenase I was used to treat control group 8 (glutaraldehyde Aldehyde cross-linked porcine pericardium), sample 44, sample 45, sample 47 and sample 51, calculate the enzymatic degradation weight loss rate of each group of samples as shown in Table 11.
  • the enzymatic degradation weight loss rate of sample 44, sample 45, sample 47, sample 51 is all lower than the control group (glutaraldehyde cross-linked pig pericardium), which shows that the stability of sample 44, sample 45, sample 47, sample 51 is higher than The stability of the control group (glutaraldehyde cross-linked porcine pericardium), that is, sample 44, sample 45, sample 47, and sample 51 was higher.
  • the results of enzyme degradation experiments show that the double bond post-crosslinking method of the present application for preparing biological valve materials can improve the stability of biological valves.
  • the biological valve material was cut into sheets of 0.80.8cm2 , sterilized and implanted under the skin of rats and taken out after 30 days. Each piece of sample was divided into two parts, one part was removed from the capsule, freeze-dried and weighed, and digested with 6M hydrochloric acid Finally, the calcium element content per gram of the sample was determined; another part of the sample was fixed by paraformaldehyde tissue fixative. After the fixation, take it out and repair it with a scalpel, then transfer it to the dehydration box. Dehydrate material samples with graded ethanol. After dehydration, the material samples were transferred to an embedding machine for embedding with melted paraffin, and then transferred to a -20°C refrigerator to cool and trim the shape.
  • Sections of 5 ⁇ m thickness were cut from the trimmed wax blocks on a microtome, transferred from the spreader to glass slides and deparaffinized and rehydrated. The sections were stained with Alizarin Red staining solution for 3 minutes, washed with water, dried and then permeabilized with xylene for 5 minutes. After the sections were sealed with neutral gum, images of staining results were collected on a pathological slide scanner.
  • Control group 8 (glutaraldehyde cross-linked pig pericardium) 74.9 ⁇ 12.3 Sample 42 15.1 ⁇ 4.7 Sample 46 8.4 ⁇ 4.6 Sample 48 12.7 ⁇ 5.1
  • samples 42, 46, 48 and control group 8 glucosealdehyde cross-linked pig pericardium implanted subcutaneously in rats for 30 days was detected to characterize the degree of calcification of samples in each group. As shown in Table 12, the calcium content of sample 42, sample 46, and sample 48 were all lower than those of the control group (glutaraldehyde cross-linked pig pericardium) after 30 days of subcutaneous implantation in rats.
  • the method of biological valve material can improve the anti-calcification performance of biological valve.
  • control group 8 (glutaraldehyde cross-linked porcine pericardium), sample 42, sample 46, and sample 48 implanted subcutaneously in rats for 30 days were stained with alizarin red to directly observe the degree of calcification of the samples in each group.
  • Figures 65-68 show the images of the Alizarin Red staining results of the sample sections 30 days after implantation into the subcutaneous tissue of rats, where the darker the color of the sample after Alizarin Red staining, the higher the degree of calcification.
  • the simple glutaraldehyde cross-linked group was set as the control group, and the pig pericardium was soaked in 0.625% (w/w) glutaraldehyde at room temperature for 72 hours to prepare the glutaraldehyde cross-linked pig pericardium, which was recorded as Control sample 9.
  • Glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water, and the double bonds of glutaraldehyde-crosslinked porcine pericardium were soaked in 5% (v/v) glycidyl methacrylate in isopropanol aqueous solution at room temperature.
  • the reaction time is 72 hours, and the solvent of the double bond solution used is 18% (v/v) isopropanol aqueous solution.
  • the glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water, and then soaked in 6% (v/v) glycidyl acrylate aqueous propanol at room temperature to modify the double bond of the glutaraldehyde-crosslinked porcine pericardium,
  • the reaction time was 72 hours, and the solvent of the double bond solution used was 20% (v/v) propanol aqueous solution.
  • Glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and soaked in isopropyl acrylate containing 2% (v/v) glycidyl acrylate and 4% (v/v) allyl glycidyl ether at room temperature.
  • the double bond modification of the glutaraldehyde cross-linked porcine pericardium was carried out in the alcohol aqueous solution, and the reaction time was 72 hours, and the solvent of the double bond solution used was 30% (v/v) ethanol aqueous solution.
  • the glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and soaked in an aqueous solution of 3% (v/v) glycidyl methacrylate and 2% (v/v) glycidyl acrylate in isopropanol at room temperature
  • the double bond modification of glutaraldehyde cross-linked porcine pericardium was carried out in , the reaction time was 48 hours, and the solvent of the double bond solution used was 25% (v/v) isopropanol aqueous solution.
  • Glutaraldehyde-crosslinked porcine pericardium was washed with deionized water, and glutaraldehyde-crosslinked porcine pericardium was soaked in 4% (v/v) glycidyl methacrylate ethanol aqueous solution at room temperature for glutaraldehyde-crosslinked pig pericardium.
  • the reaction time is 72 hours, and the solvent of the double bond solution used is 20% (v/v) ethanol aqueous solution.
  • Glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water, and the double bonds of glutaraldehyde-crosslinked porcine pericardium were soaked in 4% (v/v) glycidyl methacrylate in isobutanol aqueous solution at room temperature. modification, the reaction time was 72 hours, and the solvent of the double bond solution used was 15% (v/v) isobutanol aqueous solution.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water, and the glutaraldehyde cross-linked porcine pericardium was modified by immersing in 4% (v/v) glycidyl acrylate in isopropanol aqueous solution at room temperature , the reaction time was 48 hours, and the solvent of the double bond solution used was 20% (v/v) aqueous methanol.
  • the glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water, and the double bonds of the glutaraldehyde-crosslinked porcine pericardium were soaked in 5% (v/v) glycidyl methacrylate in ethylene glycol aqueous solution at room temperature. modification, the reaction time was 72 hours, and the solvent of the double bond solution used was 25% (v/v) ethylene glycol aqueous solution.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water, and then soaked in 7% (v/v) glycidyl acrylate aqueous propanol at room temperature to modify the double bond of the glutaraldehyde cross-linked porcine pericardium,
  • the reaction time was 60 hours, and the solvent of the double bond solution used was 30% (v/v) propanol aqueous solution.
  • glutaraldehyde-crosslinked porcine pericardium was soaked in isopropanol containing 4% (v/v) glycidyl methacrylate and 2% (v/v) glycidyl acrylate at room temperature.
  • the double bond modification of the glutaraldehyde cross-linked porcine pericardium is carried out in an aqueous solution, and the reaction time is 84 hours, and the solvent of the double bond solution used is 25% (v/v) ethanol aqueous solution.
  • the thermal stability and cross-linking degree of bio-valve material were characterized by measuring the thermal shrinkage temperature of bio-valve material; Enzyme degradation test characterizes the stability of biological valve material; characterizes the degree of calcification (anti-calcification performance) of the sample through rat subcutaneous implantation experiment; characterizes its elasticity by testing the elastic angle of biological valve material.
  • the thermal stability and cross-linking degree of biological valve materials are characterized by the determination of the heat shrinkage temperature; the higher the heat shrinkage temperature, the higher the corresponding thermal stability and cross-linking degree.

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Abstract

本申请公开一种生物瓣膜材料及其制备方法和应用，制备方法包括：步骤S100，利用生物材料上的氨基，采用化学反应的方式接入第一碳碳双键，且在所述步骤S100的反应过程中至少有醛基交联剂存在；步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。本申请的方法通过两次交联形成更多更大的聚合物交联网络，提高生物材料的交联度和提升抗钙化性能；在二次引入碳碳双键的同时还引入附加功能性基团，可赋予生物材料新的特性，进一步改善生物材料的性能。

Description

一种生物瓣膜材料及其制备方法和应用 技术领域
本发明涉及介入材料技术领域，具体涉及一种生物瓣膜材料及其制备方法和应用。
背景技术
生物心脏瓣膜通常采用猪或牛的心包膜制备而成，用于替换功能缺损的人体自有心脏瓣膜；生物心脏瓣膜相比于机械心脏瓣膜有很多优点：生物心脏瓣膜植入后患者不需要长期服用抗凝药、生物心脏瓣膜可以采用微创介入的手术方式，这些优点使得生物心脏瓣膜在临床应用当中逐步成为市场主流。
当前市场上的生物瓣膜产品几乎全部是采用戊二醛进行交联制备而成，戊二醛可以交联心包膜当中的胶原蛋白，提高膜片的力学性能以满足市场上对膜片力学性能的要求。但是，戊二醛交联生物瓣膜具有醛基，用以产生钙化位点，从而具有血液相容性不佳的缺点，导致其在体内的寿命有限。
由于传统戊二醛交联生物膜客观问题的存在，近些年，也有一些探索非戊二醛交联生物膜的研究报道，例如，利用碳二亚胺作为交联剂制备非戊二醛交联生物膜，但是，研究中发现，非戊二醛交联生物膜的力学性能难以达到要求，真正实现产业化比较困难。
因此，戊二醛交联生物膜仍是主流生物瓣膜材料，研究改善戊二醛交联膜的性能仍然是当前重要研究方向之一，常规方式下，为了增加膜片的力学性能会提高戊二醛的交联时间与浓度，但由于戊二醛自身的自聚反应，使得交联程度受到了限制，无法实现膜片所有氨基的全部交联，并且一味的增加时间和浓度只会增加膜片表面的戊二醛自聚，使膜片性能***。
发明内容
本申请提供一种生物瓣膜材料及其制备方法和应用，通过在戊二醛交联基础上引入碳碳双键作为二次交联的基础，进一步地通过引发碳碳双键的聚合从而实现二次交联，以改善戊二醛交联膜的性能。
对于引入碳碳双键作为二次交联的基础的过程，一方面，可在戊二醛交联过程中引入双键单体进行共交联，通过引入其他交联基团的交联方式重新提供一个可控的交联机会与范围；另一方面，可在戊二醛交联后通过戊二醛交联后膜片残余氨基，引入带有双键交联的单体，通过引入其他交联基团的交联方式重新提供一个可控的交联机会与范围。
一种生物瓣膜材料的制备方法，包括：
步骤S100，将生物材料依次经第一处理液和第二处理液进行处理，得到化学接枝有第一碳碳双键的预处理后的生物材料；所述第一处理液和所述第二处理液彼此相异的含有试剂A和试剂B中的其中一种，其中试剂A为带有所述第一碳碳双键的第一功能单体，试剂B为醛基交联剂；
步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
可选的，所述的步骤S100中，所述第一功能单体还带有活性基团，通过所述活性基团参与化学反应；所述第一处理液含有试剂A，所述活性基团可与醛基反应；或
所述第一处理液含有试剂B，所述活性基团可与氨基反应。
可选的，所述生物材料为动物组织，包括心包膜、瓣膜、肠膜、脑膜、肺膜、血管、皮肤或韧带的一种或多种。
可选的，所述动物组织为新鲜的动物组织或经脱细胞处理后的生物组织。
可选的，所述醛基交联剂为戊二醛或甲醛。
步骤S200中：
可选的，将引发剂加入上一步处理的体系中；或将上一步处理后的生物材料取出、直接或清洗后再浸泡于含引发剂的溶液中；
可选的，所述引发剂为单一引发剂或混合引发剂，所述混合引发剂为：
过硫酸铵和亚硫酸氢钠的混合物，或过硫酸铵和亚硫酸钠的混合物，或过硫酸钠和亚硫酸钠的混合物，或过硫酸钾和亚硫酸钠的混合物，或过硫酸钠和亚硫酸氢钠的混合物，或过硫酸钾和亚硫酸氢钠的混合物，所述混合物中各组分的浓度分别为1～100mM；
或所述混合引发剂为：
过硫酸铵和N,N,N',N'-四甲基乙二胺的混合物，或过硫酸钾和N,N,N',N'-四甲基乙二胺的混合物，或过硫酸氨和N,N,N',N'-四甲基乙二胺的混合物，或过硫酸钠和N,N,N',N'-四甲基乙二胺的混合物；混合物中过硫酸铵、过硫酸钾或过硫酸钠的质量百分浓度分别为2％～5％；四甲基乙二胺的质量百分比为0.2％～0.5％；
所述单一引发剂为各混合引发剂中的任一组分。
可选的，步骤S100包括：
AS110将生物材料与第一处理液接触进行物理渗透，所述第一处理液为含所述第一功能单体的溶液；
AS120将经AS110处理后的生物材料与第二处理液接触接触，进行共交联接入所述第一碳碳双键，所述第二处理液为醛基交联剂溶液。
可选的，所述活性基团为氨基或酰肼。
可选的，步骤S100包括：
BS110将生物材料与第一处理液接触进行交联，所述第一处理液为醛基交联剂溶液；
BS120将步骤BS110处理后的生物材料与第二处理液接触，化学反应接入所述第一碳碳双键，所述第二处理液为含所述第一功能单体的溶液。
可选的，所述活性基团为环氧乙烷基。
可选的，步骤S100中，采用非缩合的化学反应接入所述第一碳碳双键。
可选的，步骤S100中，所述生物材料在经过醛基交联剂处理之前未经过任何其他试剂参与的化学反应。
可选的，步骤S100的反应体系中通过带有活性基团的第一功能单体提供所述第一碳碳双键，且步骤S100中的反应原料仅包括所述生物材料、所述第一功能单体以及所述醛基交联剂。
可选的，步骤S100包括：
AS110将生物材料浸泡于第一处理液中进行物理渗透；所述第一处理液为含所述第一功能单体的溶液；所述第一功能单体还带有活性基团；所述活性基团为氨基或酰肼；
AS120将步骤AS110处理后的生物材料浸泡于第二处理液中进行共交联接入第一碳碳双键，所述第二处理液为醛基交联剂溶液。
可选的，所述第一功能单体还带有功能性基团A。
可选的，所述功能性基团A选自羟基、羧基、酰胺基、磺酸基、两性离子、聚乙二醇、脲基、氨基甲酸酯基、羧酸根离子、磺酸酯、亚砜、吡咯烷酮中的至少一种中的至少一种。
可选的，步骤S100还包括：
AS130将经步骤AS120处理后的生物材料浸泡于含第二功能单体的溶液中进行物理渗透引入第二碳碳双键；所述第二功能单体带有第二碳碳双键。
可选的，所述第二功能单体还带有功能性基团B。
可选的，所述功能性基团B选自羟基、羧基、羧酸胆碱、磺酸胆碱、磷酸胆碱、吡咯烷酮、磺酸基团、羧酸根离子、磺酸酯、亚砜、酰胺基团、甲氧基中的至少一种。
可选的，所述第二功能单体选自聚乙二醇二丙烯酸酯、1,4-丁二醇二丙烯酸酯、丙烯酸乙酯、N-甲基-2-丙烯酰胺、N-2,2-丙烯基-2-丙烯酰胺、N-乙基丙烯酰胺、N,N'-乙烯基双丙烯酰胺、(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯、双键化透明质酸、丙烯酰胺、2-(丙-2-烯酰氨基)乙酸、2-丙烯酰胺基-2-甲基丙磺酸、甲基丙烯酸羟乙酯、3-[[2-(甲基丙烯酰氧)乙基]二甲基铵]丙酸酯、N-异丙基丙烯酰胺、N-(羟甲基)丙烯酰胺、N-(2-羟基乙基)甲基丙烯酰胺、N,N-二甲基甲基丙烯酰胺、3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐、2-甲基丙烯酰氧乙基磷酸胆碱、双键化多聚赖氨酸中的至少一种。
可选的，步骤AS130中，将第二功能单体加入前步处理的体系中；或将前步处理后的生物材料清洗后再浸泡于含第二功能单体的溶液中；所述含第二功能单体的溶液中仅包括第二功能单体和不参与化学反应的溶剂。
可选的，所述含第二功能单体的溶液中溶剂为水、生理盐水、pH中性缓冲液或乙醇的水溶液；所述含第二功能单体的溶液中第二功能单体的质量百分浓度为1～10％；浸泡时间为2～20h。
可选的，所述第一功能单体选自DL-2-氨基-4-戊烯酸、2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯、甲基丙烯酰肼、丙烯酰肼、双键化多聚赖氨酸、2-氨基-7-烯-辛酸、6-烯-庚氨酸、2-氨基戊-4-烯酸、4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇、4-(1-氨基-乙基)-七-1,6-二烯-4-醇中的至少一种。
可选的，含所述第一功能单体的溶液中仅包括第一功能单体和不参与化学反应的溶剂。
可选的，步骤AS110中含所述第一功能单体的溶液中溶剂为水、生理盐水、异丙醇、pH中性缓冲液或乙醇的水溶液；含所述第一功能单体的溶液中第一功能单体的浓度为10～100mM；浸泡时间为2～20h。
可选的，步骤AS120中，所述醛基交联剂在AS120反应体系中的终浓度为10～800mM； 共交联时间为10～30h。
可选的，步骤S100中：
在步骤AS120之后还包括步骤AS120(M)：将经步骤AS120处理后的生物材料浸泡于含第三功能单体的溶液中，消除残留醛基；所述第三功能单体带有氨基或酰肼。
可选的，步骤S100中：
在步骤AS130之前还包括步骤AS120(M)：将经步骤AS120处理后的生物材料浸泡于含第三功能单体的溶液中，消除残留醛基；所述第三功能单体带有氨基或酰肼。
可选的，步骤AS120(M)中，所述含第三功能单体的溶液中溶剂为水、生理盐水、pH中性缓冲液或乙醇的水溶液；所述含第三功能单体的溶液中第三功能单体的浓度为10～100mM；浸泡时间为2～48h。
可选的，所述第三功能基团还带有功能性基团C。
可选的，所述功能性基团C选自羟基、羧基、酰胺基、磺酸基、两性离子、聚乙二醇、脲基、氨基甲酸酯基、羧酸根离子、磺酸酯、亚砜、吡咯烷酮中的至少一种中的至少一种。
可选的，所述第三功能单体选自DL-2-氨基-4-戊烯酸、2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯、甲基丙烯酰肼、丙烯酰肼、双键化多聚赖氨酸、2-氨基-7-烯-辛酸、6-烯-庚氨酸、2-氨基戊-4-烯酸、4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇、4-(1-氨基-乙基)-七-1,6-二烯-4-醇中的至少一种。
可选的，步骤S100包括：
BS110将生物材料与第一处理液接触进行交联，所述第一处理液为醛基交联剂溶液；
BS120将步骤BS110处理后的生物材料浸泡于第二处理液中，化学反应接入第一碳碳双键；所述第二处理液为含有所述第一功能单体的溶液；所述第一功能单体还带有活性基团；所述活性基团为环氧乙烷基。
可选的，步骤S100包括：
BS110将生物材料与第一处理液接触进行交联，所述第一处理液为醛基交联剂溶液；
BS120将步骤BS110处理后的生物材料浸泡于第二处理液中，化学反应接入第一碳碳双键；所述第二处理液为含有所述第一功能单体的溶液；所述第一功能单体还带有活性基团；所述活性基团为环氧乙烷基；
BS130将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第二功能单体具有第二碳碳双键。
可选的，所述第二功能单体选自聚乙二醇二丙烯酸酯、1,4-丁二醇二丙烯酸酯、乙烷-1,2-二基二丙烯酸酯、丙烯酸乙酯、N-甲基-2-丙烯酰胺、N-2,2-丙烯基-2-丙烯酰胺、N-乙基丙烯酰胺、N,N'-乙烯基双丙烯酰胺、(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯、N,N'-二甲基丙烯酰胺、N,N-二甲基甲基丙烯酰胺、双键化聚赖氨酸中的一种或多种。
可选的，所述第二功能单体还带有功能性基团B。
可选的，所述功能性基团B选自羟基、羧基、羧酸胆碱、磺酸胆碱、磷酸胆碱、吡咯烷酮、磺酸基团、羧酸根离子、磺酸酯、亚砜、酰胺基团、甲氧基中的至少一种。
可选的，所述第二功能单体选自丙烯酰胺、丙烯酸、丙烯酸钠、甲基丙烯酸、甲基丙烯酸钠、2-(丙-2-烯酰氨基)乙酸、2-丙烯酰胺基-2-甲基丙磺酸、甲基丙烯酸羟乙酯、3-[[2-(甲基丙烯酰氧)乙基]二甲基铵]丙酸酯、N-甲基-2-丙烯酰胺、N-异丙基丙烯酰胺、N-(羟甲基)丙烯酰胺、N-(2-羟基乙基)甲基丙烯酰胺、3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐、2-甲基丙烯酰氧乙基磷酸胆碱、N-(2-羟乙基)丙烯酰胺、N-(甲氧基甲基)甲基丙烯酰胺、2-丙烯酰胺-2-甲基丙磺酸、双键化透明质酸中的一种或多种。
步骤BS130中：
可选的，所述第二功能单体通过物理渗透进入所述生物材料中。
所述的物理渗透可以理解为当步骤S120处理后的生物材料浸泡于含第二功能单体的溶液中时，溶液中的第二功能单体通过粘附于所述生物材料的表面或嵌入生物材料内的缝隙中，该过程中，第二功能单体与生物材料之间不发生化学反应。
可选的，所述含第二功能单体的溶液中仅包括第二功能单体和不参与反应的溶剂。
可选的，所述含第二功能单体的溶液中第二功能单体的v/v浓度为0.1％-20％；浸泡时间为0.5h-120h。
进一步地，所述含第二功能单体的溶液中第二功能单体的v/v浓度为0.1％-6％。
可选的，所述第一功能单体选自烯丙基缩水甘油醚、甲基丙烯酸缩水甘油酯和丙烯酸缩水甘油酯中的至少一种。
可选的，步骤BS110中：
所述醛基交联剂溶液的w/w浓度为0.1％～5％；交联时间为0.5h-120h。
可选的，步骤BS120中：
含所述第一功能单体的溶液中仅包括第一功能单体和不参与化学反应的溶剂。
可选的，含所述第一功能单体的溶液中第一功能单体的w/w浓度为1％～10％；反应时间为2～120小时。
可选的，含所述第一功能单体的溶液中溶剂为甲醇、乙醇、乙二醇、丙醇、1,2-丙二醇、1,3-丙二醇、异丙醇、丁醇、异丁醇、1,2-丁二醇、1,3-丁二醇、1,4-丁二醇和甘油中任意一种的水溶液、水、生理盐水、pH中性缓冲液中的一种或多种。
本申请还提供一种生物瓣膜材料的制备方法，包括：
步骤BS110将生物材料与醛基交联剂溶液接触进行交联；
步骤BS120将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
本申请还提供一种生物瓣膜材料的制备方法，包括：
步骤BS110将生物材料与醛基交联剂溶液接触进行交联；
步骤BS120将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
步骤BS130将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第 二功能单体具有第二碳碳双键；
步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
本申请还提供一种生物瓣膜材料的制备方法，包括：
步骤BS110将生物材料与醛基交联剂溶液接触进行交联；
步骤BS120将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
步骤BS130将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第二功能单体具有第二碳碳双键和功能性基团B；
步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
本申请还提供一种生物瓣膜材料，由所述的制备方法制备得到。
本申请还提供一种生物瓣膜材料，包括：
步骤S100，将生物材料依次经第一处理液和第二处理液进行处理，得到化学接枝有第一碳碳双键的预处理后的生物材料；所述第一处理液和所述第二处理液彼此相异的含有试剂A和试剂B中的其中一种，其中试剂A为带有所述第一碳碳双键的第一功能单体，试剂B为醛基交联剂；
步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
本申请还提供一种生物瓣膜材料，包括：
步骤BS110将生物材料与醛基交联剂溶液接触进行交联；
步骤BS120将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
本申请还提供一种生物瓣膜材料，包括：
步骤BS110将生物材料与醛基交联剂溶液接触进行交联；
步骤BS120将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
步骤BS130将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第二功能单体具有第二碳碳双键；
步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
本申请还提供一种生物瓣膜材料，包括：
步骤BS110将生物材料与醛基交联剂溶液接触进行交联；
步骤BS120将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
步骤BS130将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第二功能单体具有第二碳碳双键和功能性基团B；
步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
本申请还提供一种生物瓣膜，包括支架和瓣叶，所述瓣叶为所述的生物瓣膜材料。
可选的，所述生物瓣膜为心脏瓣膜。
本申请还提供一种介入***，包括心脏瓣膜和导管组件，所述心脏瓣膜折叠后由导管组件输送，心脏瓣膜包括支架和瓣叶，所述瓣叶为所述的生物瓣膜材料。
与现有技术相比，本申请至少具有如下有益效果之一：
(1)本申请的方法在醛基交联时引入功能单体进行共交联，共交联的同时引入碳碳双键，作为二次交联的基础，通过两次交联制备交联生物材料，可提高生物材料的交联度，改善生物材料的机械性能。
(2)本申请在共交联过程中，功能单体引入双键的同时也可封闭生物材料上的部分残留醛基，改善生物材料的抗钙化和抗凝血性能，同时也可进一步提高交联效率。
(3)本申请的方法在引入碳碳双键的同时还可引入功能性基团，可进一步改善生物材料的性能，例如表面亲水性、生物相容性等。
(4)共交联结束后，再次浸泡于功能单体溶液中，对剩余残留醛基消除的同时引入碳碳双键，为后续的双键聚合提供更多碳碳双键，进一步提高生物材料的交联度。
(5)本申请通过在双键聚合步骤加入第二功能单体共聚，形成更多更大的聚合物交联网络，可以提高生物材料的交联度和提升抗钙化性能。
(6)本申请在二次引入碳碳双键的同时还引入附加功能性基团，可赋予生物材料新的特性，进一步改善生物材料的性能。
(7)生物材料在戊二醛交联的同时与功能单体进行共交联，改善戊二醛交联生物材料的抗钙化及抗凝血性能。
针对先将生物材料进行戊二醛交联、再引入碳碳双键、最后引发碳碳双键聚合二次交联的方案，还具有进一步的有益效果：
(8)本申请在戊二醛交联后的生物瓣膜材料的基础上，通过双键化修饰在戊二醛交联的生物瓣膜材料上引入双键作为二次交联的基础，进一步地通过引发戊二醛交联的生物瓣膜材料上双键的聚合从而实现二次交联，一方面，可进一步地提高生物瓣膜材料的交联度，从而改善生物瓣膜材料的稳定性，另一方面，可进一步地降低了结构降解引起的钙化风险，因此还具备一定的抗钙化性能。
(9)在先进行戊二醛交联处理再化学接枝第一碳碳双键的方案中，带碳碳双键的功能单体通过环氧乙烷基与戊二醛交联膜表面的氨基、羟基及羧基通过化学反应连接，将碳碳双键主要接入生物瓣膜材料的表面，能更好保护生物材料的原纤维结构，可在有效确保膜片力学性能的同时，保证生物材料原始纤维的取向方向。
附图说明
图1为本申请方案一一种较优选实施方案的工艺流程图；
图2为本申请方案一实施方案的化学原理示意图；
图3为本申请方案二一种较优选实施方案的工艺流程图；
图4为本申请方案三一种较优选实施方案的工艺流程图；
图5为本申请方案四一种较优选实施方案的工艺流程图；
图6为本申请方案五一种较优选实施方案的工艺流程图；
图7为本申请一种较优选实施方案的反应原理图；
图8为本申请另一种较优选实施方案的反应原理图；
图9为实施例1的样品1与对照组1心包膜(GA)的红外光谱图；
图10为实施例1的样品1与对照组1心包膜(GA)乳酸脱氢酶相对活性结果图；
图11为实施例1的样品1与对照组1心包膜(GA)大鼠皮下植入后挂钙量检测结果图；
图12为实施例2的样品2与对照组2心包膜(GA)水接触角检测结果图；
图13为实施例2的样品2与对照组2心包膜(GA)乳酸脱氢酶检测以及溶血率结果图；
图14为实施例2的样品2与对照组2心包膜(GA)钙离子浓度结果图；
图15为实施例3的基本原理图；
图16为对照组3切片的茜素红染色结果图；
图17为样品3大鼠皮下植入后切片的茜素红染色结果图；
图18为样品4大鼠皮下植入后切片的茜素红染色结果图；
图19为样品5大鼠皮下植入后切片的茜素红染色结果图；
图20为样品6大鼠皮下植入后切片的茜素红染色结果图；
图21为样品7大鼠皮下植入后切片的茜素红染色结果图；
图22为样品8大鼠皮下植入后切片的茜素红染色结果图；
图23为实施例10的基本原理图；
图24为对照组4的对照样植入30天后得茜素红染色切片图；
图25为10号样植入30天后得茜素红染色切片图；
图26为11号样植入30天后得茜素红染色切片图；
图27为12号样植入30天后得茜素红染色切片图；
图28为13号样植入30天后得茜素红染色切片图；
图29为14号样植入30天后得茜素红染色切片图；
图30为15号样植入30天后得茜素红染色切片图；
图31为对照样4血液接触实验扫描电镜图；
图32为12号样血液接触实验扫描电镜图；
[根据细则91更正，06.12.2022]图33为13号样血液接触实验扫描电镜图；图34为实施例17的基本原理图；
图35为对照组5的对照样植入30天后得茜素红染色切片图；
图36为样品17植入30天后得茜素红染色切片图；
图37为样品18植入30天后得茜素红染色切片图；
图38为样品19植入30天后得茜素红染色切片图；
图39为样品20植入30天后得茜素红染色切片图；
图40为样品21植入30天后得茜素红染色切片图。
图41为实施例23的基本原理图；
图42为对照组6的对照样的血液扫面电镜图；
图43为样品23的血液孵育实验扫描电镜图；
图44为样品24的血液孵育实验扫描电镜图；
图45为对照组6的对照样植入60天后得茜素红染色切片图；
图46为样品23植入60天后得茜素红染色切片图；
图47为样品24植入60天后得茜素红染色切片图；
图48为样品25植入60天后得茜素红染色切片图；
图49为样品26植入60天后得茜素红染色切片图；
图50为样品27植入60天后得茜素红染色切片图；
图51为实施例29和30的反应原理图；
图52为实施例31的反应原理图；
图53为实施例32的反应原理图；
图54为实施例33的反应原理图；
图55为对照样7的血液接触实验扫描电镜图；
图56为样品29的血液接触实验扫描电镜图；
图57为样品31血液接触实验扫描电镜图；
图58为对照样7大鼠植入30天后得茜素红染色切片图；
图59为样品30大鼠植入30天后得茜素红染色切片图；
图60为样品32大鼠植入30天后得茜素红染色切片图；
图61为样品33大鼠植入30天后得茜素红染色切片图；
图62为样品34大鼠植入30天后得茜素红染色切片图；
图63为本申请方案六的一种较优选实施方式的工艺流程图；
图64为本申请方案六一种较优选实施方案的反应原理图；
图65为对照组8(戊二醛交联猪心包)在大鼠皮下植入30天后的茜素红染色结果图；
图66为实施例42的样品42在大鼠皮下植入30天后的茜素红染色结果图；
图67为实施例46的样品46在大鼠皮下植入30天后的茜素红染色结果图；
图68为实施例48的样品48在大鼠皮下植入30天后的茜素红染色结果图；
图69为本申请方案七一种双键后共聚交联实施方式的工艺流程图；
图70为本申请方案七双键后共聚交联实施方案的反应原理图；
图71为对照组9(戊二醛交联猪心包)在大鼠皮下植入30天后的茜素红染色结果图；
图72为实施例52的样品52在大鼠皮下植入30天后的茜素红染色结果图；
图73为实施例53的样品53在大鼠皮下植入30天后的茜素红染色结果图；
图74为实施例56的样品56在大鼠皮下植入30天后的茜素红染色结果图；
图75为本申请方案八双键后功能化共聚交联实施方式的工艺流程图；
图76为本申请方案八双键后功能化共聚交联实施方案的反应原理图；
图77为对照组10(戊二醛交联猪心包)的血液黏附扫面电镜图；
图78为实施例62的样品62的血液黏附扫面电镜图；
图79为实施例63的样品63的血液黏附扫面电镜图；
图80为实施例68的样品68的血液黏附扫面电镜图；
图81为对照组10(戊二醛交联猪心包)在大鼠皮下植入30天后的茜素红染色结果图；
图82为实施例62样品62在大鼠皮下植入30天后的茜素红染色结果图；
图83为实施例63样品63在大鼠皮下植入30天后的茜素红染色结果图；
图84为实施例69样品69在大鼠皮下植入30天后的茜素红染色结果图；
图85为本申请心脏瓣膜的结构示意图；
图86为本申请介入***的结构示意图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。
为改善常规戊二醛交联膜的功能，在戊二醛交联基础上，本申请通过引入碳碳双键再引发碳碳双键的二次交联，改善基于戊二醛交联的生物瓣膜的抗凝血、抗钙化、弹性等各项性能。具体地，提供一种生物瓣膜材料的制备方法，包括：
步骤S100，将生物材料依次经第一处理液和第二处理液进行处理，得到化学接枝有第一碳碳双键的预处理后的生物材料；所述第一处理液和所述第二处理液彼此相异的含有试剂A和试剂B中的其中一种，其中试剂A为带有所述第一碳碳双键的第一功能单体，试剂B为醛基交联剂；
步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
通过化学反应引入的第一碳碳双键再在引发剂作用下进行聚合反应，进一步形成交联网络，改善基于戊二醛交联的生物瓣膜的抗凝血、抗钙化、弹性等各项性能。
本申请所采用的生物材料为现有戊二醛交联工艺中常规的生物材料，所述生物材料的胶原含量为60％～90％。进一步地，所述生物材料为动物组织，动物来源为猪、牛、马或羊，包括心包膜、瓣膜、肠膜、脑膜、肺膜、血管、皮肤或韧带的一种或多种。
可选的，所述动物组织为新鲜的动物组织或经脱细胞处理后的生物组织。
可选的，所述脱细胞处理的步骤中，利用表面活性剂对生物组织进行如下的处理：
利用离子型表面活性剂对生物组织进行脱细胞；或
利用非离子型表面活性剂对生物组织进行脱细胞。
所述离子型表面活性剂主要用于裂解细胞，非离子表面活性剂主要用于去除脂类物质(例如磷脂)。
可选的，所述离子型表面活性剂为脱氧胆酸钠、脂肪酸钾皂、十二烷基硫酸钠、胆酸钠、十六烷基三甲基溴化铵、脂肪酸钾盐、烷基二甲基磺丙基甜菜碱中的至少一种。
可选的，所述非离子型表面活性剂为曲拉通、吐温中的至少一种。
可选的，所述离子型表面活性剂为十二烷基磺酸钠，非离子型表面活性剂为曲拉通。
可选的，所述离子型表面活性剂为十二烷基磺酸钠，非离子型表面活性剂为吐温-20。
本申请的交联剂采用当前主流交联方法所用的醛基交联剂，可选的，所述醛基交联剂可选择戊二醛、甲醛中的一种。
可选的，本申请的步骤S100中采用非缩合的化学反应接入所述第一碳碳双键。
可选的，步骤S100中，所述生物材料经过醛基交联剂处理之前未经过任何其他试剂参与的化学反应。
进一步可选的，步骤S100的反应体系中通过带有活性基团的第一功能单体提供所述第一碳碳双键，且步骤S100中的反应原料仅包括所述生物材料、所述第一功能单体以及所述醛基交联剂。也可以理解为，当第一处理液含有试剂A(带有所述第一碳碳双键的第一功能单体)时，第一处理液中仅含有第一功能单体和不参与化学反应的溶剂；当第一处理液含有试剂B(醛基交联剂)时，第一处理液中仅含有醛基交联剂和不参与化学反应的溶剂。同理适用于第二处理液。
本申请中，所述将生物材料依次经第一处理液和第二处理液进行处理，可以理解为所包含的试剂与生物材料相接触的先后顺序、加料或彼此接触的方式并不严格限定，例如第一处理液处理后可以取出生物材料与第二处理液接触，也可以将第二处理液直接加入浸有生物材料的第一处理液中。
所述第一处理液和所述第二处理液彼此相异的含有试剂A和试剂B中的其中一种，也可以理解为：当第一处理液含有试剂A时，第二处理液只能含有试剂B，此处所说的第二处理液只能含有试剂B理解为强调其不含有试剂A，而非第二处理液中只有试剂B；当第一处理液含有试剂B时，第二处理液只能含有试剂A，同理，此处所说的第二处理液只能含有试剂A理解为强调其不含有试剂B，而非第二处理液中只有试剂A。
步骤S100中第一功能单体需参与化学接枝反应，可选的，所述第一功能单体还带有活性基团，通过该活性基团参与化学接枝反应。
进一步地，当第一处理液含有试剂A(带有所述第一碳碳双键的第一功能单体)、第二处理液含有试剂B(醛基交联剂)时，第一功能单体的活性基团可与醛基反应，通过化学反应将第一碳碳双键间接接入生物材料上；当第一处理液含有试剂B(醛基交联剂)、第二处理液 含有试剂A(带有所述第一碳碳双键的第一功能单体)时，第一功能单体的活性基团可与氨基反应，将第一碳碳双键直接接入生物材料上。即，步骤S100中化学接枝第一碳碳双键的过程中，第一碳碳双键可以间接通过交联剂连接在生物材料上，也可以直接与生物材料上的活性基团(至少包括氨基)反应接入生物材料中。
一种可选方案中，步骤S100中，第一处理液含有试剂A(带有所述第一碳碳双键的第一功能单体)、第二处理液含有试剂B(醛基交联剂)，该可选方案下，所述第一功能单体先物理渗透进入生物材料中；所述生物材料的氨基以及所述第一功能单体的活性基团再与所述醛基交联剂进行共交联接入第一碳碳双键。该方案中，第一功能单体先物理渗透进入生物材料中，再加入醛基交联剂，进行共交联即所述化学反应，生物材料上的氨基通过醛基交联剂间接的接入第一碳碳双键。该方案中，可选的，所述第一功能单体的活性基团为氨基或酰肼。
另一种可选方案中，步骤S100中，第一处理液含有试剂B(醛基交联剂)、第二处理液含有试剂A(带有所述第一碳碳双键的第一功能单体)，该可选方案下，所述生物材料先与醛基交联剂进行交联反应，再与所述第一功能单体的活性基团反应接入第一碳碳双键。该方案中，步骤S100中先加入醛基交联剂，醛基交联剂先与生物材料的部分氨基反应，再加入第一功能单体，利用生物材料上剩余的氨基及其他基团(例如羟基和羧基)与第一功能基团上活性基团反应直接接入第一碳碳双键。该方案中，可选的，所述第一功能单体的活性基团为环氧乙烷基，生物材料上除剩余氨基参与反应外，其羟基和羧基也可与环氧乙烷基反应，参与所述化学反应。
第一种可选方案中，在戊二醛改性过程中通过加入功能单体进行共交联，共交联的同时引入碳碳双键，作为二次交联的基础，通过两次交联制备交联生物材料，可提高生物材料的交联度，改善生物材料的机械性能；第二种可选方案中，先进行戊二醛交联处理，然后由戊二醛交联膜上的残余氨基以及羟基、羧基等活性基团化学连接带碳碳双键的功能单体，带碳碳双键的功能单体通过环氧乙烷基与戊二醛交联膜表面的氨基、羟基及羧基通过化学反应连接，将碳碳双键主要接入生物瓣膜材料的表面，在两次交联改善生物材料的交联度、机械性能的基础上，能更好保护生物材料的原纤维结构，可在有效确保膜片力学性能的同时，保证生物材料原始纤维的取向方向。
本申请中利用生物材料上的氨基理解为生物材料上的至少一部分氨基参与了引入第一碳碳双键的化学反应，步骤S100在实际操作中，可以包括多个子步骤，步骤S100的反应体系中涉及的原料参与至少其中一子步骤，并不严格限制参与所有子步骤的反应。
一方面，本申请对现有的戊二醛交联方法进行改进，在戊二醛交联的同时引入功能单体，进行共交联，改善戊二醛交联生物材料的性能，接近戊二醛交联生物材料抗钙化及抗凝血性能差的问题。
该改进的交联方案(记为方案一)中，在戊二醛交联前引入带有与醛基反应基团的功能单体，该功能单体先物理渗透进入生物材料，然后与醛基交联剂进行共交联。
具体的，包括：
AS110将生物材料与含第一功能单体的溶液接触，进行物理渗透；所述第一功能单体具有至少一个与醛基反应的基团；
S120向步骤AS110的体系中加入醛基交联剂，进行共交联。
该方案一的反应原理：
功能单体先物理渗透进入生物材料中，该功能单体带有与醛基反应的基团，功能单体渗透后加入醛基交联剂，进行共交联，共交联过程中，所发生的反应至少包括：
1)一部分交联剂两端的醛基均与生物材料的氨基反应；2)一部分交联剂其中一端的醛基与生物材料的氨基反应、另一端的醛基与第一功能单体反应；3)一部分交联剂其中一端的醛基与生物材料的氨基反应、另一端的醛基在生物材料上形成残留醛基；4)部分残留醛基与第一功能单体的氨基反应。
另一方面，当前市场上的生物瓣膜产品几乎全是采用戊二醛进行交联制备而成，戊二醛可以交联心包膜当中的胶原蛋白，但是，戊二醛交联的生物瓣膜存在着不容忽视的血栓问题，严重威胁着患者的生活质量及生命。本申请在戊二醛交联基础上通过交联手段的改进，改善戊二醛交联膜的机械性能、抗钙化及抗凝血性能。
该改进的交联方案(记为方案二)中，在戊二醛交联前引入带有碳碳双键和与醛基反应基团的第一功能单体，该第一功能单体先物理渗透进入生物材料，然后与醛基交联剂进行共交联，共交联过程中，第一功能单体与生物材料上的残留醛基反应，将第一碳碳双键引入生物材料中，再引发双键聚合，发生二次交联，完成生物材料的交联处理，二次交联后的生物材料还可带有功能性基团，进一步改善生物膜的生物相容性等。
具体的，包括：
AS110将生物材料浸泡于含第一功能单体的溶液中进行物理渗透；所述第一功能单体具有至少一个第一碳碳双键和至少一个与醛基反应的基团；
AS120向步骤AS110的体系中加入醛基交联剂，进行共交联；
S200将经步骤AS120处理后的生物材料与引发剂接触，引发双键聚合。
该方案二的原理：
第一步：第一功能单体先物理渗透进入生物材料中，引入的第一功能单体带有第一碳碳双键和与醛基反应的基团(如氨基)，功能单体充分渗透后加入醛基交联剂(如戊二醛)，进行共交联，共交联过程中，所发生的反应至少包括：
1)一部分交联剂两端的醛基均与生物材料的氨基反应；2)一部分交联剂其中一端的醛基与生物材料的氨基反应、另一端的醛基与第一功能单体的氨基反应；3)一部分交联剂其中一端的醛基与生物材料的氨基反应，另一端的醛基在生物材料上形成残留醛基；4)部分残留醛基与第一功能单体的氨基反应，将第一碳碳双键引入生物材料。
第二步：完成交联并引入碳碳双键的生物材料与含引发剂的溶液接触，引发双键聚合，发生二次交联。
再一方面，为进一步改进戊二醛交联膜的生物形容性问题，进一步地，本申请在方案二的功能单体基础上，进一步引入工功能性基团，即该改进的交联方案(记为方案三)中，在戊二醛交联前引入带有氨基、第一碳碳双键和功能性基团A的第一功能单体，该功能单体先物理渗透进入生物材料，然后与醛基交联剂进行共交联，第一功能单体的氨基与醛基反应，将第一碳碳双键和功能性基团A同时引入生物材料中，再引发双键聚合，发生二次交联，二次交联后的生物材料带有功能性基团A，可进一步改善生物材料的生物相容性等。
具体的，包括：
AS110将生物材料浸泡于含第一功能单体的溶液中，物理渗透；所述第一功能单体具有至少一个氨基、至少一个第一碳碳双键和至少一个功能性基团A；
AS120向步骤AS110处理后的生物材料所浸泡的溶液中加入醛基交联剂，进行共交联；
S200将经步骤AS120处理后的生物材料与引发剂接触，引发双键聚合。
该方案三的原理：
第一步：第一功能单体先物理渗透进入生物材料中，引入的第一功能单体带有氨基、第一碳碳双键和功能性基团A，第一功能单体充分渗透后加入醛基交联剂(如戊二醛)，进行共交联，共交联过程中，所发生的反应至少包括：
1)一部分交联剂两端的醛基均与生物材料的氨基反应；2)一部分交联剂其中一端的醛基与生物材料的氨基反应、另一端的醛基与第一功能单体的氨基反应；3)一部分交联剂其中一端的醛基与生物材料的氨基反应、另一端的醛基在生物材料上形成残留醛基；4)部分残留醛基与第一功能单体的氨基反应，将第一碳碳双键引入生物材料。
第二步：完成交联并引入碳碳双键后的生物材料与含引发剂的溶液接触，引发生物材料上的碳碳双键聚合，发生二次交联。
该方案的方法在醛基交联时引入第一功能单体进行共交联，共交联的同时引入第一碳碳双键作为二次交联的基础，通过两次交联制备交联生物材料，可提高生物材料的交联度。本申请的方法在引入第一碳碳双键的同时还引入了功能性基团A，可进一步改善生物材料的性能，例如表面亲水性、生物相容性等。
再一方面，为进一步改善戊二醛交联膜的机械性能、抗钙化及抗凝血性能，进一步地，本申请在方案三的基础上进一步改进交联手段。
该改进的交联方案(记为方案四)中，在戊二醛交联前引入带有第一碳碳双键和与醛基反应基团的第一功能单体，该第一功能单体先物理渗透进入生物材料，然后与醛基交联剂进行共交联，第一功能单体的氨基与醛基反应，将第一碳碳双键和功能性基团A同时引入生物材料中；再进一步通过第二功能单体物理渗透引入一部分第二碳碳双键，最后引发生物材料上的第一碳碳双键和第二功能单体的第二碳碳双键聚合，形成交联网络，进一步提高生物材料的交联度。
具体的，包括：
AS110将生物材料浸泡于含第一功能单体的溶液中进行物理渗透；所述第一功能单体具有至少一个第一碳碳双键和至少一个与生物材料上的残留醛基反应的基团；
AS120向步骤AS110的体系中加入醛基交联剂，进行共交联；
AS130将经步骤AS120处理后的生物材料浸泡于含第二功能单体的溶液中进行物理渗透；所述第二功能单体具有至少一个第二碳碳双键；
S200向步骤AS130的体系中加入引发剂，引发双键聚合。
该方案四的反应原理：
第一步反应原理同方案三的第一步，再此不再赘述。
第二步：第二功能单体先物理渗透进入完成共交联并一次引入碳碳双键后的生物材料，进一步引入第二碳碳双键，该步骤引入第二碳碳双键为物理渗透，待第二功能单体渗透后，引发第二功能单体的第二碳碳双键与生物材料表面的第一碳碳双键共聚合，进行二次交联，形成交联网络。
该方案的方法通过醛基共交联和双键聚合二次交联对生物材料进行交联处理，两次交联处理得到的生物材料其交联度好；该方案中共交联引入第一碳碳双键后再次通过第二功能单体进一步引入第二碳碳双键，第二次通过物理渗透引入的碳碳双键使在双键聚合过程中有额外的功能单体参与共聚，形成更大的聚合物交联网络，有利于提升生物瓣膜交联度和抗钙化性能。
再一方面，为进一步改善生物材料的性能，如生物相容性等，进一步地，在方案四的基础上，进一步通过第二功能单体引入功能基团B。
该改进的交联方案(记为方案五)中，在戊二醛交联前引入带有第一碳碳双键和残留氨基的第一功能单体，该第一功能单体先物理渗透进入生物材料，然后与醛基交联剂进行共交联，第一功能单体的氨基与生物膜上氨基的通过与戊二醛交联剂醛基反应将第一碳碳双键引入生物材料中，并同时引入功能性基团B；最后引发第二功能单体与生物材料上的碳碳双键共聚合，形成交联网络的同时引入功能性官能团B，进一步提高生物材料的交联度和性能。
具体的，包括：
AS110将生物材料浸泡于含第一功能单体的溶液中及逆行物理渗透；所述第一功能单体具有至少一个氨基和至少一个第一碳碳双键；
AS120向步骤AS110的体系中加入醛基交联剂，进行共交联；
AS130将经步骤AS120处理后的生物材料浸泡于含第二功能单体的溶液中进行物理渗透；所述第二功能单体具有至少一个第二碳碳双键和至少一个功能性基团B；
S200向步骤AS130的体系中加入引发剂，引发双键聚合。
该方案的原理同方案四，区别在于第二次引入第二碳碳双键的同时还引入功能性基团B，第二功能单体的功能性基团B可赋予生物材料新的特性。
该方案通过醛基共交联和双键聚合二次交联对生物材料进行交联处理，两次交联处理得到的生物材料其交联度好；交联过程中第一功能单体可反应掉部分残留醛基；共交联引入第 一碳碳双键后再次通过第二功能单体渗透进一步引入第二碳碳双键，两步引入碳碳双键可在双键聚合过程中有额外的功能单体参与共聚，形成更大的聚合物交联网络，有利于提升生物瓣膜交联度和抗钙化性能；第二次引入碳碳双键的同时还引入功能性基团B，可赋予生物材料新的特性，进一步改善生物材料的性能。
较为优选的实施方式中，可选的，在方案一、方案二级方案3的步骤AS120之后、方案四和方案五的步骤AS130之前还包括步骤AS120(M)：将经步骤AS120处理后的生物材料浸泡于含第三功能单体的溶液中，消除剩余部分的残留醛基；该步骤的第三功能单体具有至少一个与醛基反应的基团。
方案一中，第一功能单体和第三功能单体均具有至少一个与醛基反应的基团，第一功能单体在共交联过程中通过该基团与醛基反应，发生共交联；在包含AS120(M)的方案中，第三功能单体通过该基团与生物材料上的残留醛基反应，消除残留醛基。可选的，第一功能单体和第三功能单体中所述与醛基反应的基团各自独立地选自氨基或酰肼。
可选的，与醛基反应的基团选择氨基的方案中，所述第一功能单体和第三功能单体各自独立地选自至少一个氨基取代的烷烃、至少一个氨基取代的环烷烃、至少一个氨基取代的烯烃或含氨基的聚合物。
与醛基反应的基团选择氨基的方案中，进一步可选的，所述第一功能单体和第三功能单体各自独立地选自乙二胺、2-甲基丙胺、1,4-丁二胺、正己胺、油胺、1,10-二氨基癸烷、辛胺、正十一胺、十二胺、十四胺、十六胺、十七烷-9-胺、环十二胺、环庚烷胺、环辛胺、6-甲基庚烷-1-胺、十九烷-10-胺、3-乙基戊-1-胺、2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯中的一种。
与醛基反应的基团选择酰肼的方案中，可选的，所述第一功能单体和第三功能单体各自独立地选择甲基丙烯酰肼或丙烯酰肼。
方案一的第一功能单体和第三功能单体除带有氨基外，还可带有功能性基团A；可选的，所述功能性基团A为亲水基团。进一步可选的，所述第一功能单体和第三功能单体的功能性基团A各自独立地选自羟基、羧基、酰胺基、磺酸基、两性离子、聚乙二醇、脲基、氨基甲酸酯基、羧酸根离子、磺酸酯、亚砜、吡咯烷酮中的至少一种。
所述功能性基团A中：
羟基：作为亲水的基团，提升共交联生物材料的表面亲水性以实现抗凝血的效果；
羧基：作为亲水的基团，提升共交联生物材料的表面亲水性以实现抗凝血的效果；
酰胺基：作为亲水的基团，提升共交联生物材料的表面亲水性以实现抗凝血的效果；
羧酸根离子、磺酸基：通过离子水合作用提升共交联生物材料的表面亲水性以实现抗凝血的效果；
亚砜、吡咯烷酮：作为亲水的基团，提升共交联生物材料的表面亲水性以实现抗凝血的效果；
两性离子：通过离子水合作用提升共交联生物材料的表面亲水性以实现抗凝血的效果；有利于形成共交联生物瓣膜电中性表面进而降低对钙离子的吸附而达到抗钙化效果；
聚乙二醇：作为亲水的基团，提升共交联生物材料的表面亲水性；增加钙离子与胶原间结合的空间位阻，提升共交联生物瓣膜材料表面亲水性；
氨基甲酸酯基、脲基：作为亲水的基团，提升共交联生物材料的表面亲水性，以实现抗凝血的效果
氨基甲酸酯基：作为亲水的基团，提升共交联生物材料的表面亲水性，以实现抗凝血的效果。
对于同时带氨基和亲水功能基团的方案中，可选的，述第一功能单体和第二功能单体各自独立地选自2-氨基-4-戊酸、2-氨基-辛酸、2-氨基-5-羟基戊酸、2-氨基-2,3-二甲基丁酰胺、2-氨基十四烷酸、2-氨基-4-甲基戊酸、三羟甲基氨基甲烷、氨基封端的聚乙二醇及聚乙二醇结构衍生物、氨基油酸、天然氨基酸、非天然氨基酸、、聚天然氨基酸(如聚赖氨酸)、DL-2-氨基-4-戊烯酸、2-氨基-7-烯-辛酸、6-烯-庚氨酸、2-氨基戊-4-烯酸、4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇、4-(1-氨基-乙基)-七-1,6-二烯-4-醇、双键化多聚赖氨酸中的一种。
关于方案一的第一功能单体和第二功能单体，可以理解为第一功能单体和第三功能单体各自独立地选自上述范围，可相同也可不同。
方案二的第一功能单体具有至少一个与醛基反应的基团，在共交联过程中，该功能单体通过该基团与生物材料上的残留醛基反应，将碳碳双键引入生物材料中；在包含AS120(M)的方案中，第三功能单体具有至少一个与醛基反应的基团，通过该基团与生物材料上的残留醛基反应，消除残留醛基。第一功能单体和第三功能单体中所述与醛基反应的基团包括但不限于氨基和酰肼。可选的，第三功能单体还可具有至少一个碳碳双键，再次用第三功能单体溶液处理生物材料时，通过第三功能单体上的氨基与生物材料上的残留醛基反应，封闭剩余残留醛基的同时再次引入碳碳双键，增加用于后续双键聚合的碳碳双键基数，有利于提高交联度。即，第一功能单体和第三功能单体中所述与醛基反应的基团各自独立地选自氨基和酰肼中的一种，可相同也可不同。满足带有至少一个氨基和至少一个碳碳双键的功能单体的一种方案中，可直接采用市购产品，可选的，方案二中所述第一功能单体和第三功能单体各自独立地选自DL-2-氨基-4-戊烯酸、2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯、甲基丙烯酰肼和丙烯酰肼中的至少一种。
方案三的第一功能单体具有至少一个活性基团，在共交联过程中，该功能单体通过其活性基团与生物材料上的残留醛基反应，将第一碳碳双键引入生物材料中，第一功能单体的活性基团可为氨基或酰肼。
在包含AS120(M)的方案中，第三功能单体具有至少一个活性基团，通过该活性基团与生物材料上的残留醛基反应，消除残留醛基。第三功能单体的活性基团可为氨基或酰肼。可选的，第三功能单体还可具有至少一个碳碳双键，再次用第三功能单体溶液处理生物材料 时，通过第三功能单体上的氨基与生物材料上的残留醛基反应，封闭剩余残留醛基的同时再次引入碳碳双键，增加用于后续双键聚合的碳碳双键基数，有利于提高交联度。
该方案中，所述第一功能单体中除带有碳碳双键和氨基外，还可带有功能性基团A，所述第三功能单体除除带有碳碳双键和氨基外，也可带有功能性基团C。可选的，所述功能性基团A和功能性基团C各自独立地选自羟基、羧基、酰胺基和磺酸基中的至少一种。即，第一功能单体的功能性基团A为羟基、羧基、酰胺基和磺酸基中的至少一种；第三功能单体的功能性基团C也为羟基、羧基、酰胺基和磺酸基中的至少一种；可相同也可不同。
引入羟基可改善生物瓣膜的亲水性；引入羧基可维持步骤AS110反应体系pH中性；引入酰胺基可通过水分子与酰胺基之间的氢键相互作用增加生物瓣膜亲水性；引入磺酸基可通过水分子与磺酸基之间的离子水合作用增加生物瓣膜亲水性。
关于所述第一功能单体和第三功能单体，一种方案中，可直接采用市购产品，可选的，所述第一功能单体和第三功能单体各自独立地选自DL-2-氨基-4-戊烯酸、2-氨基-7-烯-辛酸、6-烯-庚氨酸、2-氨基戊-4-烯酸、4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇和4-(1-氨基-乙基)-七-1,6-二烯-4-醇中的一种。
方案四的第一功能单体具有至少一个与醛基反应的基团，在共交联过程中，第一功能单体通过该基团与生物材料上的部分残留醛基反应，将第一碳碳双键引入生物材料中。可选的，所述第一功能单体中与醛基反应的基团包括但不限于氨基和酰肼。在包含AS120(M)的方案中，第三功能单体具有至少一个与醛基反应的基团，浸泡过程中与生物材料上的剩余残留醛基反应。可选的，所述第三功能单体中与醛基反应的基团包括但不限于氨基和酰肼。
该方案的第一功能单体具有至少一个氨基和至少一个第一碳碳双键，一种方案中，可直接采用市购产品，可选的，所述第一功能单体为2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯、甲基丙烯酰肼、丙烯酰肼中的一种。
该方案的第三功能单体具有至少一个氨基，较优选的方案中，第三功能单体也具有至少一个碳碳双键，再次用第三功能单体溶液处理生物材料时，通过功能单体上的氨基与生物膜上的残留醛基反应，封闭剩余残留醛基的同时可再次引入碳碳双键，增加后续双键聚合的双键基数。
可选的，所述第三功能单体为2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯、甲基丙烯酰肼、丙烯酰肼中的一种。
该方案中，所述第一功能单体中除带有碳碳双键和氨基外，还可带有功能性基团A，所述第三功能单体除除带有碳碳双键和氨基外，也可带有功能性基团C。可选的，所述功能性基团A和功能性基团C各自独立地选自羟基、羧基、酰胺基、磺酸基中的至少一种。
引入羟基可改善生物材料的亲水性；引入羧基可使生物材料呈现电中性；引入羟基可改善生物瓣膜的亲水性；引入羧基可维持步骤AS110反应体系pH中性；引入酰胺基可通过水分子与酰胺基之间的氢键相互作用增加生物瓣膜亲水性；引入磺酸基可通过水分子与磺酸基之间的离子水合作用增加生物瓣膜亲水性。
关于同时满足带有至少一个氨基、至少一个碳碳双键和至少一个如前所述的功能性基团的功能单体，一种方案中，可直接采用市购产品。可选的，所述第一功能单体和第三功能单体各自独立地选自2-氨基-7-烯-辛酸、6-烯-庚氨酸、2-氨基戊-4-烯酸、4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇、4-(1-氨基-乙基)-七-1,6-二烯-4-醇中的一种。
方案四的步骤AS120或步骤AS120(M)结束后，通过第二功能单体进一步引入第二碳碳双键，该引入过程为物理渗透，第二功能单体在该步骤中不与生物材料发生反应，一种方案中，可选的，所述第二功能单体为聚乙二醇二丙烯酸酯、1,4-丁二醇二丙烯酸酯、丙烯酸乙酯、N-甲基-2-丙烯酰胺、N-2,2-丙烯基-2-丙烯酰胺、N-乙基丙烯酰胺、N,N'-乙烯基双丙烯酰胺、(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯中的一种。
方案五中，第一功能单体具有至少一个氨基和至少一个第一碳碳双键，一种方案中，可直接采用市购产品，可选的，所述第一功能单体为2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯、甲基丙烯酰肼、丙烯酰肼中的一种。
方案五的第三功能单体具有至少一个氨基，较优选的方案中，第三功能单体也具有至少一个碳碳双键，再次用第三功能单体溶液处理生物材料时，通过功能单体上的氨基与生物膜上的残留醛基反应，封闭剩余残留醛基的同时可再次引入碳碳双键。
可选的，所述第三功能单体为2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯、甲基丙烯酰肼、丙烯酰肼中的一种。
方案五中第一功能单体和第三功能单体除带有碳碳双键和氨基外，还可带有功能性基团，可选的，所述第一功能单体还具有至少一个功能性基团A，第三功能单体还具有至少一个功能性基团C，；该方案中所述功能性基团A和功能性基团B各自独立地选自羟基、羧基、酰胺基、磺酸基中的一种。
引入羟基可改善生物材料的亲水性；引入羧基可使生物材料呈现电中性；引入羟基可改善生物瓣膜的亲水性；引入羧基可维持步骤AS110反应体系pH中性；引入酰胺基可通过水分子与酰胺基之间的氢键相互作用增加生物瓣膜亲水性；引入磺酸基可通过水分子与磺酸基之间的离子水合作用增加生物瓣膜亲水性。
第一功能单体和第三功能单体带有功能性基团的一种方案中，可直接采用市购产品。可选的，所述第一功能单体和第三功能单体各自独立地选自DL-2-氨基-4-戊烯酸、2-氨基-7-烯-辛酸、6-烯-庚氨酸、2-氨基戊-4-烯酸、4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇、4-(1-氨基-乙基)-七-1,6-二烯-4-醇中的一种。
方案五中，步骤AS120或步骤AS120(M)结束后，生物材料与含第二功能单体溶液接触，第二功能单体物理渗透进入生物材料中，第二功能单体除带有双键外，还带有可改善生物材料性能的功能性基团B，可选的，所述功能性基团B为羟基、羧基、羧酸胆碱、磺酸胆碱、磷酸胆碱、吡咯烷酮、磺酸基团、羧酸根离子、磺酸酯、亚砜、酰胺基团、甲氧基中的一种。
针对第二功能单体，一种方案中，所述第二功能单体为聚乙二醇二丙烯酸酯、丙烯酰胺、2-(丙-2-烯酰氨基)乙酸、2-丙烯酰胺基-2-甲基丙磺酸、甲基丙烯酸羟乙酯、3-[[2-(甲基丙烯酰氧)乙基]二甲基铵]丙酸酯、N-甲基-2-丙烯酰胺、N-异丙基丙烯酰胺、N-(羟甲基)丙烯酰胺、N-(2-羟基乙基)甲基丙烯酰胺、N,N-二甲基甲基丙烯酰胺、3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐、2-甲基丙烯酰氧乙基磷酸胆碱中的一种。可市购获得。
对于方案四和方案五，所述第二功能单体除上述市购途径外，也可自行双键改性制备，可选的，所述第二功能单体为双键化透明质酸或双键化多聚赖氨酸。
对于方案一～方案五，第一功能单体和第三功能单体处如前所示的市购途径外，也可通过双键改性制备得到，例如双键化多聚赖氨酸。
即，所述第一功能单体、第二功能单体和第三功能单体可独立地选择双键化透明质酸或双键化多聚赖氨酸。
对于方案一～方案五，第一功能单体和第三功能单体各自独立地选自上述可选范围(包括市购和改性制备)，可相同也可不同。
双键改性透明质酸的一种实施方案，包括：
称取2g分子量为10000的透明质酸钠，并使用20ml PBS溶解，再依次加入6-12ml甲基丙烯酸缩水甘油酯以及4-8ml三乙胺。在37℃摇床上放置5-10天。最后使用截留分子量为5000的透析袋透析5-7天，冷冻干燥得到双键化的透明质酸(根据实际需要可以等比例放大制备)；
双键化多聚赖氨酸制备的一种实施方案，包括：
将多聚赖氨酸溶解在去离子水中，然后以1:1.5-1:5(甲基丙烯酸缩水甘油酯：氨基)的摩尔比加入甲基丙烯酸缩水甘油酯。将混合物在37℃摇床上放置5-10天。最后使用截留分子量为1000的透析袋透析5-7天，冷冻干燥得到部分双键化的多聚赖氨酸。
本申请的生物材料在引入功能单体前，需先进行常规的预处理，可选的，所述预处理包括常规的清洗操作：获取生物材料，并于4℃低温湿润状态下保存；将新鲜的生物材料采用柔和摩擦和流体压力在4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，直至没有可见的粘附的非心包或非胶原组织。
预处理后的生物材料与含第一功能单体的溶液接触，可选的，所述接触过程可为静态接触也可为动态接触；采用静态接触时，将生物材料置于含第一功能单体的溶液中浸泡即可；动态接触时可在浸泡的过程中摇床震动。与第一功能单体接触过程中，温度在20～50℃均可，优选的，接触过程终温度无需特别控制，室温环境均可，以不超过人体适应温度为宜，优选在36～37℃进行。
步骤AS110中第一功能单体的浓度以及生物材料与含第一功能单体溶液的接触时间以保证更多的第一功能单体渗透进入生物材料中为宜，一般地，第一功能单体的浓度较高、相应的接触时间可较短，第一功能单体的浓度较低、相应的接触时间适应延长。
可选的，步骤AS110中所述溶液的溶剂为水、生理盐水、pH中性缓冲液或乙醇的水溶液其中，乙醇的水溶液中，乙醇和水可按任意比例混合，常用为50％左右乙醇；所述溶液中功能单体的浓度为10～100mM。
可选的，在第一功能单体浓度为10～100mM条件下，接触时间为2～20h，以使第一功能单体充分渗透进入生物材料。
进一步可选的，步骤AS110中所述溶液中第一功能单体的浓度为10～30mM，浸泡时间为2～5h。
第一功能单体渗透后，向反应体系中加入交联剂，可选的，所述交联剂的浓度为10～800mM。
共交联过程中，温度在20～50℃均可，优选的，所述共交联过程中温度无需特别控制，室温环境均可，以不超过人体适应温度为宜，可选的，在36～37℃进行；所述共交联的反应时间以交联反应尽量彻底为宜，可选的，在交联剂浓度为10～800mM条件下，共交联时间为10～30h。
进一步可选的，步骤AS120中交联剂的浓度为50～500mM；进一步地，步骤AS120中交联剂的浓度为50～150m M，共交联时间为20～30h。
可选的，共交联时生物材料与交联剂溶液之间可为静态接触也可为动态接触，动态接触过程可选择在浸泡的同时震荡反应体系，加快交联进程。
步骤AS120(M)中第三功能单体的浓度及浸泡时间以更多的封闭残留醛基为宜，可选的，步骤AS120(M)中，所述溶液中第三功能单体的浓度为10～100mM；浸泡时间为2～48h。
进一步可选的，步骤AS120(M)中所述溶液中溶剂为水、生理盐水、pH中性缓冲液或乙醇的水溶液其中，乙醇的水溶液中，乙醇和水可按任意比例混合，常用为50％左右乙醇；所述含第三功能单体的溶液中第三功能单体的浓度为10～100mM；浸泡时间为2～48h。
进一步地：
方案一中第三功能单体的浓度为30～50mM；浸泡时间为10～20h
方案二中第三功能单体的浓度为10～30mM；浸泡时间为3～8h。
方案三申请2中第三功能单体的浓度为30～50mM；浸泡时间为3～8h。
方案四申请3中第三功能单体的浓度为20～50mM；浸泡时间为3～8h。
方案五中所述溶液中第三功能单体的浓度为20～40mM；浸泡时间为2～4h。该步骤AS120(M)中，将经步骤AS120处理后的生物材料清洗后再浸泡于第三功能单体溶液中；或将经步骤AS120处理后的生物材料直接转移至第三功能单体溶液中。
该步骤AS120(M)中，在20～50℃下进行均可，优选的，浸泡温度无需特别控制，室温环境均可，以不超过人体适应温度为宜，优选在36～37℃进行。
本申请的方案四～方案五中，在共交联或步骤AS120(M)完成后再次引入碳碳双键(即 方案四和方案五中的步骤AS130)，并通过双键聚合完成二次交联。一种可选的方案中，在所述共交联或步骤AS120(M)完成后直接引入第二功能单体。该方案俗称一锅法，即在共交联完成后直接向共交联或步骤AS120(M)的反应体系中加入第二功能单体，第二功能单体渗透进入生物材料后，再直接向反应体系中加入引发剂引发双键聚合，无需将生物材料取出清洗的过程。
另一种可选的方案中，还包括共交联或步骤AS120(M)完成后清洗生物材料的步骤。该方案中，共交联或步骤AS120(M)后取出生物材料，对生物材料进行清洗操作，去除残余功能单体、交联剂等，再浸泡于含第二功能单体溶液接触，引发双键聚合。
共交联后的生物材料与含第二功能单体溶液接触，进一步引入碳碳双键，第二功能单体的终浓度以及生物材料与含第二功能单体溶液的接触时间以保证更多的第二功能单体渗透进入生物材料中为宜，一般地，第二功能单体的浓度较高、相应的接触时间可较短，第二功能单体的浓度较低、相应的接触时间适应延长。
可选的，所述含第二功能单体溶液中溶剂为水、生理盐水或pH中性缓冲液或乙醇的水溶液其中，乙醇的水溶液中，乙醇和水可按任意比例混合，常用为50％左右乙醇；第二功能单体的质量百分浓度为1～10％。
可选的，在第二功能单体质量百分浓度为1～10％条件下，接触时间为2～20h。以使第二功能单体充分渗透进入生物材料。
进一步可选的，所述含第二功能单体溶液中第二功能单体的质量百分浓度为2～5％；浸泡时间为10～15h。
可选的，生物材料与含第二功能单体溶液的接触过程可为静态接触也可为动态接触；接触过程在20～50℃下均可，优选的，温度无需特别控制，室温环境均可，以不超过人体适应温度为宜，优选在36～37℃进行。
在方案二和方案三中，在完成共交联后，生物材料中引入了第一碳碳双键，进一步地，引发碳碳双键聚合，完成二次交联，即方案二和方案三中的步骤AS130。一种可选的方案中，在所述共交联完成后直接引发双键聚合。该方案俗称一锅法，即在共交联完成后直接向反应体系中加入引发剂，无需将生物材料取出清洗的过程。另一种可选的方案中，还包括共交联完成后清洗生物材料的步骤。该方案中，共交联后取出生物材料，对生物材料进行清洗操作，去除残余功能单体、交联剂等，再浸泡于含引发剂的溶液中。
在增设步骤AS120(M)的优选方案中，向步骤AS120(M)浸泡后的体系中直接加入引发剂；或将步骤AS120(M)浸泡后的生物材料清洗后再浸泡于含引发剂的溶液中。
在方案四和方案五中，第二功能单体渗入后，加入引发剂，引发碳碳双键发生自由基聚合，进行二次交联(即方案四和方案五中的步骤S200)。
一种可选的引发方案中，所述引发剂为过硫酸铵和亚硫酸氢钠的混合物；所述溶液中过硫酸铵和亚硫酸氢钠的浓度分别为10～100mM；进一步地，过硫酸铵和亚硫酸氢钠的浓度分别为20～40mM。
另一种可选的引发方案中，所述引发剂为过硫酸铵和N,N,N',N'-四甲基乙二胺的混合物；所述溶液中过硫酸铵和N,N,N',N'-四甲基乙二胺的质量百分浓度分别为2％～5％和0.2％～0.5％。
可选的，所述含引发剂的溶液中溶剂为水、生理盐水或pH中性缓冲液。
如前所述的引发剂的浓度，在一锅法中，该浓度可以理解为硫酸铵和亚硫酸氢钠在步骤AS120反应体系所含溶液中的浓度，在分步法中，该浓度可以理解为含引发剂的溶液中的浓度。
可选的，双键聚合过程在20～50℃下进行均可，优选的，双键聚合过程中温度无需特别控制，室温环境均可，以不超过人体适应温度为宜，优选在36～37℃进行。双键聚合时间以2～48h为宜，优选20～25h。
可选的，还包括双键聚合结束后的后处理过程，所述后处理包括常规的清洗、柔顺、干燥等操作。
对于制备湿膜的需求，柔顺处理后溶剂保存即可，例如可采用甘油保存。对于制备干膜的需求，柔顺处理后再对生物材料进行干燥处理：干燥过程为室温干燥、鼓风干燥、真空干燥、冷冻干燥中的一种或几种组合方式。干燥时间为1h～10天，室温干燥温度为10℃～30℃，鼓风干燥或真空干燥温度为15℃～100℃，冷冻干燥温度为-20℃～-80℃。
对于方案一，以图1所示较为优选的流程为例进行说明，包括如下步骤：
S1、获取生物材料，并于4℃低温湿润状态下保存；
S2、将步骤S1中的生物材料采用柔和摩擦和流体压力在4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，直至没有可见的粘附的非心包或非胶原组织；
S3、然后将步骤S2清洗后的生物材料浸泡于摩尔浓度为10-100mM的精氨酸水溶液中，在37℃条件下浸泡12小时，确保精氨酸充分的物理渗透；
S4、向步骤S3处理后的生物材料所浸泡的溶液中加入戊二醛发生共交联，溶液体系中的戊二醛摩尔浓度为10-800mM，在37℃条件下反应24小时。
S5、将步骤S4处理后的生物材料再次浸泡于精氨酸溶液(10-100mM)中2～48时间。
S6、向步骤S5处理后的生物材料用蒸馏水浸泡清洗，清除没有反应的精氨酸和戊二醛。
在需要干态保存的方案中，还包括：
S7、对生物材料进行常规干燥处理。
该实施方案的化学原理示意图如图2所示。
对于方案二，以图3所示较为优选的流程为例进行说明，包括如下步骤：
步骤一，摘取生物瓣膜材料，对生物瓣膜材料进行常规的预处理操作；
步骤二，第一功能单体(氨基-双键化合物)溶液浸泡生物材料；
步骤三，向步骤二的反应体系中加入交联剂(戊二醛)，对第一功能单体(氨基-双键化合物)和生物瓣膜材料进行共交联，引入自由基(碳碳双键)；
步骤四，将经步骤三处理后的生物材料再次浸泡于第三功能单体溶液中。
步骤五，引发自由基聚合的二次交联。
步骤六，二次交联后对生物材料进行清洗、甘油处理，干态或湿态保存生物瓣膜。
对于方案三，以图4所示较为优选的流程为例进行说明，包括如下步骤：
步骤一，摘取生物瓣膜材料，对生物瓣膜材料进行常规的预处理操作；
步骤二，氨基-双键化合物溶液(即功能单体，同时带有功能性基团)溶液浸泡生物瓣膜材料；
步骤三，向步骤二的反应体系中加入戊二醛(交联剂)，对氨基-双键化合物(功能单体)和生物瓣膜材料进行共交联，引入碳碳双键(自由基)和功能性基团；
步骤四，将经步骤三处理后的生物材料再次浸泡于氨基-双键化合物(功能单体)溶液中。
步骤五，引发自由基聚合的二次交联。
步骤六，二次交联后对生物材料进行清洗、甘油处理，干态或湿态保存生物瓣膜。
对于方案四，以图5所示较为优选的流程为例进行说明，包括如下步骤：
步骤一，摘取生物瓣膜材料，对生物瓣膜材料进行常规的预处理操作；
步骤二，第一功能单体(氨基-双键化合物)溶液浸泡生物材料；
步骤三，向步骤二的反应体系中加入交联剂(戊二醛)，对功能单体(氨基-双键化合物)和生物瓣膜材料进行共交联，引入自由基(碳碳双键)，也可进一步引入功能性基团；
步骤四，将经步骤三处理后的生物材料再次浸泡于氨基-双键化合物(第三功能单体)溶液中。
步骤五，自由基聚合单体(第二功能单体)浸泡处理；
步骤六，引发自由基聚合的二次交联。
步骤七，二次交联后对生物材料进行清洗、甘油处理，干态或湿态保存生物瓣膜。
对于方案五，以图6所示较为优选的流程为例进行说明，包括如下步骤：
步骤一，摘取生物瓣膜材料，对生物瓣膜材料进行常规的预处理操作；
步骤二，氨基-双键化合物(第一功能单体)溶液浸泡生物材料；
步骤三，向步骤二的反应体系中加入戊二醛(交联剂)，对氨基-双键化合物(第一功能单体)和生物瓣膜材料进行共交联，引入碳碳双键(自由基)，也可进一步引入功能性基团；
步骤四，将经步骤三处理后的生物材料再次浸泡于氨基-双键化合物(第三功能单体)溶液中。
步骤五，自由基聚合单体(第二功能单体)浸泡处理；
步骤六，引发自由基聚合的二次交联。
步骤七，二次交联后对生物材料进行清洗、甘油处理，干态或湿态保存生物瓣膜。
一种更具体的实施方案，包括如下步骤：
S1、获取生物材料，并于4℃低温湿润状态下保存；
S2、将步骤S1中的生物材料采用柔和摩擦和流体压力在4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，直至没有可见的粘附的非心包或非胶原组织；
S3、然后将步骤S2清洗后的生物材料浸泡于摩尔浓度为10-100mM的DL-2-氨基-4-戊烯酸水溶液中，在37℃条件下浸泡12小时，确保DL-2-氨基-4-戊烯酸充分的物理渗透；
S4、向步骤S3处理后的生物材料所浸泡的溶液中加入戊二醛发生共聚合，溶液体系中的戊二醛摩尔浓度为10-500mM，在37℃条件下反应24小时。
S5、向步骤S4处理后的生物材料用蒸馏水浸泡清洗，清除没有反应的DL-2-氨基-4-戊烯酸和戊二醛。
S6、将步骤S5处理后的生物材料浸泡于5％的聚乙二醇二丙烯酸酯的水溶液中，在37℃条件下浸泡12小时，确保聚乙二醇二丙烯酸酯充分的物理渗透。
S7、将步骤S6处理后的生物材料加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度为10-100mM。
该实施方案的化学原理示意图如图7所示。
该实施方式中，在步骤S3中，采用DL-2-氨基-4-戊烯酸/戊二醛/心包膜共交联引入可自由基聚合烯丙基的方法相比于文献已报道的类似研究具有更高的引入可自由基聚合基团的效率，且本方案在引入烯丙基基团的同时，可以进一步提高心包膜的交联度。
该实施方式制备得到抗钙化和抗凝血性能优异的生物瓣膜材料，并满足乳酸脱氢酶相对活性为0.1～0.25，挂钙量为30～50μg/mg的性能指标。采用的共聚交联方法是使用DL-2-氨基-4-戊烯酸和戊二醛为共聚交联剂，以在生物材料上引入碳碳双键，然后再加入聚乙二醇二丙烯酸酯，在过硫酸铵和亚硫酸氢钠引发剂引发下，在心包膜表面通过共聚交联获得与心包膜共价形式结合的聚乙二醇修饰的材料，从而能够提升生物瓣膜材料的结构稳定性、抗钙化和抗凝血性能，潜在地延长其使用寿命。
另一种更具体的实施方案中，包括如下步骤：
S1、获取生物材料，并于4℃低温湿润状态下保存；
S2、将步骤S1中的生物材料采用柔和摩擦和流体压力在4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，直至没有可见的粘附的非心包或非胶原组织，同时通过渗压休克实现对心包组织有效脱细胞；
S3、称取2g分子量为10000的透明质酸钠，并使用20ml PBS溶解，再依次加入6-12ml甲基丙烯酸缩水甘油酯以及4-8ml三乙胺。在37℃摇床上放置5-10天。最后使用截留分子量为5000的透析袋透析5-7天，冷冻干燥得到双键化的透明质酸(根据实际需要可以等比例放大制备)；
S4、将多聚赖氨酸溶解在去离子水中，然后以1:1.5-1:5(甲基丙烯酸缩水甘油酯：氨基)的摩尔比加入甲基丙烯酸缩水甘油酯。将混合物在37℃摇床上放置5-10天。最后使用截留分 子量为1000的透析袋透析5-7天，冷冻干燥得到部分双键化的多聚赖氨酸；
S5、将S2中的心包膜浸泡于S4中制备的部分双键化的多聚赖氨酸(摩尔浓度为100mM-500mM)水溶液1-3天，确保其达到接近饱和的物理渗透，从而尽可能多地引入部分双键化的多聚赖氨酸，然后向水溶液中加入戊二醛至其质量浓度为2.5％。
S6、将步骤S5处理后的生物材料与S3中制备的双键化透明质酸在过硫酸铵以及/N,N,N',N'-四甲基乙二胺的引发下进行自由基共聚反应，使用的双键化透明质酸浓度为20mg/ml-60mg/ml。置于37℃下反应12-24小时；
S7、最后用蒸馏水浸泡清洗，将清除没有接枝上的双键化透明质酸。
该实施方案中，透明质酸以及多聚赖氨酸的改性示意图和部分双键化多聚赖氨酸修饰心包膜以及双键化透明质酸自由基聚合原理示意图如图8所示。
该优选实施方案与已经报道的类似心包膜多糖亲水处理研究的对比，优点包括：
1)采用甲基丙烯酸化的多聚赖氨酸/戊二醛/心包膜共同交联的同时引入可自由基聚合的甲基丙烯基，与其他已经报道的方法(先将心包与戊二醛反应，然后利用残基引入双键)相比，本方法具有更高的引入双键的效率；
2)本研究策略是采用双重交联，包括戊二醛交联和自由基聚合交联，材料交联度较高；
3)与大部分使用多糖进行表界面亲水改性的研究相比，本方法中透明质酸与心包材料的结合方式为化学共价结合，具有更高的稳定性。
综上所述，该方案中利用甲基丙烯酸缩水甘油酯分别对多聚赖氨酸以及透明质酸进行修饰得到部分双键化的多聚赖氨酸和双键化的透明质酸，随后在戊二醛的作用下对心包膜和部分双键化的多聚赖氨酸(同时具备氨基和双键)进行共聚交联以同时实现心包膜的交联和双键化修饰。最后利用双键化的戊二醛瓣膜与双键化的透明质酸自由基共聚得到透明质酸改性的戊二醛心包材料。能够提升生物材料的血液相容性及抗钙化性能，并得到水接触角为42.66～60.44°、乳酸脱氢酶活性为0.22～0.26、溶血率为0.26～0.50％、钙离子浓度为8.28～65.62μg/mg的生物瓣膜材料，潜在地延长其使用寿命。
当前市场上的生物瓣膜产品几乎全部是采用戊二醛进行交联制备而成，戊二醛可以在一定程度提升心包膜的力学性能并降低其免疫原性，但是戊二醛交联的生物瓣膜的稳定性和交联度仍然存在较低的问题，这将导致其在植入后发生组分的降解，使得其结构收到破坏而发生结构性退化。再者，生物瓣膜成分的降解将进一步诱导其机械损伤及钙化，影响瓣膜正常的功能并降低其使用寿命。戊二醛交联仍是当前生物瓣膜产品的主流方法，因此，在戊二醛交联基础上进一步地对生物瓣膜进行交联和改性以提升其交联度和稳定性，对于科学研究以及相关产业领域的发展具有重大意义。
本申请在戊二醛交联基础上通过进一步引入双键并引发进行后交联，即在戊二醛交联膜基础上通过第一功能单体(含第一碳碳双键和环氧乙烷基)与戊二醛交联生物膜化学反应引入第一碳碳双键，该方案记为方案六，这将改善戊二醛交联生物瓣膜材料膜的交联度、稳定性、机械性能及抗钙化。
具体包括(参见图63)：
BS110将生物瓣膜材料浸泡于醛基交联剂溶液中交联；制备戊二醛交联的生物瓣膜材料；
BS120将步骤BS110所制备戊二醛交联的生物瓣膜材料浸泡于含双键化试剂(第一功能单体)的溶液中进行双键化修饰，制备双键化的生物瓣膜材料；所述双键化试剂(第一功能单体)具有至少一个第一碳碳双键和环氧乙烷基。
S200将经步骤BS120处理后的生物瓣膜材料与引发剂接触，引发双键聚合。
该方案的反应原理：
该双键交联方案中，生物瓣膜材料在戊二醛交联后，进一步通过用第一功能单体即双键化试剂溶液以引入第一碳碳双键实现戊二醛交联生物瓣膜材料的双键化，所用第一功能单体即双键化试剂同时具备第一碳碳双键和环氧乙烷基。
为便于理解该方案涉及的化学原理，以如图64所示为例进一步说明：利用该第一功能单体即双键化试剂对戊二醛交联生物瓣膜材料进行改性，通过第一功能单体即双键化试剂中环氧乙烷基与戊二醛交联生物瓣膜材料上的羟基、羧基以及戊二醛交联后剩余少量的氨基发生开环反应，进而在戊二醛交联生物瓣膜材料中直接引入第一碳碳双键；进一步地，引发这些在戊二醛交联生物瓣膜材料上的双键聚合，实现二次交联，完成生物瓣膜材料的后交联处理。二次交联后的生物瓣膜材料的交联度将进一步提升，同时其稳定性、机械性能和抗钙化性能也将进一步提升。
该方案的步骤BS120中：
可选的，所述双键化试剂即第一功能单体选自烯丙基缩水甘油醚、甲基丙烯酸缩水甘油酯和丙烯酸缩水甘油酯的至少一种。
可选的，所述含第一功能单体即双键化试剂溶液中双键化试剂的浓度为1％～10％(w/w)；双键化修饰的反应时间为2～120小时。
可选的，所述含第一功能单体即双键化试剂的溶液中溶剂为水、生理盐水、pH中性缓冲液或甲醇、乙醇、乙二醇、丙醇、1,2-丙二醇、1,3-丙二醇、异丙醇、丁醇、异丁醇、1,2-丁二醇、1,3-丁二醇、1,4-丁二醇、甘油的水溶液的一种或多种。
可选的，将经S110处理后的生物膜材料取出，经清洗后或直接置于含双键化试剂(第一功能单体)的溶液中。
该方案的步骤S200中：
步骤BS120处理后的生物瓣膜材料用去离子水洗涤后再浸入引发剂溶液中进行步骤S200的处理或直接向步骤BS120的反应体系中加入引发剂引发聚合反应，后者俗称一锅法。
可选的，所述含引发剂的溶液中溶剂为水、生理盐水或pH中性缓冲液。
如前所述的引发剂的浓度，在一锅法中，该浓度可以理解为引发剂在步骤BS120反应体系所含溶液中的浓度，在分步法中，该浓度可以理解为含引发剂的溶液中的浓度。
可选的，所述引发剂为过硫酸铵和亚硫酸氢钠的混合物，或过硫酸铵和亚硫酸钠的混合物，或过硫酸钠和亚硫酸钠的混合物，或过硫酸钾和亚硫酸钠的混合物，或过硫酸钠和亚硫酸氢钠的混合物，或过硫酸钾和亚硫酸氢钠的混合物，或过硫酸钾和四甲基乙二胺，或过硫 酸氨和四甲基乙二胺，或过硫酸钠和四甲基乙二胺；所述混合物中各组分的浓度分别为1～100mM。
可选的，双键聚合时间以3～24h为宜。
该方案与现有技术相比，至少具有如下有益效果之一：
(1)该方案在戊二醛交联后的生物瓣膜材料的基础上，通过双键化修饰在戊二醛交联的生物瓣膜材料上引入双键作为二次交联的基础，进一步地通过引发戊二醛交联的生物瓣膜材料上双键的聚合从而实现二次交联，可进一步地提高生物瓣膜材料的交联度，从而改善生物瓣膜材料的稳定性。
(2)该方案通过在戊二醛交联的生物瓣膜材料上引入双键后，进一步引发双键的聚合，提高了戊二醛交联材料的稳定性，进一步地降低了结构降解引起的钙化风险，因此还具备一定的抗钙化性能。
当前临床植入使用的生物瓣膜产品几乎全是采用戊二醛交联的生物瓣膜材料制备而成，通过戊二醛与生物瓣膜材料中的胶原蛋白基质反应可以交联生物瓣膜材料当中的胶原蛋白并进一步降低生物瓣膜材料自身的免疫原性、提升生物瓣膜材料的机械强度；然而，生物瓣膜材料在经过戊二醛交联后仍然存在着交联度不高的问题并面临结构降解退化的风险，这将直接地导致其在植入后发生组分的降解，使得其结构完整性受到破坏而发生结构性退化和衰败。再者，生物瓣膜成分的降解将进一步促使其瓣叶结构的机械损伤并诱导钙化的发生，这将影响瓣膜正常的开合运动并随着结构退化而降低生物瓣膜使用年限。
当前，戊二醛交联的生物心脏瓣膜仍然是临床上使用的主流生物心脏瓣膜，鉴于戊二醛交联的生物心脏瓣膜仍存在稳定性和交联度低的问题以及其面临的结构降解和破坏带来的结构性退化和失效的风险，基于戊二醛交联后进行一系列后交联和改性既符合现实生产需求又对科学研究具有重大意义。
因此，进一步地，本申请将生物心脏瓣膜材料在基于戊二醛交联的条件下，通过双键化处理戊二醛交联生物瓣膜材料进一步在戊二醛交联生物瓣膜材料上引入碳碳双键作为二次交联的平台，通过引发双键化的戊二醛交联生物瓣膜材料中的双键与功能单体的双键发生共聚反应，在戊二醛交联生物瓣膜材料上引入功能单体的聚合物网络，进一步地扩大交联网络，即在方案六的基础上，进一步通过第二功能单体(含第二碳碳双键)物理渗透引入第二碳碳双键，记为方案七，这将提高戊二醛交联生物瓣膜材料膜的交联度、提升其结构稳定性、更进一步地降低的材料的钙化程度以改善其抗钙化性能。
具体的，包括(参见图69)：
BS110将生物瓣膜材料浸泡于醛基交联剂溶液中交联；制备戊二醛交联的生物瓣膜材料；
BS120将步骤BS110所制备戊二醛交联的生物瓣膜材料浸泡于双键化试剂(第一功能单体)的溶液中进行双键化修饰，制备双键化的生物瓣膜材料；所述双键化试剂(第一功能单体)具有至少一个第一碳碳双键和环氧乙烷基。
BS130将步骤BS120所得双键化的生物瓣膜材料用功能单体(第二功能单体)溶液浸 泡，所述第二功能单体具有至少一个第二碳碳双键；
S200向步骤BS130浸泡结束后的溶液中加入引发剂，使其与生物瓣膜材料和功能单体溶液接触，引发双键聚合。
该方案的反应原理：
该双键交联方案中，生物瓣膜材料在戊二醛交联后，进一步通过用双键化试剂(第一功能单体)溶液以引入第一碳碳双键，通过实现戊二醛交联生物瓣膜材料的双键化以作为二次交联的平台，所用双键化试剂(第一功能单体)同时具备碳碳双键和环氧乙烷基。
为便于理解该方案涉及的化学原理，以如图70所示为例进一步说明：利用该双键化试剂(第一功能单体)对戊二醛交联生物瓣膜材料进行改性，通过双键化试剂(第一功能单体)中环氧乙烷基与戊二醛交联生物瓣膜材料上的羟基、羧基以及戊二醛交联后剩余少量的氨基发生开环反应，进而在戊二醛交联生物瓣膜材料中引入第一碳碳双键；进一步地，第二功能单体通过物理渗透进一步引入第二碳碳双键；再进一步地，通过引发双键化的戊二醛交联生物瓣膜材料中的双键与功能单体的双键发生共聚反应，引入功能单体聚合物作为交联网络，实现进一步二次交联，完成对生物瓣膜材料的双键共聚后交联处理。
由于生物瓣膜材料上更多的官能团(除氨基以外的羟基、羧基)被用于交联，且通过与功能单体的共聚进一步引入功能单体的聚合物作为交联网络，扩大了生物瓣膜材料的交联网络，双键共聚后交联后的生物瓣膜材料的交联度将显著提升，其结构稳定性和抗钙化性能随着功能单体聚合物网络的引入也将得以显著提升。
该方案的步骤BS120中：双键化试剂(第一功能单体)选择、双键化试剂(第一功能单体)溶液浓度、双键化试剂(第一功能单体)溶液溶剂选择、双键化修饰的反应时间以及操作过程均与方案六相同。在此不再赘述。
该方案的步骤BS130中：
将经步骤BS120处理后的生物瓣膜材料直接或经清洗后浸泡于第二功能单体溶液中。
可选的，所述第二功能单体具有至少一个第二碳碳双键。
进一步可选的，所述第二功能单体为聚乙二醇二丙烯酸酯、1,4-丁二醇二丙烯酸酯、乙烷-1,2-二基二丙烯酸酯、丙烯酸乙酯、N-甲基-2-丙烯酰胺、N-2,2-丙烯基-2-丙烯酰胺、N-乙基丙烯酰胺、N,N'-乙烯基双丙烯酰胺、(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯、N,N'-二甲基丙烯酰胺、N,N-二甲基甲基丙烯酰胺、双键化聚赖氨酸中的一种或多种。
可选的，所述第二功能单体溶液的浓度为0.1％～20％(v/v)；进一步地，所述第二功能单体溶液的浓度为0.1％～6％(v/v)。
可选的，所述第二功能单体溶液的溶剂为水、生理盐水、乙醇、异丙醇或pH中性缓冲溶液中的一种或几种混合物。
可选的，在所述第二功能单体溶液中的浸泡时间为0.5h-120h。
该方案的步骤S200中：
步骤BS120处理后的生物瓣膜材料用去离子水洗涤后再浸入引发剂溶液中进行步骤S200的处理或直接向步骤BS120的反应体系中加入引发剂引发聚合反应，后者俗称一锅法。
可选的，所述含引发剂的溶液中溶剂为水、生理盐水或pH中性缓冲液。
如前所述的引发剂的浓度，在一锅法中，该浓度可以理解为引发剂在步骤BS120反应体系所含溶液中的浓度，在分步法中，该浓度可以理解为含引发剂的溶液中的浓度。
可选的，所述引发剂为过硫酸铵和亚硫酸氢钠的混合物，或过硫酸铵和亚硫酸钠的混合物，或过硫酸钠和亚硫酸钠的混合物，或过硫酸钾和亚硫酸钠的混合物，或过硫酸钠和亚硫酸氢钠的混合物，或过硫酸钾和亚硫酸氢钠的混合物，或过硫酸钾和四甲基乙二胺，或过硫酸氨和四甲基乙二胺，或过硫酸钠和四甲基乙二胺；所述混合物中各组分的浓度分别为1～100mM。
步骤S200的反应时间为3～24小时。
该方案与现有技术相比至少具有如下有益效果之一：
(1)该方案在戊二醛交联后的生物瓣膜材料的基础上，通过双键化修饰在戊二醛交联的生物瓣膜材料上引入碳碳双键作为二次交联的基础，进一步地通过引发戊二醛交联的生物瓣膜材料上碳碳双键和功能单体上的碳碳双键之间的聚合从而引入功能单体聚合物交联网络实现二次交联，可进一步地提高生物瓣膜材料的交联度。
(2)该方案通过在戊二醛交联的生物瓣膜材料上引入碳碳双键后，进一步引发双键化生物瓣膜材料上的碳碳双键和功能单体上的碳碳双键之间的聚合，通过引入功能单体聚合物交联网络，该交联网络能在一定程度上通过物理阻断的方式进一步减少体内的胶原酶与生物瓣膜材料上胶原基质的结合，保护生物瓣膜材料胶原基质提高戊二醛交联生物瓣膜材料的稳定性，进一步地降低了生物瓣膜材料结构降解引起的钙化风险，因此还具备一定的抗钙化性能。
(3)该方案通过在戊二醛交联的生物瓣膜材料上引入碳碳双键后，进一步引发双键化戊二醛交联生物瓣膜材料上的碳碳双键和功能单体上的碳碳双键之间的聚合，通过引入功能单体聚合物交联网络，该交联网络能够作为聚合物屏障进一步减少环境中的钙离子与生物瓣膜材料上易与钙离子结合的矿化区的结合，降低钙化风险，进而起到抗钙化的作用。
(4)该方案通过在戊二醛交联的生物瓣膜材料上引入碳碳双键后，进一步引发双键化戊二醛交联生物瓣膜材料上的碳碳双键和功能单体上的碳碳双键之间的聚合，通过引入功能单体聚合物交联网络使得生物瓣膜材料上交联度增加，导致生物瓣膜材料结构更加刚性而使得弹性增加；另一方面，功能单体聚合物交联网络使得生物瓣膜材料上胶原基质间间隙得到填充以抑制胶原纤维的形变，使生物瓣膜材料质地***而弹性得以提升。
当前临床上使用的生物瓣膜产品几乎全是经戊二醛交联生物瓣膜材料制备而成。戊二醛与生物瓣膜材料中的胶原蛋白基质反应可以交联生物瓣膜材料当中的胶原蛋白并进一步降低生物瓣膜材料自身的免疫原性、提升生物瓣膜材料的机械强度；然而，生物瓣膜材料在经过戊二醛交联后仍然存在着交联度不高的问题并面临结构降解退化的风险，这将直接地导致其在植入后发生组分的降解，使得其结构完整性受到破坏而发生结构性退化和衰败。再者，生物瓣膜成分的降解将进一步促使其瓣叶结构的机械损伤并诱导钙化的发生，这将影响瓣膜正常的开合运动并随着结构退化而降低生物瓣膜使用年限。虽然相比机械瓣膜具有较低的致栓 性，但生物瓣膜血栓仍然存在，将破坏生物瓣膜的正常功能，带来二次置换瓣膜的风险。另一方面，钙化的发生将直接导致生物瓣膜的衰败。
因此，生物瓣膜的交联度、稳定性、抗血栓和抗钙化性能仍待提升。当前，戊二醛交联制备的生物瓣膜仍然是临床最常用的生物瓣膜，鉴于戊二醛交联的生物心脏瓣膜仍存在稳定性、交联度低、血栓和钙化等问题以及这些问题带来的结构降解和失效的风险，对戊二醛交联生物心脏瓣膜进行一系列功能性后交联既符合现实生产实际需求又具备较高的科学价值。
本申请将生物心脏瓣膜材料在基于戊二醛交联的条件下，通过双键化处理戊二醛交联生物瓣膜材料进一步在戊二醛交联生物瓣膜材料上引入碳碳双键作为功能化共聚交联的平台，通过引发双键化戊二醛交联生物瓣膜材料中双键与功能单体的双键发生共聚反应，在戊二醛交联生物瓣膜材料上引入功能性聚合物网络，进一步地扩大交联网络，实现生物瓣膜材料的双键后功能化共聚交联，即在方案七的基础上，第二功能单体还带有功能基团B，记为方案八。这将提高戊二醛交联生物瓣膜材料膜的交联度、提升其结构稳定性；功能性聚合交联网络的引入使得生物瓣膜材料功能化，将进一步地改善其抗钙化性能、抗血栓性能和生物相容性。功能性聚合交联网络的引入通过提升生物瓣膜材料的交联度并使得胶原基质间间隙得到填充以抑制胶原纤维的形变，进一步使生物瓣膜材料质地相对***而弹性得以提升。
具体的，包括(参见图75)：
BS110将生物瓣膜材料浸泡于醛基交联剂溶液中交联；制备戊二醛交联的生物瓣膜材料；
BS120将步骤BS110所制备戊二醛交联的生物瓣膜材料浸泡于双键化试剂(第一功能单体)的溶液中进行双键化修饰，制备双键化的生物瓣膜材料；所述双键化试剂(第一功能单体)具有至少一个第一碳碳双键和环氧乙烷基。
BS130将步骤BS120所得双键化的生物瓣膜材料用第二功能单体溶液浸泡，所述第二功能单体具有至少一个第二碳碳双键和至少一个功能性基团B；
S200向步骤BS130浸泡结束后的溶液中加入引发剂，使其与生物瓣膜材料和功能单体溶液接触，引发双键聚合。
该方案的原理：
该双键交联方案中，生物瓣膜材料在戊二醛交联后，进一步通过用双键化试剂(第一功能单体)溶液以引入第一碳碳双键，通过实现戊二醛交联生物瓣膜材料的双键化以作为二次交联的平台，所用双键化试剂(第一功能单体)同时具备碳碳双键和环氧乙烷基，第二功能单体带有第二碳碳双键和功能性基团B。
为便于理解该方案涉及的化学原理，以如图76所示为例进一步说明：利用该双键化试剂(第一功能单体)对戊二醛交联生物瓣膜材料进行改性，通过双键化试剂(第一功能单体)中环氧乙烷基与戊二醛交联生物瓣膜材料上的羟基、羧基以及戊二醛交联后剩余少量的氨基发生开环反应，进而在戊二醛交联生物瓣膜材料中引入碳碳双键；进一步地，第二功能单体通过物理渗透进一步引入碳碳双键和功能性基团B；再进一步地，进一步引发双键化戊二醛交联生物瓣膜材料上双键和功能单体上双键之间的聚合引入功能性聚合物交联网络，实现进一步二次交联，完成对生物瓣膜材料的双键后功能化共聚交联处理。
由于生物瓣膜材料上更多的官能团(除氨基以外的羟基、羧基)被用于交联，且通过与功能单体的共聚进一步引入功能单体的聚合物作为交联网络，扩大了生物瓣膜材料的交联网络，双键后功能化共聚交联处理的生物瓣膜材料的交联度将显著提升，其结构稳定性和抗钙化性能随着功能性聚合物网络的引入也将得以显著提升。进一步地，通过双键后功能化共聚在生物瓣膜处理上引入功能性聚合物网络，使得生物瓣膜材料上富含功能性基团，从而赋予了生物瓣膜材料功能性基团对应的性能；功能性基团B可选自羟基、羧基、羧酸胆碱、磺酸胆碱、磷酸胆碱、吡咯烷酮、磺酸基团、羧酸根离子、磺酸酯、亚砜、酰胺基团、甲氧基，这些基团能够与水分子通过氢键、离子水合作用结合水分子，这进一步提升生物瓣膜材料表面的亲水性，在生物瓣膜上形成一定的水合层来抵御体内蛋白和细胞的过度黏附，提升抗血栓性能和生物相容性。
对于引入的功能性基团B：
羟基：作为亲水的基团，提升生物材料的表面亲水性以实现抗凝血的效果；
羧基：作为亲水的基团，提升生物材料的表面亲水性以实现抗凝血的效果；
羧酸根离子、磺酸基：通过离子水合作用提升生物材料的表面亲水性以实现抗凝血的效果；
亚砜、吡咯烷酮：作为亲水的基团，提升生物材料的表面亲水性以实现抗凝血的效果；
两性离子：通过离子水合作用提升生物材料的表面亲水性以实现抗凝血的效果；有利于形成生物瓣膜电中性表面进而降低对钙离子的吸附而达到抗钙化效果；
聚乙二醇：作为亲水的基团，提升生物材料的表面亲水性；增加钙离子与胶原间结合的空间位阻，提升生物瓣膜材料表面亲水性；
氨基甲酸酯基、脲基：作为亲水的基团，提升生物材料的表面亲水性，以实现抗凝血的效果；
氨基甲酸酯基：作为亲水的基团，提升生物材料的表面亲水性，以实现抗凝血的效果。
酰胺：作为亲水的基团，提升生物材料的表面亲水性以实现抗凝血的效果；作为增韧的基团，可动态调整生物材料的弹性，以提高生物材料利用率，其制备的瓣膜流体力学性能优异。
该方案的步骤BS120中：双键化试剂(第一功能单体)选择、双键化试剂(第一功能单体)溶液浓度、双键化试剂(第一功能单体)溶液溶剂选择、双键化修饰的反应时间以及操作过程均与方案六相同。在此不再赘述。
该方案的步骤BS130中：
将经步骤BS120处理后的生物瓣膜材料直接或经清洗后浸泡于第二功能单体溶液中。
可选的，所述第二功能单体具有至少一个第二碳碳双键和至少一个功能性基团B。
可选的，所述第二功能单体为丙烯酰胺、丙烯酸、丙烯酸钠、甲基丙烯酸、甲基丙烯酸钠、2-(丙-2-烯酰氨基)乙酸、2-丙烯酰胺基-2-甲基丙磺酸、甲基丙烯酸羟乙酯、3-[[2-(甲基丙烯酰氧)乙基]二甲基铵]丙酸酯、N-甲基-2-丙烯酰胺、N-异丙基丙烯酰胺、N-(羟甲基)丙烯酰胺、N-(2-羟基乙基)甲基丙烯酰胺、3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1- 磺酸内盐、2-甲基丙烯酰氧乙基磷酸胆碱、N-(2-羟乙基)丙烯酰胺、N-(甲氧基甲基)甲基丙烯酰胺、2-丙烯酰胺-2-甲基丙磺酸、2-丙烯酰胺-2-甲基丙磺酸、双键化透明质酸(可由如前所述方法制备)中的一种或多种。
可选的，所述第二功能单体溶液浓度为0.1％～6％(v/v)。
可选的，所述第二功能单体溶液的溶剂为水、生理盐水、乙醇、异丙醇或pH中性缓冲溶液中的一种或几种混合物。
可选的，在所述第二功能单体溶液中的浸泡时间为0.5h-120h。
该方案的步骤S200中：
该方案的步骤S200处理与方案七的步骤S200处理相同，在此不再赘述。
该方案与现有技术相比至少具有如下有益效果之一：
(1)该方案在戊二醛交联生物瓣膜材料的基础上进行改性，通过双键化化试剂与戊二醛交联生物瓣膜材料反应在戊二醛交联生物瓣膜材料上引入碳碳双键，所得双键化戊二醛交联生物瓣膜材料作为功能化共聚交联的平台，进一步地通过引发戊二醛交联生物瓣膜材料上碳碳双键和功能单体上的碳碳双键之间的聚合以引入功能单体聚合物作为功能化交联网络实现功能化共聚交联，可进一步地提高生物瓣膜材料的交联度并引入功能性基团。通过提升交联度，生物瓣膜材料的稳定性会得以提升。
(2)该方案是在戊二醛交联生物瓣膜材料上引入碳碳双键后，进一步引发双键化生物瓣膜材料上碳碳双键和功能单体上碳碳双键之间的聚合从而在生物瓣膜材料上引入功能性聚合物交联网络，该功能***联网络能作为一种聚合物屏障在一定程度上减少体内的胶原酶与生物瓣膜材料上胶原基质的接触和相互作用，显著减少胶原酶对生物瓣膜材料上胶原基质的降解作用，提高戊二醛交联生物瓣膜材料的稳定性，进一步地降低了生物瓣膜材料结构降解引起的生物瓣膜结构性退化风险。
(3)该方案通过在戊二醛交联的生物瓣膜材料上引入双键后，进一步引发双键化戊二醛交联生物瓣膜材料上碳碳双键和功能单体上碳碳双键之间的聚合引入功能性聚合物交联网络，该功能性聚合物交联网络能够作为聚合物屏障进一步减少钙离子与生物瓣膜材料上易与钙离子结合的矿化区的结合，降低钙化风险，进而起到抗钙化的作用。
(4)该方案通过在戊二醛交联的生物瓣膜材料上引入碳碳双键后，进一步引发双键化戊二醛交联生物瓣膜材料碳碳上双键和功能单体上碳碳双键之间的聚合引入功能性聚合物交联网络，通过引入功能性聚合物交联网络使得生物瓣膜材料上交联度增加，导致生物瓣膜材料结构更加刚性而使得弹性上升；再者，功能性聚合物交联网络使得生物瓣膜材料上胶原基质间间隙得到填充以抑制胶原纤维的形变，使生物瓣膜材料质地相对***而弹性得以提升。
(5)该方案通过在戊二醛交联的生物瓣膜材料上引入碳碳双键后，进一步引发双键化戊二醛交联生物瓣膜材料上碳碳双键和功能单体上碳碳双键之间的聚合引入功能性聚合物交联网络，由于功能性聚合物交联网络中还具备功能性官能团，因此，功能性聚合物交联网络引入不仅实现了生物瓣膜材料的再交联还实现了生物瓣膜材料的功能化。功能化共聚后交联的生物瓣膜材料具备功能性官能团而表现出功能性官能团对应的性能。所述功能性基团羟基、 羧基、羧酸胆碱、磺酸胆碱、磷酸胆碱、吡咯烷酮、磺酸基团、羧酸根离子、磺酸酯、亚砜、酰胺基团、甲氧基，其能够与水分子通过氢键、离子水合作用结合水分子，这进一步提升生物瓣膜材料表面的亲水性，在与体内形成一定的水合层来抵御蛋白和细胞的过度黏附，提升抗血栓性能和生物相容性。
在方案六～方案八中，BS100和BS200的所有反应过程如无特殊说明在0～50℃下进行均可，优选的，温度无需特别控制，室温环境均可，以不超过人体适应温度为宜，优选在36～37℃进行。
在方案六～方案八中，BS100和BS200的所有反应如无特殊说明既可静置反应也可动态反应，动态反应可以是在蠕动泵等可使溶液循环的设备作用下进行，也可以在10rpm-150rpm的转速下摇晃进行，所述蠕动循环或摇晃时间可持续进行，也可间断进行。
在方案六～方案八的BS110中，所述戊二醛溶液的浓度为0.1％～5％(w/w)，交联时间可为0.5h-120h中的任意时间。
对于方案六～方案八，可选的，还包括双键聚合结束后的脱水和干化处理，制成干态膜。双键聚合结束后对生物瓣膜材料进行常规的清洗、柔顺后进行脱水和干化处理。
清洗溶液可以是水、生理盐水、乙醇、异丙醇或pH中性缓冲溶液中的一种或几种混合物，使用前和使用过程中可调pH至5.0-9.5之间，也可选择不调。
可选的，所述脱水处理是将双键聚合完的膜片或该膜片缝制好的瓣膜暴露于脱水溶液中。
可选的，所述脱水溶液是醇类溶液与水的混合溶液，醇类溶液占比20-90％(v/v)，该醇类试剂可以是乙醇、异丙醇中的一种或两种混合物。
可选的，所述的干化处理是将脱水后的膜片或瓣膜暴露于柔顺剂溶液中，处理时间20min-10h。
可选的，所述柔顺剂溶液主要成分为甘油、聚乙二醇中的一种或两种的混合溶液，甘油浓度为10-100％(v/v)，其他成份为水，乙醇，异丙醇中的一种或几种，占比0-90％(v/v)。
可选的，干化处理后的瓣膜灭菌方式可以是环氧乙烷灭菌或电子束灭菌中的一种。
上述方法制备得到的生物瓣膜材料，可以用于介入生物瓣膜，例如通过微创介入；也可用于外科生物瓣膜，例如通过外科手术植入。
如图85所示，在一实施例中提供了一种人工心脏瓣膜，包括支架1以及连接在支架1内的瓣叶2，支架整体上为筒状，侧壁为镂空的网格结构，支架内部为血流通道，多片瓣叶相互配合控制支架内血流通道的开闭程度。
支架根据释放模式的不同，加工时选用相应的材质，例如具有形状记忆可体内自膨的镍钛合金，或利用球扩释放的不锈钢材质等等，支架本身可利用管材切割或线材编织的方式成型，瓣叶可以采用缝缀、粘结或一体模具成型的方式连接于支架。
为了在体内的定位还可以在支架外周设置可与周边原生组织相作用的定位结构，例如锚 刺、臂部等等，为了防止周漏还可以在支架的内侧和/或外侧设置裙边或防周漏材料等。其中瓣叶、裙边或防周漏材料均可以采用上文各实施例的生物瓣膜材料。
如图86，采用导管介入时，人工心脏瓣膜3与相应的输送***组成瓣膜介入***，输送***包括导管组件4以及控制导管组件的手柄，人工心脏瓣膜在体内输送时呈径向压缩状态，在体内解除导管组件的束缚或进行球扩并径向扩张释放。
以下以具体实施例进行进一步说明：
实施例1：
在本实施例中，新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在30mM DL-2-氨基-4-戊烯酸水溶液在37℃当中12小时，然后加入戊二醛使其浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时后，采用蒸馏水进行清洗。清洗后浸泡于5％的聚乙二醇二丙烯酸酯的水溶液中，在37℃条件下浸泡12小时，确保聚乙二醇二丙烯酸酯充分的物理渗透，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为40mM，在37℃条件下反应24小时，记为样品1。
在处理过程中，设置戊二醛处理组为对照组1，即将心包膜浸泡于0.625％的戊二醛当中24小时。
对照例1
将新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2h，直至没有可见的粘附的非心包或非胶原组织，然后将清洗后的生物材料浸泡在0.625％的戊二醛中，于37℃浸泡24小时，制得生物瓣膜材料，记为对照例1。
化学结构测定
任意选取上述实施例1的生物瓣膜材料(GA-PEG)和对照例1的生物瓣膜材料(GA)进行红外光谱分析，将上述实施例1和对照例1的样品裁剪成1cm×1cm，冷冻干燥，采用衰减全反射红外光谱(Thermo Scientific Nicolet iS50 FT-IR,USA)分析，扫描波长为4000-400cm -1。
样品1和对照组1样品的心包膜(GA)的红外光谱图如图9所示，2919cm -1和2867cm -1处-CH2-的C-H伸缩振动峰，1724cm -1对应酯的C＝O伸缩振动峰，和1100cm -1醚键的C-O伸缩振动峰在实施例1的红外光谱中的强度明显，可以看出，聚乙二醇成功地化学接枝在生物瓣膜材料上。
乳酸脱氢酶相对活性测量
为获得生物瓣膜材料的抗凝血性能，选取上述实施例1、对照例1的生物瓣膜材料进行乳酸脱氢酶相对活性测量。
测量方法为：将待测样品(直径为6mm原片)用0.9％生理盐水冲洗5分钟后，与100μL 富血小板血浆在96孔板，37℃条件下共孵育1小时后，将血清吸出，使用PBS清洗3次样品表面，每次五分钟。阳性对照为100μL富血小板血浆。使用乳酸脱氢酶释放检测试剂盒(Beyotime Biotechnology，Shanghai,China)，根据说明书操作检测样品表面黏附的血小板相对含量。490nm的吸光度值使用酶标仪(BioTek Synergy H1,USA)测定。
挂钙量测定
为获得生物瓣膜材料与钙离子的结合力，判断不同生物瓣膜材料的抗钙化性能，任意选取上述实施例1和对比例1的生物瓣膜材料进行挂钙量测定。
挂钙量测定方法为：将待测样品(1cm×1cm大小)在0.9％生理盐水中冲洗5分钟。在45-50g雄性Sprague-Dawley大鼠的中央背壁区域的两个皮下袋中通过手术植入样品(每个大鼠每组一个样品)。30天后，将植入的样品从大鼠背壁取出。将样品周围的纤维囊去除后，冷冻干燥并称量干重。而后在95℃水浴中使用6M盐酸(Adamas，Shanghai,China)提取钙离子24小时，使用电感耦合原子发射光谱仪(Agilent 720)测量钙元素含量。
实施例1以及戊二醛对照组1乳酸脱氢酶相对活性的测量结果如图10所示、分析结果如表1所示，挂钙量检测结果如图11和表2所示。
表1
  乳酸脱氢酶相对活性
戊二醛对照组1 0.410±0.072
实施例1 0.100±0.019
表2
  挂钙量μg/mg
戊二醛对照组1 168.595±9.973
实施例1 43.220±10.873
稳定性测定
任意选取上述实施例1的生物瓣膜材料(GA-PEG)和对照例1的生物瓣膜材料(GA)进行酶降解测试，将上述实施例1和对比例1的样品裁剪成直径为1cm圆片，冷冻干燥，称量干重。将每个样品浸泡于500μL浓度为100U/mLⅠ型胶原酶(Invitrogen,NY,USA)的Tris缓冲液(0.1M Tris,50mM CaCl 2,pH＝7.4)中，在37℃条件下浸泡24小时。样品浸泡结束后用去离子水清洗3次，冷冻干燥并称量干重，计算样品的失重率。
分析结果为如下：
实施例1的失重率(％)：3.234±0.125；对照例1的失重率(％)：8.036±0.760。
对照例1在经过24小时Ⅰ型胶原酶作用下失重率达8.0％，而实验例1仅为3.2％，说明通过本专利制备的生物瓣膜材料结构稳定性明显提高。
实施例2
改性透明质酸的制备：称取2g分子量为10000的透明质酸钠，并使用20ml PBS溶解，再依次加入6.5ml甲基丙烯酸缩水甘油酯以及4.5ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸；
改性多聚赖氨酸的制备：将多聚赖氨酸溶解在去离子水中，然后以(1:1.5)的摩尔比加入甲基丙烯酸缩水甘油酯(甲基丙烯酸缩水甘油酯：氨基)。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸；
在本实施例中，新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在180mM改性多聚赖氨酸水溶液在室温当中12小时，然后加入戊二醛溶液至质量浓度为2.5％，在37℃摇床上反应24小时，取出心包材料清洗后浸泡在50mg/ml改性透明质酸水溶液在室温当中12小时，然后采用2.5％过硫酸铵和0.25％N,N,N',N'-四甲基乙二胺在37℃下浸泡12小时，最后采用蒸馏水进行清洗，记为样品2。
对实施例2制备的样品2与对照组2样品分别进行水接触角测试、乳酸脱氢酶活性测试、溶血率测试以及钙化测试。
对照组2：将新鲜采集的猪心包于4℃100RPM转速振荡条件下用蒸馏水清洗2小时，然后浸泡在质量浓度为0.625％戊二醛溶液中24小时，反应完成后取出浸泡在质量分数为0.2％的戊二醛溶液中保存，记为对照样2。
(1)水接触角测试
将对照组与实施例1材料裁剪成1*1cm的方片，放在两片玻璃片中间压平，真空冷冻干燥后进行水接触角测试。
(2)乳酸脱氢酶活性测试：采集新鲜兔血，1500rpm离心15min，获得富血小板血浆。将对照组以及实施例1材料剪成直径为10mm的圆片并用PBS冲洗3次，放入48孔板中，加入100μL富血小板血浆在37℃浸泡1h。选取100μL富血小板血浆作为阳性对照进行定量检测。孵育后，用PBS冲洗3次。用乳酸脱氢酶测定试剂盒测定血小板粘附的相对量。用酶标仪记录各组在490nm处的吸光度，并计算每组乳酸脱氢酶相对活性，血小板相对数量用乳酸脱氢酶相对活性表示。
(3)溶血率测试
采集新鲜兔血，1500rpm离心15min，弃上清液，取红细胞。将对照组以及实施例1样品放入2ml离心管中，加入PBS稀释红细胞(9/1,PBS/RBC)在37℃孵育1小时。PBS和去离子水稀释10倍的红细胞设阴性对照和阳性对照。在3000rpm转速下离心5min，把上清转移到96孔板。用酶标仪记录545nm处的吸光度值并计算溶血率。
(4)钙化测试
在45-50g雄性SD大鼠背部做切口，并用钝器分离皮下组织创建空腔，将对照组以及实 施例1的样品放入空腔中，然后缝合皮肤，在30天后取出样品，冷冻干燥并称重，使用1ml6M的盐酸在100℃下消解，然后将消解的溶液用去离子水稀释至10ml，进行电感耦合等离子体原子发射光谱测定钙浓度。
水接触角检测结果如图12中a～c所示，对照组为戊二醛处理组即对照例2，实验组为亲水处理组即实施例2，实验组水接触角降低。进一步地，实施例2以及戊二醛对照组2最终的水接触角结果如表3所示。
表3
  水接触角(°)
戊二醛对照组2 84.29
实施例2 55.26
乳酸脱氢酶活性及溶血率检测结果如图13所示，其中a为乳酸脱氢酶活性比较结果，b为溶血率比较结果，c为钙离子浓度比较结果，对照组为戊二醛处理组即对照例2，实验组为亲水处理组即实施例2，实验组乳酸脱氢酶活性及溶血率均减低。进一步的，实施例2以及戊二醛对照组2最终的乳酸脱氢酶活性以及溶血率结果如表4所示。
表4
  乳酸脱氢酶活性 溶血率(％)
戊二醛对照组2 0.41 1.54
实施例2 0.24 0.38
钙离子浓度检测结果如图14所示，对照组为戊二醛实验组即对照例2，实验组为亲水处理组即实施例2，实验组钙离子含量均减小。进一步地，实施例2以及戊二醛对照组2最终的钙离子浓度结果如表5所示。
表5
  钙离子浓度(μg/mg)
戊二醛对照组2 188.39
实施例2 36.95
结合表1、表2和表3可以发现采用实施例2的方法对生物材料进行处理后，生物材料水接触角均下降，乳酸脱氢酶活性均降低，钙离子含量均减少。
该实施例提供的方法能够提升生物材料的亲水性能及血液相容性和抗钙化能力，潜在地延长其使用寿命。
对照组3
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在 100mM的戊二醛溶液中，室温下，100RPM转速振荡条件之下交联24小时得对照样3。
实施例3
制备流程见图3，根据制备流程图一种共交联联合双键交联制备生物瓣膜材料的方法，基本原理见图15。新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在20mM的2-甲基烯丙胺水溶液在37℃中2小时，然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。取出猪心包，采用蒸馏水进行清洗。清洗后浸泡于20mM的2-甲基烯丙胺水溶液在37℃中4小时，取出猪心包，采用蒸馏水进行清洗。清洗后浸泡于蒸馏水中，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。为方便区分各实施例制备的样品，本实施例所得样品记为样品3。
实施例4
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在20mM的3-丁烯-1-胺水溶液在37℃当2小时，然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。取出猪心包，采用蒸馏水进行清洗。清洗后浸泡于去离子水中，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。本实施例所得样品记为样品4。
实施例5
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后在37℃下浸泡在20mM的甲基丙烯酸2-氨基乙酯水溶液中2小时，然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。取出猪心包，采用蒸馏水进行清洗。清洗后浸泡于去离子水中，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。本实施例所得样品记为样品5。
实施例6
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在含有10mM的甲基丙烯酸2-氨基乙酯和10mM的2-甲基烯丙胺水溶液在37℃当中2小时，然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。取出猪心包，采用蒸馏水进行清洗。清洗后浸泡于去离子水中，加入等物质的量的过硫酸钾和亚硫酸氢钠引发剂引发聚合反应，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时后用蒸馏水清洗并转移至甘油中脱水得干燥瓣膜样品。本实施例所得样品记为样品6。
实施例7
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后在37℃下浸泡在20mM的甲基丙烯酸2-氨基乙酯水溶液中2小时，然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。取出猪心包，采用蒸馏水进行清洗。清洗后浸泡于20mM的2-氨基-4-戊烯酸水溶液在37℃当中24小时。取出猪心包，采用蒸馏水进行清洗。清洗后浸泡于蒸馏水中，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。本实施例所得样品记为样品7。
实施例8
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后在37℃下浸泡在20mM的甲基丙烯酸2-氨基乙酯水溶液中2小时，然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。随后直接向溶液中继续加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。本实施例所得样品记为样品8。
实施例9
改性透明质酸的制备：称取2g分子量为10000的透明质酸钠，并使用20ml PBS溶解，再依次加入6.5ml甲基丙烯酸缩水甘油酯以及4.5ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸；
改性多聚赖氨酸的制备：将多聚赖氨酸溶解在去离子水中，然后以(1：1.5)的摩尔比加入甲基丙烯酸缩水甘油酯(甲基丙烯酸缩水甘油酯：氨基)。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸；
在本实施例中，新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在60mM(按赖氨酸算)改性多聚赖氨酸水溶液在室温当中12小时，然后加入戊二醛溶液至质量浓度为250mM，在37℃摇床上反应24小时，取出心包材料清洗后浸泡在50mg/ml改性透明质酸水溶液在室温当中12小时，然后采用2.5％过硫酸铵和0.25％N,N,N',N'-四甲基乙二胺在37℃下浸泡12小时，最后采用蒸馏水进行清洗，记为样品9。相对于对照样2，样品9的水接触角下降，乳酸脱氢酶活性降低，钙离子含量减少，可提升生物材料的亲水性能及血液相容性和抗钙化能力，潜在地延长其使用寿命。
酶降解实验(表征交联度)：
将样品5、样品6、样品7、样品8和对照组3裁剪成直径为1cm的圆形片材，每组设置6个平行样。将所有圆形片材样本放置于48孔板，负80℃冷冻过夜然后转移到真空冻干机中冻干48小时。在十万分之一天平上称取每片样品的重量记为初始重量(W0)放回48孔板。用移液枪向48孔板中各孔加入0.5mL胶原酶Ⅰ的PBS溶液并保证生物瓣膜样品完全浸没于胶原酶的PBS溶液中(100U/mL)，将48孔板转移到37℃恒温孵育箱中孵育24小时。 孵育结束后弃去孔板中的溶液，用胶头滴管吸取去离子水反复吹打孔板中的生物瓣膜样品。经过反复吹洗3次后在负80℃下冷冻过夜然后转移到真空冻干机中冻干48小时。在十万分之一天平上称取每片样品经过胶原酶溶液降解后的重量记为最终重量(Wt)。酶降解失重率计算公式如下：
Figure PCTCN2022132374-appb-000001
表6
样品 酶降解失重率(％)
对照组3 8.31
样品5 5.27
样品6 4.88
样品7 4.11
样品8 3.23
对样品5、样品6、样品7、样品8和对照组3进行酶降解实验以表征各组样品的交联效率，利用胶原酶Ⅰ处理样品5、样品6、样品7、样品8和对照组3后计算各组样品的酶降解失重率如表6所示。样品5、样品6、样品7、样品8的酶降解失重率均低于对照组3，这表明样品5、样品6、样品7、样品8的酶降解稳定性均高于对照组3，即样品5、样品6、样品7、样品8的交联效率更高。酶降解实验结果表明本申请的共交联联合双键交联制备生物瓣膜材料的方法能够提高生物瓣膜材料的交联度。
对对照组3及实施例3～8所得样品进行茜素红染色实验，茜素红染色实验：
将样品3、样品4、样品5、样品6、样品7、样品8和对照组3植入到大鼠皮下30天后取出，经过多聚甲醛组织固定液固定。固定结束后取出用手术刀进行修理平整后转移到脱水盒中。用50％、75％、85％、95％(v/v)和无水乙醇对材料样品进行梯度脱水。脱水结束后将材料样品转移至包埋机用融化的石蜡进行包埋，然后转移到-20℃冰箱冷却、修整形状。在切片机上从修整好的蜡块切取3-5μm厚的切片，从摊片机转移至载玻片上并进行脱蜡和复水。用茜素红染液对切片进行染色3分钟，经水洗、烘干后用二甲苯通透5分钟。切片用中性树胶封片后在病理切片扫描仪上采集染色结果图像。
通过茜素红染色对植入到大鼠皮下30天后的样品3、样品4、样品5、样品6、样品7、样品8和对照组3以表征各组样品的钙化程度。植入到大鼠皮下30天后的样品切片的茜素红染色结果图像如图16-22所示，其中茜素红染色后样品的颜色越深表明钙化程度越高。相比于对照样3切片的茜素红染色结果(图16)，样品3、样品4、样品5、样品6、样品7和样品8切片的茜素红染色结果图颜色明显较淡，这表明样品3、样品4、样品5、样品6、样品7和样品8的钙化程度低于对照组3，即样品3、样品4、样品5、样品6、样品7和样品8相比于对照组3具有一定的抗钙化效果。对植入到大鼠皮下30天后的样品3、样品4、样品5、 样品6、样品7、样品8和对照组3的茜素红染色结果表明本申请的共交联联合双键交联制备生物瓣膜材料的方法能够提升生物瓣膜的抗钙化性能。
对照组4
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在100mM的戊二醛溶液中，室温下，100RPM转速振荡条件之下交联24小时得对照样4。
实施例10
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
然后在37℃下将猪心包膜浸泡在30mM的2-氨基-7-烯-辛酸溶液中4小时，然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
将猪心包膜浸泡在30mM的2-氨基-7-烯-辛酸溶液中4小时；
清洗后浸泡于去离子水中，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时得样品记为10号样。
该实施例的反应原理如图23所示。
实施例11
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
然后在37℃下将猪心包膜浸泡在20mM的4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇水溶液中24小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
将猪心包膜浸泡在30mM的4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇水溶液4小时；
清洗后浸泡于去离子水中，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时得样品记为11号样。
实施例12
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
然后在37℃下浸泡在20mM的2-氨基戊-4-烯酸水溶液中4小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
清洗后浸泡于去离子水中，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时，转移至甘油中脱水得到干态的瓣 膜样品记为12号样。
实施例13
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后在37℃下浸泡在20mM的2-氨基戊-4-烯酸水溶液当中4小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时得样品记为13号样。
酶降解实验(表征交联度)：
采用同如前所述的酶降解实验相同的方法对12号样、13号样和对照组4进行酶降解实验，结果如表7所示。
表7
  酶降解失重率
对照组4 7.06％
12号样 5.81％
13号样 4.53％
对12号样、13号样和对照组4进行酶降解实验以表征各组样品的交联度，利用胶原酶Ⅰ处理12号样、13号样和对照组4后计算各组样品的酶降解失重率如表7所示。12号样、13号样酶降解失重率均低于对照组4，这表明样12号样、13号样的酶降解稳定性均高于对照组4，即12号样、13号样的交联度更高。酶降解实验结果表明该实施例的双键后交联制备生物瓣膜材料的方法能够提高生物瓣膜材料的交联度。
实施例14
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后在37℃下浸泡在20mM的2-氨基戊-4-烯酸水溶液当中4小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
将猪心包膜转移至50mM的2-氨基戊-4-烯酸水溶液当中浸泡4小时；
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时得样品记为14号样。
实施例15
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后在37℃ 下浸泡在20mM的2-氨基戊-4-烯酸水溶液当中4小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
在50mM的2-氨基戊-4-烯酸水溶液当中4小时；
取出猪心包，采用蒸馏水进行清洗；
将心包浸泡于蒸馏水中，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时，随后用蒸馏水清洗，用甘油脱水得干膜样品记为15号样。
实施例16
改性透明质酸的制备：称取2g分子量为10000的透明质酸钠，并使用20ml PBS溶解，再依次加入6.5ml甲基丙烯酸缩水甘油酯以及4.5ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸；
改性多聚赖氨酸的制备：将多聚赖氨酸溶解在去离子水中，然后以(1：1.5)的摩尔比加入甲基丙烯酸缩水甘油酯(甲基丙烯酸缩水甘油酯：氨基)。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸；
在本实施例中，新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在60mM(按赖氨酸算)改性多聚赖氨酸水溶液在室温当中12小时，然后加入戊二醛溶液至质量浓度为250mM，在37℃摇床上反应24小时，取出心包材料清洗后浸泡在50mg/ml改性透明质酸水溶液在室温当中12小时，然后采用2.5％过硫酸铵和0.25％N,N,N',N'-四甲基乙二胺在37℃下浸泡12小时，最后采用蒸馏水进行清洗，记为样品16。相对于对照样2，样品16的水接触角下降，乳酸脱氢酶活性降低，钙离子含量减少，可提升生物材料的亲水性能及血液相容性和抗钙化能力，潜在地延长其使用寿命。
对对照例4及实施例10～15所得样品(10号样、11号样、12号样、13号样、14号样、15号样和对照样4)进行大鼠皮下植入实验，植入30天后取出各组样品进行茜素红染色实验以表征各组样品在大鼠皮下植入30天后的钙化程度。
茜素红染色实验：
采用同如前所述茜素红染色实验相同的方法对10号样、11号样、12号样、13号样、14号样、15号样和对照样4进行茜素红染色实验。
通过茜素红染色实验对植入到大鼠皮下30天后的10号样、11号样、12号样、13号样、14号样、15号样和对照样4进行染色以表征各组样品的钙化程度。对照样4及10号样、11号样、12号样、13号样、14号样、15号样植入到大鼠皮下30天后样品切片的茜素红染色结果图像如图24-30所示，其中茜素红染色后样品的颜色越深表明钙化程度越高。相比于对照 样4切片的茜素红染色结果(图24)，10号样、11号样、12号样、13号样、14号样、15号样的茜素红染色结果图颜色明显较淡，这表明10号样、11号样、12号样、13号样、14号样、15号样的钙化程度低于对照样3，即10号样、11号样、12号样、13号样、14号样、15号样相比于对照组4具有一定的抗钙化效果。对植入到大鼠皮下30天后的10号样、11号样、12号样、13号样、14号样、15号样和对照组4的茜素红染色结果表明本申请双键后交联制备生物瓣膜材料的方法能够提升生物瓣膜的抗钙化性能。
血液接触实验：
将表面和厚度均匀的对照样4、12号样与13号样样品裁剪成直径为1cm的片材用生理盐水冲洗后沥干水分置于24孔板，向各孔加入300μL兔血并在37℃下以70bpm的转速摇晃孵育1小时。孵育结束后，将兔血弃去并向各孔加入500μL生理盐水在摇床轻微摇动下冲洗掉未黏附的血液。冲洗结束，将样品转移至2.5％(w/w)戊二醛溶液中固定4小时。固定后的样品用梯度乙醇(25％、50％、75％和100％，v/v)脱水，每个梯度20分钟。干燥的样品用导电胶固定在测试台上并进行喷金处理，在场发射扫描电镜上观察并拍摄各组样品上血液黏附的图像。
对照样4、12号样与13号样血液接触实验扫描电镜图如图31～图33所示。与兔血接触孵育后对照样4的扫描电镜图上观察到较多的血细胞黏附和聚集，而12号样和13号样上黏附的血细胞较少，仅有少许血细胞分散地黏附于表面。结果表明，12号样和13号样能够一定程度抑制血细胞的黏附从而降低凝血的风险，具有抗凝血的效果。
对照组5
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在100mM的戊二醛溶液中，室温下，100RPM转速振荡条件之下交联24小时为对照组5。
实施例17
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
然后在37℃下浸泡在20mM的2-甲基烯丙胺水溶液中2小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。
取出猪心包，采用蒸馏水进行清洗；
将猪心包浸泡在20mM的2-甲基烯丙胺水溶液中2小时；
清洗后浸泡于5wt％的N,N'-乙烯基双丙烯酰胺的水溶液中，在37℃条件下浸泡12小时，确保N,N'-乙烯基双丙烯酰胺充分地物理渗透。
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。为方便区分各实施例制备的样品，本实施例所得样品记为样品17。
该实施例的反应原理图如图34所示。
实施例18
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
然后在37℃下浸泡在20mM的2-甲基烯丙胺水溶液中2小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
清洗后浸泡于5wt％的1,4-丁二醇二丙烯酸酯的水溶液中，在37℃条件下浸泡12小时，确保1,4-丁二醇二丙烯酸酯充分地物理渗透；
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。为方便区分各实施例制备的样品，本实施例所得样品记为样品18。
实施例19
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
然后在37℃下浸泡在20mM的2-氨基戊-4-烯酸水溶液中2小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
清洗后浸泡于2wt％的(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯的水溶液中，在37℃条件下浸泡12小时，确保(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯充分地物理渗透。
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时；
用蒸馏水洗涤，浸泡于甘油中，脱水得干膜。为方便区分各实施例制备的样品，本实施例所得样品记为样品19。
实施例20
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，
然后在37℃下浸泡在20mM的甲基丙烯酸2-氨基乙酯水溶液中2小时，
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。
加入N,N'-乙烯基双丙烯酰胺使其终浓度为5％，在37℃条件下浸泡12小时，确保N,N'-乙烯基双丙烯酰胺充分地物理渗透。
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM， 在37℃条件下反应24小时。为方便区分各实施例制备的样品，本实施例所得样品记为样品20。
实施例21
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
然后在37℃下浸泡在20mM的2-氨基戊-4-烯酸水溶液中2小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
将心包膜浸泡在50mM的2-氨基戊-4-烯酸水溶液中4小时；
清洗后浸泡于2wt％的(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯的水溶液中，在37℃条件下浸泡12小时，确保(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯充分地物理渗透。
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。为方便区分各实施例制备的样品，本实施例所得样品记为样品21。
实施例22
改性透明质酸的制备：称取2g分子量为10000的透明质酸钠，并使用20ml PBS溶解，再依次加入6.5ml甲基丙烯酸缩水甘油酯以及4.5ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸；
改性多聚赖氨酸的制备：将多聚赖氨酸溶解在去离子水中，然后以(1：1.5)的摩尔比加入甲基丙烯酸缩水甘油酯(甲基丙烯酸缩水甘油酯：氨基)。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸；
在本实施例中，新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在60mM(按赖氨酸算)改性多聚赖氨酸水溶液在室温当中12小时，然后加入戊二醛溶液至质量浓度为250mM，在37℃摇床上反应24小时，取出心包材料清洗后浸泡在50mg/ml改性透明质酸水溶液在室温当中12小时，然后采用2.5％过硫酸铵和0.25％N,N,N',N'-四甲基乙二胺在37℃下浸泡12小时，最后采用蒸馏水进行清洗，记为样品22。相对于对照样2，样品22的水接触角下降，乳酸脱氢酶活性降低，钙离子含量减少，可提升生物材料的亲水性能及血液相容性和抗钙化能力，潜在地延长其使用寿命。
酶降解实验：
采用同如前所述酶降解实验相同的方法对对照例5及实施例17～20所得样品17、样品18、样品19、样品20、样品21进行胶原酶降解失重率测定。
结果如表8所示，由表8的结果可知，本申请的制备方法能够显著提高生物材料的交联 度。
表8胶原酶降解失重率。
样品编号 胶原酶降解失重率
对照组5 8.6％
样品17 2.3％
样品18 3.7％
样品19 1.9％
样品20 3.8％
样品21 3.1％
对样品17、样品18、样品19、样品20、样品21和对照组5进行酶降解实验以表征各组样品的交联度，利用胶原酶Ⅰ处理样品17、样品18、样品19、样品20、样品21和对照组5后计算各组样品的酶降解失重率，结果如表8。样品17、样品18、样品19、样品20、样品21的酶降解失重率均低于对照组5，表明样样品17、样品18、样品19、样品20的酶降解稳定性均高于对照组5，即样品17、样品18、样品19、样品20的交联度更高。酶降解实验结果表明本申请共交联后双键交联生物瓣膜材料的制备方法能够提高生物瓣膜材料的交联度。
茜素红染色实验：
采用与如前所述茜素红染色实验相同的试验方法对样品17、样品18、样品19、样品20、样品21和对照组5进行茜素红染色实验。
通过茜素红染色实验对植入到大鼠皮下30天后的对照组5、样品17、样品18、样品19、样品20、样品21进行染色以表征各组样品的钙化程度。植入到大鼠皮下30天后样品切片的茜素红染色结果图像如图35-40所示，其中茜素红染色后样品的颜色越深表明钙化程度越高。相比于对照样5切片的茜素红染色结果(图35)，样品17、样品18、样品19、样品20、样品21的茜素红染色结果图颜色明显较淡，这表明样品17、样品18、样品19、样品20、样品21的钙化程度低于对照样5，即样品17、样品18、样品19、样品20、样品21相比于对照组5具有一定的抗钙化效果。对植入到大鼠皮下30天后的样品17、样品18、样品19、样品20、样品21和对照组5的茜素红染色结果表明本申请共交联后双键交联生物瓣膜材料的制备方法能够提升生物瓣膜的抗钙化性能。
对照组6
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在100mM的戊二醛溶液中，室温下，100RPM转速振荡条件之下交联24小时得对照样6。
实施例23
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，
然后在37℃下浸泡在30mM的2-氨基戊-4-烯酸水溶液中2小时，
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。
取出猪心包，采用蒸馏水进行清洗。
将猪心包浸泡在30mM的2-氨基戊-4-烯酸水溶液中2小时
清洗后浸泡于5wt％的3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐的水溶液中，在37℃条件下浸泡12小时，确保其充分地物理渗透。
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。为方便区分各实施例制备的样品，本实施例所得样品记为样品23。
该实施例的反应原理如图41所示。
实施例24
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，
然后在37℃下浸泡在20mM的2-氨基戊-4-烯酸水溶液中2小时，
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。
取出猪心包，采用蒸馏水进行清洗。
清洗后浸泡于3wt％的3-[[2-(甲基丙烯酰氧)乙基]二甲基铵]丙酸酯的水溶液中，在37℃条件下浸泡12小时，确保3-[[2-(甲基丙烯酰氧)乙基]二甲基铵]丙酸酯充分地物理渗透。
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。为方便区分各实施例制备的样品，本实施例所得样品记为样品24。
血液接触实验
采用同如前所述血液接触实验相同的试验方法对对照样6、样品23和样品24进行血液接触实验。
对照样6、样品23和样品24的血液接触实验扫描电镜图如图42、图43和图44所示。与兔血接触孵育后对照样6的扫描电镜图上观察到较多的血细胞黏附和聚集，而样品23和样品24上黏附的血细胞较少，仅有少许血细胞分散地黏附于表面。结果表明，样品23和样品24能够一定程度抑制血细胞的黏附从而降低凝血的风险，具有抗凝血的效果。
实施例25
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，
然后在37℃下浸泡在20mM的甲基丙烯酸2-氨基乙酯水溶液中2小时，
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。
取出猪心包，采用蒸馏水进行清洗。
清洗后浸泡于5wt％的N-异丙基丙烯酰胺的水溶液中，在37℃条件下浸泡12小时，确保N-异丙基丙烯酰胺充分地物理渗透。
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。为方便区分各实施例制备的样品，本实施例所得样品记为样品25。
酶降解实验(表征交联度)
采用同如前所述酶降解实验相同的方法对样品23、样品24、样品25和对照组6进行胶原酶降解失重率测定。
结果如表9所示，由表9的结果可知，本申请的制备方法能够显著提高生物材料的交联度。
表9胶原酶降解失重率
样品编号 胶原酶降解失重率
对照组6 8.6％
样品23 2.9％
样品24 1.7％
样品25 2.4％
对样品23、样品24、样品25和对照组6进行酶降解实验以表征各组样品的交联度，利用胶原酶Ⅰ处理样品23、样品24、样品25和对照组6后计算各组样品的酶降解失重率如上表。样品23、样品24、样品25的酶降解失重率均低于对照组6，这表明样样品23、样品24、样品25的酶降解稳定性均高于对照组6，即样品23、样品24、样品25的交联度更高。酶降解实验结果表明本申请制备生物瓣膜材料的方法能够提高生物瓣膜材料的交联度。
实施例26
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，
然后在37℃下浸泡在20mM的甲基丙烯酸2-氨基乙酯水溶液中2小时，
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。
加入N-异丙基丙烯酰胺使其终浓度为5wt％，在37℃条件下浸泡12小时，确保N-异丙基丙烯酰胺充分地物理渗透。
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM， 在37℃条件下反应24小时。为方便区分各实施例制备的样品，本实施例所得样品记为样品26。
实施例27
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，
然后在37℃下浸泡在30mM的2-氨基戊-4-烯酸水溶液中2小时，
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时。
取出猪心包，采用蒸馏水进行清洗。
将猪心包浸泡在30mM的2-氨基戊-4-烯酸水溶液中2小时
清洗后浸泡于5wt％的3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐的水溶液中，在37℃条件下浸泡12小时，确保其充分地物理渗透。
加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为30mM，在37℃条件下反应24小时。
反应结束，用蒸馏水洗猪心包后用甘油浸泡，脱水得干膜，本实施例所得样品记为样品27。
实施例28
改性透明质酸的制备：称取2g分子量为10000的透明质酸钠，并使用20ml PBS溶解，再依次加入6.5ml甲基丙烯酸缩水甘油酯以及4.5ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸；
改性多聚赖氨酸的制备：将多聚赖氨酸溶解在去离子水中，然后以(1：1.5)的摩尔比加入甲基丙烯酸缩水甘油酯(甲基丙烯酸缩水甘油酯：氨基)。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸；
在本实施例中，新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在60mM(按赖氨酸算)改性多聚赖氨酸水溶液在室温当中12小时，然后加入戊二醛溶液至质量浓度为250mM，在37℃摇床上反应24小时，取出心包材料清洗后浸泡在50mg/ml改性透明质酸水溶液在室温当中12小时，然后采用2.5％过硫酸铵和0.25％N,N,N',N'-四甲基乙二胺在37℃下浸泡12小时，最后采用蒸馏水进行清洗，记为样品28。相对于对照样2，样品28的水接触角下降，乳酸脱氢酶活性降低，钙离子含量减少，可提升生物材料的亲水性能及血液相容性和抗钙化能力，潜在地延长其使用寿命。
茜素红染色实验
采用与如前所述茜素红染色实验相同的试验方法对样品23、样品24、样品25、样品26、样品27和对照样6进行茜素红染色实验。
通过茜素红染色实验对植入到大鼠皮下30天后的对照样6、样品23、样品24、样品25、 样品26、样品27进行染色以表征各组样品的钙化程度。植入到大鼠皮下30天后样品切片的茜素红染色结果图像如图45-50所示，其中茜素红染色后样品的颜色越深表明钙化程度越高。相比于对照样6切片的茜素红染色结果(图45)，样品23、样品24、样品25、样品26和样品27的茜素红染色结果图颜色明显较淡，这表明样品23、样品24、样品25、样品26和样品27的钙化程度低于对照样6，即样品23、样品24、样品25、样品26和样品27相比于对照组6具有一定的抗钙化效果。对植入到大鼠皮下30天后的样品23、样品24、样品25、样品26和样品27和对照组6的茜素红染色结果表明本申请方法制备生物瓣膜材料的方法能够提升生物瓣膜的抗钙化性能。
对照例7
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在100mM的戊二醛溶液中，随后于室温、100RPM转速振荡条件之下交联24小时得对照样7。
实施例29
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在30mM的精氨酸水溶液中2小时；
然后加入戊二醛使其终浓度为150mM，在37℃、100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
在37℃下，将猪心包浸泡在50mM的精氨酸水溶液中12小时，随后用蒸馏水清洗得样品记为样品29。
实施例30
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时
在37℃下浸泡在30mM的精氨酸水溶液中2小时，
然后加入戊二醛使其终浓度为150mM，在37℃，100RPM转速振荡条件之下浸泡24小时。
取出猪心包，采用蒸馏水进行清洗；
浸泡于甘油中得到干膜记为样品30。
实施例29和实施例30的反应原理如图51所示。
实施例31
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在30mM的三羟甲基氨基甲烷水溶液中2小时；
然后加入戊二醛使其终浓度为150mM，在37℃、100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
在37℃下，将猪心包浸泡在50mM的三羟甲基氨基甲烷水溶液中12小时，随后用蒸馏水清洗得样品记为样品31。
实施例31的反应原理图如图52所示。
实施例32
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在20mM的油胺的乙醇水溶液(50％乙醇，v/v)中2小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
随后在37℃下，将猪心包浸泡在20mM的油胺的乙醇水溶液(50％乙醇，v/v)中12小时；随后用乙醇水溶液(50％乙醇，v/v)清洗得样品记为样品32。
实施例32的反应原理图如图53所示。
实施例33
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在20mM的十二胺的乙醇水溶液(50％乙醇，v/v)中2小时；
然后加入戊二醛使其终浓度为100mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
随后在37℃下，将猪心包浸泡在20mM的十二胺的乙醇水溶液(50％乙醇，v/v)中12小时；随后用乙醇水溶液(50％乙醇，v/v)清洗得样品记为样品33。
实施例33的反应原理图如图54所示。
实施例34
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在30mM的2-氨基-7-烯-辛酸的乙醇水溶液(40％乙醇，v/v)中2小时；
然后加入戊二醛使其终浓度为120mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
随后在37℃下，将猪心包浸泡在30mM的2-氨基-7-烯-辛酸的乙醇水溶液(40％乙醇，v/v)中12小时；随后用乙醇水溶液(40％乙醇，v/v)清洗得样品记为样品34。
实施例35
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在20mM的戊-4-烯-1-胺的溶液中2小时；
然后加入戊二醛使其终浓度为120mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
随后在37℃下，将猪心包浸泡在20mM的戊-4-烯-1-胺的溶液中12小时；随后用水溶液清洗得样品记为样品35。
实施例36
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在30mM的3-丁烯-1-胺水溶液中2小时；
然后加入戊二醛使其终浓度为120mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
随后在37℃下，将猪心包浸泡在30mM的3-丁烯-1-胺水溶液中12小时；随后用蒸馏水清洗得样品记为样品36。
实施例37
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在20mM的戊-4-烯-1-胺溶液中2小时；
然后加入戊二醛使其终浓度为120mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
随后在37℃下，将猪心包浸泡在油胺的乙醇水溶液(50％乙醇，v/v)中12小时；随后用蒸馏水清洗得样品记为样品37。
实施例38
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在30mM的2-氨基-7-烯-辛酸的乙醇水溶液(40％乙醇，v/v)中2小时；
然后加入戊二醛使其终浓度为120mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
随后在37℃下，将猪心包浸泡在油胺的乙醇水溶液(50％乙醇，v/v)中12小时；随后用乙醇水溶液(50％乙醇，v/v)清洗得样品记为样品38。
实施例39
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在30mM的2-氨基-7-烯-辛酸的乙醇水溶液(40％乙醇，v/v)中2小时；
然后加入戊二醛使其终浓度为120mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
随后在37℃下，将猪心包浸泡在30mM的2-氨基戊-4-烯酸水溶液中12小时；随后用蒸馏水清洗得样品记为样品39。
实施例40
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时；
在37℃下浸泡在30mM的精氨酸溶液中2小时；
然后加入戊二醛使其终浓度为120mM，在37℃，100RPM转速振荡条件之下浸泡24小时；
取出猪心包，采用蒸馏水进行清洗；
随后在37℃下，将猪心包浸泡在50mM的三羟甲基氨基甲烷水溶液中12小时；随后用蒸馏水清洗得样品记为样品40。
实施例41
改性透明质酸的制备：称取2g分子量为10000的透明质酸钠，并使用20ml PBS溶解，再依次加入6.5ml甲基丙烯酸缩水甘油酯以及4.5ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸；
改性多聚赖氨酸的制备：将多聚赖氨酸溶解在去离子水中，然后以(1：1.5)的摩尔比加入甲基丙烯酸缩水甘油酯(甲基丙烯酸缩水甘油酯：氨基)。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸；
在本实施例中，新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在60mM(按赖氨酸算)改性多聚赖氨酸水溶液在室温当中12小时，然后加入戊二醛溶液至质量浓度为250mM，在37℃摇床上反应24小时，取出心包材料清洗后浸泡在50mg/ml改性透明质酸水溶液在室温当中12小时，然后采用2.5％过硫酸铵和0.25％N,N,N',N'-四甲基乙二胺在37℃下浸泡12小时，最后采用蒸馏水进行清洗，记为样品41。相对于对照样2，样品41的水接触角下降，乳酸脱氢酶活性降低，钙离子含量减少，可提升生物材料的亲水性能及血液相容性和抗钙化能力，潜在地延长其使用寿命。
血液接触实验：
采用同如前所述血液接触实验相同的试验方法对对照样7、样品29和样品31进行血液接触实验。
对照样7、样品29和样品31的血液接触实验扫描电镜图如图55、图56和图57所示。与兔血接触孵育后对照样7的扫描电镜图上观察到较多的血细胞黏附和聚集，而样品29和样品31上黏附的血细胞较少，仅有少许血细胞分散地黏附于表面。结果表明，样品29和样品 31能够一定程度抑制血细胞的黏附从而降低凝血的风险，具有抗凝血的效果。血液接触结果表明本申请方法制备生物瓣膜材料的方法能够提升生物瓣膜的抗凝血性能。
样品35～40的血液接触实验结果显示，也具有与样品29和样品31相似的性能，能够提升生物瓣膜材料的抗凝血性能。
大鼠皮下植入30天后茜素红染色实验：
采用与如前所述茜素红染色实验相同的试验方法样品30、样品32、样品33、样品34和对照样7进行茜素红染色实验。
通过茜素红染色实验对植入到大鼠皮下30天后的对照样7、样品30、样品32、样品33、样品34进行染色以表征各组样品的钙化程度。对照样7、样品30、样品32、样品33、样品34植入到大鼠皮下30天后样品切片的茜素红染色结果图像如图58-62所示，其中茜素红染色后样品的颜色越深表明钙化程度越高。相比于对照样7切片的茜素红染色结果(图58)，样品30、样品32、样品33、样品34的茜素红染色结果图颜色明显较淡，这表明样品30、样品32、样品33、样品34的钙化程度低于对照样7，即样品30、样品32、样品33、样品34相比于对照组7具有一定的抗钙化效果。对植入到大鼠皮下30天后的样品30、样品32、样品33、样品34和对照样7的茜素红染色结果表明本申请方法制备生物瓣膜材料的方法能够提升生物瓣膜的抗钙化性能。
样品35～40的茜素红染色实验结果显示，也具有与样品29和样品32相似的性能，能够提升生物瓣膜材料的抗钙化性能。
对照组8
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，然后浸泡在0.30％(w/w)的戊二醛溶液中，室温下，100RPM转速振荡条件之下交联48小时得对照样8。
实施例42
在本实施例中，新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于5％(v/v)甲基丙烯酸缩水甘油酯的丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为20％(v/v)丙醇水溶液。
双键化修饰结束后，将双键化戊二醛交联猪心包用去离子水洗涤；随后将双键化戊二醛交联猪心包浸泡于20mM过硫酸钾和10mM亚硫酸氢钠的混合液中进一步引发双键化戊二醛交联猪心包上的双键的聚合反应，37℃下反应8小时后得到双键后交联的猪心包，记为样品42。
实施例43
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于6％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为20％(v/v)异丙醇水溶液。
双键化修饰结束后，将双键化戊二醛交联猪心包用去离子水洗涤；随后将双键化戊二醛交联猪心包浸泡于20mM过硫酸铵和5mM亚硫酸氢钠的混合液中进一步引发双键化戊二醛交联猪心包上的双键的聚合反应，37℃下反应8小时后得到双键后交联的猪心包，记为样品43。
实施例44
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于同时含有4％(v/v)丙烯酸缩水甘油酯和4％(v/v)烯丙基缩水甘油醚的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为30％(v/v)乙醇水溶液。
双键化修饰结束后，将双键化戊二醛交联猪心包用去离子水洗涤；随后将双键化戊二醛交联猪心包浸泡于20mM过硫酸铵和10mM亚硫酸氢钠的混合液中进一步引发双键化戊二醛交联猪心包上的双键的聚合反应，37℃下反应7小时后得到双键后交联的猪心包，，记为样品44，编号为GAGA-PP-3。
实施例45
新鲜采集的猪心包于4℃下100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于5％(v/v)甲基丙烯酸缩水甘油酯和2％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为35％(v/v)异丙醇水溶液。
双键化修饰结束后，将双键化戊二醛交联猪心包用去离子水洗涤；随后将双键化戊二醛交联猪心包浸泡于20mM过硫酸钠和5mM亚硫酸氢钠的混合液中进一步引发双键化戊二醛交联猪心包上的双键的聚合反应，37℃下反应8小时后得到双键后交联的猪心包，记为样品45。
实施例46
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
去离子水洗涤戊二醛交联猪心包，并在室温下将戊二醛交联猪心包浸泡于4％(v/v)甲基丙烯酸缩水甘油酯的乙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为20％(v/v)乙醇水溶液。
双键化修饰结束后，加入过硫酸铵和亚硫酸氢钠引发双键化戊二醛交联猪心包上的双键的聚合反应，其中过硫酸铵浓度为20mM，亚硫酸氢钠浓度为5mM；加入引发剂后在37℃下反应8小时后得到双键后交联的猪心包，记为样品46。
实施例47
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于4％(v/v)甲基丙烯酸缩水甘油酯的异丁醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为15％(v/v)异丁醇水溶液。
双键化修饰结束后，将双键化戊二醛交联猪心包用去离子水洗涤；随后将双键化戊二醛交联猪心包浸泡于20mM过硫酸铵和5mM亚硫酸氢钠的混合液中进一步引发双键化戊二醛交联猪心包上的双键的聚合反应，37℃下反应8小时后得到双键后交联的猪心包，记为样品47。
实施例48
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于4％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为48小时，所用双键化溶液的溶剂为20％(v/v)甲醇水溶液。
双键化修饰结束后，将双键化戊二醛交联猪心包用去离子水洗涤；随后将双键化戊二醛交联猪心包浸泡于20mM过硫酸铵和6.5mM亚硫酸钠的混合液中进一步引发双键化戊二醛交联猪心包上的双键的聚合反应，37℃下反应10小时后得到双键后交联的猪心包，记为样品48。
实施例49
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于4％(v/v)甲基丙烯酸缩水甘油酯的乙二醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为20％(v/v)乙二醇水溶液。
双键化修饰结束后，将双键化戊二醛交联猪心包用去离子水洗涤；随后将双键化戊二醛交联猪心包浸泡于40mM过硫酸铵和15mM亚硫酸氢钠的混合液中进一步引发双键化戊二醛交联猪心包上的双键的聚合反应，37℃下反应7小时后得到双键后交联的猪心包，记为样品49。
实施例50
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于7％(v/v)丙烯酸缩水甘油酯的丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为60小时，所用双键化溶液的溶剂为40％(v/v)丙醇水溶液。
双键化修饰结束后，将双键化戊二醛交联猪心包用去离子水洗涤；随后将双键化戊二醛交联猪心包浸泡于30mM过硫酸钠和10mM亚硫酸氢钠的混合液中进一步引发双键化戊二醛交联猪心包上的双键的聚合反应，37℃下反应8小时后得到双键后交联的猪心包，记为样品50。
实施例51
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
用去离子水洗涤后，在室温下将戊二醛交联猪心包浸泡于含有6％(v/v)甲基丙烯酸缩水甘油酯和3％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为84小时，所用双键化溶液的溶剂为50％(v/v)乙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于40mM过硫酸铵和10mM亚硫酸钠的混合液中进一步引发双键化戊二醛交联猪心包上的双键的聚合反应，37℃下反应12小时后得到双键后交联的猪心包，记为样品51。
对实施例42～51及对照组8的样品进行性能表征：
为表征戊二醛交联生物瓣膜材料在双键后交联处理前后的交联度变化，通过对生物瓣膜材 料的热收缩温度的测定表征生物瓣膜材料的热稳定性和交联度；通过酶降解实验表征生物瓣膜材料的稳定性；通过大鼠皮下植入实验表征样品的钙化程度(抗钙化性能)。
热收缩温度测定：
将生物瓣膜材料裁剪成直径为0.6cm的圆形片材，干燥后置于坩埚中，在差示扫描量热仪上以10℃/min的加热速度在40-120℃区间生物瓣膜材料的热收缩温度。通过对热收缩温度的测定以表征生物瓣膜材料的热稳定性和交联度；热收缩温度越高，对应热稳定性和交联度越高。
表10各组样品的热收缩温度
样品 热收缩温度(℃)
对照组8(戊二醛交联猪心包) 84.7
实施例42 88.9
实施例43 89.3
实施例50 91.5
实施例51 92.0
对实施例42、实施例43、实施例50、实施例51和对照组8(戊二醛交联猪心包)进行热收缩温度测定发现：如表10所示，实施例42、实施例43、实施例50、实施例51的热收缩温度均高于对照组8(戊二醛交联猪心包)，即实施例42、实施例43、实施例50、实施例51的热稳定性和交联度均高于对照组8(戊二醛交联猪心包)。热收缩温度测定实验结果表明本申请的双键后交联制备生物瓣膜材料的方法能够提升生物瓣膜的热稳定性和交联度。
酶降解实验
采用同如前所述酶降解实验相同的方法对样品44、样品47、样品45、样品51和对照组8进行胶原酶降解失重率测定。
表11各组样品的酶降解失重率
样品 酶降解失重率(％)
对照组8(戊二醛交联猪心包) 7.45±1.33
样品44 5.31±0.30
样品45 4.47±1.05
样品47 5.12±0.97
样品51 3.06±0.59
对对照组8(戊二醛交联猪心包)、样品44、样品45、样品47、样品51进行酶降解实验以表征各组样品的交联效率，利用胶原酶Ⅰ处理对照组8(戊二醛交联猪心包)、样品44、样品45、样品47和样品51后计算各组样品的酶降解失重率如表11所示。样品44、样品45、样品47、样品51的酶降解失重率均低于对照组(戊二醛交联猪心包)，这表明样品44、样品45、样品47、样品51的稳定性均高于对照组(戊二醛交联猪心包)，即样品44、样品45、 样品47、样品51的稳定性更高。酶降解实验结果表明本申请的双键后交联制备生物瓣膜材料的方法能够提升生物瓣膜的稳定性。
抗钙化测试
将生物瓣膜材料裁剪成0.8◇0.8cm 2的片材，灭菌后植入到大鼠皮下30天后取出，每片样品分为两部分，一部分去除包囊后冻干称重，用6M盐酸消解后测定每克样品的钙元素含量；另一部分样品经过多聚甲醛组织固定液固定。固定结束后取出用手术刀进行修理平整后转移到脱水盒中。用梯度乙醇对材料样品进行脱水。脱水结束后将材料样品转移至包埋机用融化的石蜡进行包埋，然后转移到-20℃冰箱冷却、修整形状。在切片机上从修整好的蜡块切取5μm厚的切片，从摊片机转移至载玻片上并进行脱蜡和复水。用茜素红染液对切片进行染色3分钟，经水洗、烘干后用二甲苯通透5分钟。切片用中性树胶封片后在病理切片扫描仪上采集染色结果图像。
表12大鼠皮下植入30天后各组样品钙元素含量
样品 钙元素含量(mg/g)
对照组8(戊二醛交联猪心包) 74.9±12.3
样品42 15.1±4.7
样品46 8.4±4.6
样品48 12.7±5.1
通过对植入到大鼠皮下30天后的样品42、样品46、样品48和对照组8(戊二醛交联猪心包)进行钙元素含量检测以表征各组样品的钙化程度。如表12所示，样品42、样品46、样品48在大鼠皮下植入30天后的钙元素含量均低于对照组(戊二醛交联猪心包)，这个结果表明双键后交联制备生物瓣膜材料的方法能够提升生物瓣膜的抗钙化性能。
通过茜素红染色对植入到大鼠皮下30天后的对照组8(戊二醛交联猪心包)、样品42、样品46、样品48以直接观察各组样品的钙化程度。植入到大鼠皮下30天后的样品切片的茜素红染色结果图像如图65～68所示，其中茜素红染色后样品的颜色越深表明钙化程度越高。相比于对照组8(戊二醛交联猪心包)切片的茜素红染色结果(图65)，样品42(图66)、样品46(图67)、样品48(图68)切片的茜素红染色图颜色明显变浅变淡，这直接地表明样品42、样品46、样品48的钙化程度低于对照组8，即样品42、样品46、样品48相比于对照组8具有较强的抗钙化效果。对植入到大鼠皮下30天后的生物瓣膜材料的茜素红染色结果表明本申请的双键后交联制备生物瓣膜材料的方法能够提升生物瓣膜的抗钙化性能。
对照例9
在处理过程中，设置单纯戊二醛交联组为对照组，在室温下将猪心包浸泡于0.625％(w/w)的戊二醛当中72小时制备戊二醛交联猪心包，记为对照样9。
实施例52
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于5％(v/v)甲基丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为18％(v/v)异丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于2％(v/v)聚乙二醇二丙烯酸酯溶液中2小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和聚乙二醇二丙烯酸酯上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包，记为样品52。
实施例53
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于6％(v/v)丙烯酸缩水甘油酯的丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为20％(v/v)丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于2.5％(v/v)N-甲基-2-丙烯酰胺溶液中1小时；
向上述溶液中加入引发剂，其中过硫酸钾浓度为20mM和亚硫酸钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N-甲基-2-丙烯酰胺上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包，记为样品53。
实施例54
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于同时含有2％(v/v)丙烯酸缩水甘油酯和4％(v/v)烯丙基缩水甘油醚的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为30％(v/v)乙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于2.5％(v/v)的(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯溶液中1 小时；
向上述溶液中加入引发剂，其中过硫酸钾浓度为20mM和亚硫酸钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包，记为样品54。
实施例55
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于3％(v/v)甲基丙烯酸缩水甘油酯和2％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为48小时，所用双键化溶液的溶剂为25％(v/v)异丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于1.5％(v/v)的乙烷-1,2-二基二丙烯酸酯溶液中1小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和乙烷-1,2-二基二丙烯酸酯上的双键之间的聚合，37℃下反应7小时后得到双键共聚后交联的猪心包，记为样品55。
实施例56
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
去离子水洗涤戊二醛交联猪心包，并在室温下将戊二醛交联猪心包浸泡于4％(v/v)甲基丙烯酸缩水甘油酯的乙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为20％(v/v)乙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于1.4％(v/v)的N,N'-二甲基丙烯酰胺溶液中1小时；
向上述溶液中加入引发剂，其中过硫酸钠浓度为20mM和亚硫酸氢钠浓度为7mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N,N'-二甲基丙烯酰胺上的双键之间的聚合，37℃下反应7小时后得到双键共聚后交联的猪心包，记为样品56。
实施例57
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于4％(v/v)甲基丙烯酸缩水甘油酯的异丁醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为15％(v/v)异丁醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于1.4％(v/v)的N,N'-二甲基甲基丙烯酰胺溶液中5小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N,N'-二甲基甲基丙烯酰胺上的双键之间的聚合，37℃下反应12小时后得到双键共聚后交联的猪心包，记为样品57。
实施例58
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于4％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为48小时，所用双键化溶液的溶剂为20％(v/v)甲醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于含有1.0％(v/v)的N,N'-二甲基丙烯酰胺和0.5％(v/v)N,N'-二甲基甲基丙烯酰胺溶液中1小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键与N,N'-二甲基丙烯酰胺和N,N'-二甲基甲基丙烯酰胺上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包，记为样品58。
实施例59
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于5％(v/v)甲基丙烯酸缩水甘油酯的乙二醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为25％(v/v)乙二醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于1.25％(v/v)的N,N'-二甲基甲基丙烯酰胺溶液中5小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N,N'-二甲基甲基丙烯酰胺上的双键之间的聚合，37℃下反应12小时后得到双键共聚后交联的猪心包，记为样品59。
实施例60
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于7％(v/v)丙烯酸缩水甘油酯的丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为60小时，所用双键化溶液的溶剂为30％(v/v)丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于1.0％(v/v)的N-乙基丙烯酰胺、溶液中5小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和四甲基乙二胺为1mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N,N'-二甲基甲基丙烯酰胺上的双键之间的聚合，37℃下反应12小时后得到双键共聚后交联的猪心包，记为样品60。
实施例61
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
用去离子水洗涤后，在室温下将戊二醛交联猪心包浸泡于含有4％(v/v)甲基丙烯酸缩水甘油酯和2％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为84小时，所用双键化溶液的溶剂为25％(v/v)乙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于1.50％(v/v)的N,N'-二甲基甲基丙烯酰胺溶液中3小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N,N'-二甲基甲基丙烯酰胺上的双键之间的聚合，37℃下反应7小时后得到双键共聚后交联的猪心包，记为样品61。
对实施例52～61的样品及对照例9的样品进行性能表征：
为表征戊二醛交联生物瓣膜材料在双键后共聚交联处理前后的交联度变化，通过对生物瓣膜材料的热收缩温度的测定表征生物瓣膜材料的热稳定性和交联度；通过酶降解实验表征生物瓣膜材料的稳定性；通过大鼠皮下植入实验表征样品的钙化程度(抗钙化性能)；通过测试生物瓣膜材料的弹性角度以表征其弹性。
热收缩温度测定：
将生物瓣膜材料裁剪成直径为0.6cm的圆形片材，干燥后置于坩埚中，在差示扫描量热仪上以10℃/min的加热速度在40-120℃区间生物瓣膜材料的热收缩温度。通过对热收缩温度的测定以表征生物瓣膜材料的热稳定性和交联度；热收缩温度越高，对应热稳定性和交联度 越高。
表13各组生物瓣膜材料的热收缩温度
样品名称 热收缩温度(℃)
对照组9(戊二醛交联猪心包) 85.2
样品52 91.3
样品53 92.8
样品54 90.7
样品56 91.4
样品561 90.1
对对照组9(戊二醛交联猪心包)、样品52、样品53、样品54、样品56、样品61、进行热收缩温度测定发现：如表13所示，样品52、样品53、样品54、样品56、样品61的热收缩温度均高于对照组9(戊二醛交联猪心包)，即样品52、样品53、样品54、样品56、样品61的热稳定性和交联度均高于对照组(戊二醛交联猪心包)。热收缩温度测定实验结果表明本申请的双键后共聚交联制备生物瓣膜材料的方法能够提升生物瓣膜的热稳定性和交联度。
弹性测试实验
将厚度均匀的生物瓣膜材料裁剪成1*4.6cm 2的长方形样品，沿着长方形样品的长边中线水平夹持，测试样品相对于中线水平面下垂的角度以表征样品的弹性，角度越小则弹性越高。
表14各组生物瓣膜材料的弹性角度
样品名称 弹性角度(°)
对照组9(戊二醛交联猪心包) 65
样品52 35
样品53 45
样品54 56
样品56 47
样品61 50
对样品52、样品53、样品54、样品56、样品61和对照组9(戊二醛交联猪心包)进行弹性测试实验以表征其弹性。弹性实验结果如表14所示，相比于对照组9(戊二醛交联猪心包)，样品52、样品53、样品54、样品56、样品61的弹性角较低，表明其弹性相对于对照组(戊二醛交联猪心包)均有大幅提升。双键后共聚交联制备生物瓣膜材料的方法能够提升生物瓣膜的弹性，生物瓣膜材料弹性增强有利于在经导管植入后迅速恢复形态。
酶降解实验
采用同如前所述酶降解实验相同的方法对样品52、样品53、样品56、样品61和对照组9进行胶原酶降解失重率测定，结果如表15所示。
表15各组生物瓣膜材料的酶降解失重率
样品名称 酶降解失重率(％)
对照组(戊二醛交联猪心包) 7.27±1.18
实施例52 1.12±0.40
实施例53 3.61±0.22
实施例56 2.90±0.45
实施例61 3.27±0.55
对样品52、样品53、样品56、样品61和对照组9(戊二醛交联猪心包)进行酶降解实验以表征各组样品的交联效率，利用胶原酶Ⅰ处理样品52、样品53、样品56、样品61和对照组9(戊二醛交联猪心包)后计算各组样品的酶降解失重率如表15所示。样品52、样品53、样品56、样品61酶降解失重率均低于对照组9(戊二醛交联猪心包)，这表明样品52、样品53、样品56、样品61的稳定性均高于对照组(戊二醛交联猪心包)，即样品52、样品53、样品56、样品61的稳定性更高。酶降解实验结果表明本申请的双键后共聚交联制备生物瓣膜材料的方法能够提升生物瓣膜的稳定性。
抗钙化测试
对样品52、样品53、样品56和对照组9(戊二醛交联猪心包)将生物瓣膜材料裁剪成1◇1cm 2的片材，采用同如前所述抗钙化测试相同的方法进行抗钙化测试。
表16大鼠皮下植入30天后各组生物瓣膜材料钙元素含量
样品名称 钙元素含量(mg/g)
对照组9(戊二醛交联猪心包) 67.3±10.5
样品52 5.4±2.7
样品53 8.1±3.6
样品56 13.9±4.7
通过对植入到大鼠皮下30天后的样品52、样品53、样品56和对照组9(戊二醛交联猪心包)进行钙元素含量检测以表征各组样品的钙化程度。如表16所示，样品52、样品53、样品56在大鼠皮下植入30天后的钙元素含量均低于对照组9(戊二醛交联猪心包)，这个结果表明双键后共聚交联制备生物瓣膜材料的方法能够提升生物瓣膜的抗钙化性能。
通过茜素红染色对植入到大鼠皮下30天后的对照组9(戊二醛交联猪心包)、样品52、样品53、样品56以直接观察各组样品的钙化程度。植入到大鼠皮下30天后的样品切片的茜素红染色结果图像如图71-74所示，其中茜素红染色后样品的颜色越深表明钙化程度越高。相比于对照组9(戊二醛交联猪心包)切片的茜素红染色结果(图71)，样品52(图72)、样品53(图73)、样品56(图74)、切片的茜素红染色图颜色明显变浅变淡，这直接地表明样品52、样品53、样品56的钙化程度低对照组9，即样品52、样品53、样品56相比于对照组9具有较强的抗钙化效果。对植入到大鼠皮下30天后的生物瓣膜材料的茜素红染色结果表明本申请的双键后共聚交联制备生物瓣膜材料的方法能够提升生物瓣膜的抗钙化性能。
对照例10
在处理过程中，设置单纯戊二醛交联组为对照组，在室温下将猪心包浸泡于0.25％(w/w)的戊二醛当中72小时制备戊二醛交联猪心包，记为对照样10。
实施例62
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于5％(v/v)甲基丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为48小时，所用双键化溶液的溶剂为20％(v/v)异丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于3％(w/v)3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐溶液中2小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包，记为样品62。
实施例63
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于6％(v/v)丙烯酸缩水甘油酯的丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为20％(v/v)丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于5％(w/v)2-甲基丙烯酰氧乙基磷酸胆碱溶液中1小时；
向上述溶液中加入引发剂，其中过硫酸钾浓度为20mM和亚硫酸钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和2-甲基丙烯酰氧乙基磷酸胆碱上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包，记为样品63。
实施例64
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制 备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于同时含有2％(v/v)丙烯酸缩水甘油酯和4％(v/v)烯丙基缩水甘油醚的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为30％(v/v)乙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于5％(v/v)的丙烯酰胺溶液中3小时；
向上述溶液中加入引发剂，其中过硫酸钾浓度为20mM和亚硫酸钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和丙烯酰胺上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包，记为样品64。
实施例65
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于1.0％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于3％(v/v)甲基丙烯酸缩水甘油酯和2％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为48小时，所用双键化溶液的溶剂为25％(v/v)异丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于含有1％(v/v)的丙烯酰胺和1.5％(v/v)的N-异丙基丙烯酰胺溶液中1小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键与丙烯酰胺和N-异丙基丙烯酰胺上的双键之间的聚合，37℃下反应7小时后得到双键共聚后交联的猪心包，记为样品65。
实施例66
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
去离子水洗涤戊二醛交联猪心包，并在室温下将戊二醛交联猪心包浸泡于4％(v/v)甲基丙烯酸缩水甘油酯的乙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为20％(v/v)乙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于1.5％(v/v)的N-异丙基丙烯酰胺溶液中1小时；
向上述溶液中加入引发剂，其中过硫酸钠浓度为20mM和亚硫酸氢钠浓度为7mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N-异丙基丙烯酰胺上的双键之间的聚合，37℃下反应7小时后得到双键共聚后交联的猪心包，记为样品66。
实施例67
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于4％(v/v)甲基丙烯酸缩水甘油酯的异丁醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为15％(v/v)异丁醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于2.0％(w/v)的丙烯酸钠溶液中5小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和丙烯酸钠上的双键之间的聚合，37℃下反应12小时后得到双键共聚后交联的猪心包，记为样品67。
实施例68
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于4％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为48小时，所用双键化溶液的溶剂为20％(v/v)甲醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于含有1.0％(v/v)的甲基丙烯酸羟乙酯和0.5％(w/v)的3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐溶液中1小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键与甲基丙烯酸羟乙酯和3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包，记为样品68。
实施例69
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于5％(v/v)甲基丙烯酸缩水甘油酯的乙二醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为25％(v/v)乙二醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于5％(v/v)的N-(羟甲基)丙烯酰胺溶液中5小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N-(羟甲基)丙烯酰胺上的双键之间的聚合，37℃下反应12小时后得到双键共聚后交联的猪心包，记为样品69。
实施例70
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于7％(v/v)丙烯酸缩水甘油酯的丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为60小时，所用双键化溶液的溶剂为30％(v/v)丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于1.0％(v/v)的N-(甲氧基甲基)甲基丙烯酰胺、溶液中5小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和四甲基乙二胺为1mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N-(甲氧基甲基)甲基丙烯酰胺上的双键之间的聚合，37℃下反应12小时后得到双键共聚后交联的猪心包，记为样品70。
实施例71
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
用去离子水洗涤后，在室温下将戊二醛交联猪心包浸泡于含有4％(v/v)甲基丙烯酸缩水甘油酯和2％(v/v)丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为84小时，所用双键化溶液的溶剂为25％(v/v)乙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于1％(v/v)的丙烯酰胺和1.50％(w/v)2-甲基丙烯酰氧乙基磷酸胆碱溶液中3小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键与丙烯酰胺和2-甲基丙烯酰氧乙基磷酸胆碱上的双键之间的聚合，37℃下反应7小时后得到双键共聚后交联的猪心包，记为样品71。
对实施例62～实施例71以及对照例10的样品进行性能表征：
为表征戊二醛交联生物瓣膜材料在双键后共聚交联处理前后的交联度变化，通过对生物瓣膜材料的热收缩温度的测定表征生物瓣膜材料的热稳定性和交联度；通过酶降解实验表征 生物瓣膜材料的稳定性；通过大鼠皮下植入实验表征样品的钙化程度(抗钙化性能)；通过测试生物瓣膜材料的弹性角度以表征其弹性；通过水接触角测试表征生物瓣膜材料的亲水性；通过血液黏附实验表征材料的抗血栓性能。
热收缩温度测定
将生物瓣膜材料裁剪成直径为0.6cm的圆形片材，干燥后置于坩埚中，在差示扫描量热仪上以10℃/min的加热速度在40-120℃区间生物瓣膜材料的热收缩温度。通过对热收缩温度的测定以表征生物瓣膜材料的热稳定性和交联度；热收缩温度越高，对应热稳定性和交联度越高。
表17各组生物瓣膜材料的热收缩温度
样品名称 热收缩温度(℃)
对照组10(戊二醛交联猪心包) 86.7
样品62 89.4
样品64 91.8
样品67 90.5
样品69 92.4
对样品62、样品64、样品67、样品69和对照组10(戊二醛交联猪心包)进行热收缩温度测定发现：如表17所示，样品62、样品64、样品67、样品69的热收缩温度均高于对照组10(戊二醛交联猪心包)，即样品62、样品64、样品67、样品69的热稳定性和交联度均高于对照组(戊二醛交联猪心包)。热收缩温度测定实验结果表明本申请的双键后功能化共聚交联制备生物瓣膜材料的方法能够提升生物瓣膜的热稳定性和交联度。
水接触角测试
将生物瓣膜材料裁剪为1*1cm 2的片材，80℃冷冻过夜后转移至冻干机，冻干48小时，取出片材置于水接触角测试仪上测定不同材料的水接触角以表征材料的亲水性，所得水接触角越小，生物瓣膜材料越亲水。
表18各组生物瓣膜材料的水接触角
样品名称 水接触角(°)
对照组10(戊二醛交联猪心包) 67
样品62 26
样品63 42
样品68 45
样品71 33
水接触角测试结果如表18所示，相比于对照组10(戊二醛交联猪心包)，样品62、样品63、样品68、样品71的水接触角均有明显的下降，即样品62、样品63、样品68、样品71较对照组10(戊二醛交联猪心包)更亲水，这表明通过双键后功能化共聚交联制备生物瓣 膜材料的方法能够提升生物瓣膜的亲水性。
血液黏附实验
将生物瓣膜材料裁剪为直径1cm的圆形片材，转移至48孔板，随后向材料表面加入0.5mL的新鲜兔血使其充分与血液接触以进行血液黏附实验。在与血液接触1.5小时后，将生物瓣膜材料从血液中移出，用生理盐水清洗3次。将清洗后的生物瓣膜材料浸泡于2.5％(w/v)的戊二醛溶液中固定2小时。固定结束，将生物瓣膜材料用梯度浓度(50％、75％、90％和100％，v/v)的乙醇脱水，随后进行喷金，最后置于扫描电镜上对血液黏附进行观察和拍照以表征抗血栓性能。
结果分析：如图77～80所示：通过与血液接触后，在对照组10(戊二醛交联猪心包)上观察到大量的红细胞和血小板的黏附(图77)，而在样品62(图78)、样品63(图79)、样品68(图80)仅观察到少量的红细胞黏附；样品62、样品63、样品68上血细胞黏附的较低，减少了血液与生物瓣膜材料的相互作用，这进一步降低了生物瓣膜材料上血栓形成的可能，即样品62、样品63、样品68相比于对照组10(戊二醛交联猪心包)具有较好的抗血栓性能；血液黏附实验表明，通过双键后功能化共聚交联制备生物瓣膜材料的方法能够提升生物瓣膜的抗血栓性能。
弹性测试实验
将厚度均匀的生物瓣膜材料裁剪成1◇4.6cm 2的长方形样品，沿着长方形样品的长边中线水平夹持，测试样品相对于中线水平面下垂的角度以表征样品的弹性，角度越小则弹性越高。
表19各组生物瓣膜材料的弹性角度
样品名称 弹性角度(°)
对照组10(戊二醛交联猪心包) 60
样品62 50
样品64 35
样品67 45
样品69 30
对样品62、样品64、样品67、样品69和对照组3(戊二醛交联猪心包)进行弹性测试实验以表征其弹性。弹性实验结果如表19所示，相比于对照组10(戊二醛交联猪心包)，样品62、样品64、样品67、样品69的弹性角较低，表明其弹性相对于对照组(戊二醛交联猪心包)均有大幅提升。双键后功能化共聚交联制备生物瓣膜材料的方法能够提升生物瓣膜的弹性，生物瓣膜材料弹性增强有利于在经导管植入后迅速恢复形态。
酶降解实验
采用同如前所述酶降解实验相同的方法对样品62、样品64、样品67、样品69和对照组10进行胶原酶降解失重率测定。
表20各组生物瓣膜材料的酶降解失重率
测试样品 酶降解失重率(％)
对照组(戊二醛交联猪心包) 6.35±0.89
实施例62 3.12±0.30
实施例64 2.65±0.47
实施例67 4.90±0.45
实施例69 1.15±0.26
如表20所示，对样品62、样品64、样品67、样品69和对照组10(戊二醛交联猪心包)进行酶降解实验以表征各组样品的交联效率，利用胶原酶Ⅰ处理样品62、样品64、样品67、样品69和对照组10(戊二醛交联猪心包)后计算各组样品的酶降解失重率如表20所示。样品62、样品64、样品67、样品69的酶降解失重率均低于对照组10(戊二醛交联猪心包)，这表明样品62、样品64、样品67、样品69的稳定性均高于对照组(戊二醛交联猪心包)，即样品62、样品64、样品67、样品69的稳定性更高。酶降解实验结果表明本申请的双键后功能化共聚交联制备生物瓣膜材料的方法能够提升生物瓣膜的稳定性。
抗钙化测试
对样品62、样品63、样品69和对照组10(戊二醛交联猪心包)将生物瓣膜材料裁剪成1*1cm 2的片材，采用同如前所述抗钙化测试相同的方法进行抗钙化测试。
表21大鼠皮下植入30天后各组生物瓣膜材料钙元素含量
样品名称 钙元素含量(mg/g)
对照组10(戊二醛交联猪心包) 73.8±11.3
样品62 21.3±2.7
样品63 14.1±5.4
样品69 5.9±0.87
通过对植入到大鼠皮下30天后的样品62、样品63、样品69和对照组10(戊二醛交联猪心包)进行钙元素含量检测以表征各组样品的钙化程度。如表21所示，样品62、样品63、样品69在大鼠皮下植入30天后的钙元素含量均低于对照组(戊二醛交联猪心包)，这个结果表明本申请的一种双键后功能化共聚交联制备功能化生物瓣膜材料的方法的抗钙化性能。
通过茜素红染色对植入到大鼠皮下30天后的对照组10(戊二醛交联猪心包)、样品62、样品63、样品69以直接观察各组样品的钙化程度。植入到大鼠皮下30天后的样品切片的茜素红染色结果图像如图81-84所示，其中茜素红染色后样品的颜色越深，表明钙化程度越高。相比于对照组10(戊二醛交联猪心包)切片的茜素红染色结果(图81)，实施例62(图82)、实施例63(图83)、实施例69(图84)切片的茜素红染色图颜色明显变浅变淡，这直接地表明样品62、样品63、样品69的钙化程度低对照组10，即样品62、样品63、样品69相比于对照组具有较强的抗钙化效果。对植入到大鼠皮下30天后的生物瓣膜材料的茜素红染色结果表明本申请的一种双键后功能化共聚交联制备功能化生物瓣膜材料的方法能够提升生物瓣膜 的抗钙化性能。
实施例72
新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2小时，室温下浸泡于0.30％(w/w)的戊二醛溶液中，室温下浸泡处理48小时对生物瓣膜处理进行戊二醛交联处理得到戊二醛交联猪心包。
去离子水洗涤戊二醛交联猪心包，并在室温下将戊二醛交联猪心包浸泡于4％(v/v)甲基丙烯酸缩水甘油酯的乙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为20％(v/v)乙醇水溶液。
双键化修饰结束后，加入过硫酸铵和亚硫酸氢钠引发双键化戊二醛交联猪心包上的双键的聚合反应，其中过硫酸铵浓度为20mM，亚硫酸氢钠浓度为5mM；加入引发剂后在37℃下反应8小时后得到双键后交联的猪心包。
将双键共聚后交联的猪心包材料放置在70％乙醇水溶液中浸泡20min，随后放置在干化溶液(80％甘油、2％水、18％乙醇)中室温浸泡1.5h。清除猪心包材料表面多余甘油，环氧乙烷灭菌，记为样品72。
实施例73
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.625％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于5％(v/v)甲基丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为18％(v/v)异丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于2％(v/v)聚乙二醇二丙烯酸酯溶液中2小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和聚乙二醇二丙烯酸酯上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包。
将双键共聚后交联的猪心包材料放置在60％异丙醇水溶液中浸泡45min，随后放置在10％甘油、3％聚乙二醇(Mn＝200)、87％乙醇溶液中室温浸泡3h。清除猪心包材料表面多余甘油，环氧乙烷灭菌，记为样品73。
实施例74
新鲜摘取的猪心包浸泡于生理盐水中，摇晃清洗2小时，随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于5％(v/v)甲基丙烯酸缩水甘油酯的乙二醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为72小时，所用双键化溶液的溶剂为25％(v/v)乙二醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于5％(v/v)的N-(羟甲基)丙烯酰胺溶液中5小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和N-(羟甲基)丙烯酰胺上的双键之间的聚合，37℃下反应12小时后得到双键共聚后交联的猪心包。将双键共聚后交联的猪心包材料放置在60％异丙醇水溶液中浸泡45min，随后放置在10％甘油、3％聚乙二醇(Mn＝200)、87％乙醇溶液中室温浸泡3h。清除猪心包材料表面多余甘油，环氧乙烷灭菌，记为样品74。
实施例75
新鲜的猪心包膜放在质量分数为0.5％的脱氧胆酸钠(表面活性剂)的PBS溶液中，室温条件下震荡处理4h，然后用质量分数为0.9％的氯化钠水溶液(即生理盐水)清洗三次。
随后在室温下将猪心包浸泡于0.25％(w/w)的戊二醛溶液中，浸泡摇晃处理72小时对猪心包处理进行戊二醛交联处理制备戊二醛交联猪心包。
进一步用去离子水洗涤戊二醛交联猪心包，并在室温下浸泡于5％(v/v)甲基丙烯酸缩水甘油酯的异丙醇水溶液中进行戊二醛交联猪心包的双键化修饰，反应时间为48小时，所用双键化溶液的溶剂为20％(v/v)异丙醇水溶液。
双键化修饰结束后，用去离子水洗涤双键化戊二醛交联猪心包；随后将双键化戊二醛交联猪心包浸泡于3％(w/v)3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐溶液中2小时；
向上述溶液中加入引发剂，其中过硫酸铵浓度为20mM和亚硫酸氢钠浓度为10mM，进一步引发双键化戊二醛交联生物瓣膜材料上的双键和3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐上的双键之间的聚合，37℃下反应8小时后得到双键共聚后交联的猪心包，记为样品75。
实施例76
将新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2h，直至没有可见的粘附的非心包或非胶原组织，然后将清洗后的生物材料浸泡在10mM DL-2-氨基-4-戊烯酸水溶液中，于37℃浸泡12小时，DL-2-氨基-4-戊烯酸充分的物理渗透，然后加入戊二醛，控制其浓度为10mM，在37℃、100RPM转速振荡条件之下浸泡24小时后，采用蒸馏水浸泡清洗，清除没有反应的DL-2-氨基-4-戊烯酸和戊二醛。将上述清洗后的生物材料浸泡于1％的聚乙二醇二丙烯酸酯水溶液中，于37℃浸泡12小时，确保聚乙二醇二丙烯酸酯充分的物理渗透，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为10mM，在37℃条件下反应24小时，既得生物瓣膜材料，记为样品76。
实施例77
将新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2h，直至没有可见的粘附的非心包或非胶原组织，然后将清洗后的生物材料浸泡在100mM DL-2-氨基-4-戊烯酸水溶液中，于37℃浸泡12小时，DL-2-氨基-4-戊烯酸充分的物理渗透，然后加入戊二醛，控制其浓度为500mM，在37℃、100RPM转速振荡条件之下浸泡24小时后，采用蒸馏水浸泡清洗，清除没有反应的DL-2-氨基-4-戊烯酸和戊二醛。将上述清洗后的生物材料浸泡于10％的聚乙二醇二丙烯酸酯水溶液中，于37℃浸泡12小时，确保聚乙二醇二丙烯酸酯充分的物理渗透，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为100mM，在37℃条件下反应24小时，既得生物瓣膜材料，记为样品77。
实施例78
将新鲜采集的猪心包于4℃、100RPM转速振荡条件之下蒸馏水清洗2h，直至没有可见的粘附的非心包或非胶原组织，然后将清洗后的生物材料浸泡在50mM DL-2-氨基-4-戊烯酸水溶液中，于37℃浸泡12小时，DL-2-氨基-4-戊烯酸充分的物理渗透，然后加入戊二醛，控制其浓度为220mM，在37℃、100RPM转速振荡条件之下浸泡24小时后，采用蒸馏水浸泡清洗，清除没有反应的DL-2-氨基-4-戊烯酸和戊二醛。将上述清洗后的生物材料浸泡于6％的聚乙二醇二丙烯酸酯水溶液中，于37℃浸泡12小时，确保聚乙二醇二丙烯酸酯充分的物理渗透，加入过硫酸铵和亚硫酸氢钠引发剂引发，过硫酸铵和亚硫酸氢钠的摩尔浓度均为60mM，在37℃条件下反应24小时，既得生物瓣膜材料，记为样品78。
选取上述实施例76～78和对照例1的生物瓣膜材料进行乳酸脱氢酶相对活性测量，检测方法同实施例1，检测结果如表22。
表22
Figure PCTCN2022132374-appb-000002
实施例79
分别制备改性透明质酸和改性多聚赖氨酸：
称取2g分子量为10000的透明质酸钠，并使用20mlPBS溶解，再依次加入6.5ml甲基丙烯酸缩水甘油酯以及4.5ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸。
将多聚赖氨酸溶解在去离子水中，然后以摩尔比1︰1.5(甲基丙烯酸缩水甘油酯︰氨基)加入甲基丙烯酸缩水甘油酯。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸。
取新鲜采集的猪心包于4℃、100RPM转速振荡条件下蒸馏水清洗2小时，直至没有可见的粘附的非心包或非胶原组织，然后浸泡在180mM改性多聚赖氨酸水溶液中，室温浸泡24小时，确保其达到接近饱和的物理渗透，从而尽可能多地引入部分双键化的多聚赖氨酸。然后加入戊二醛溶液至质量浓度为2.5％，在37℃摇床上反应24小时。取出心包材料清洗后浸泡在10mg/ml改性透明质酸水溶液中，室温浸泡12小时，确保其达到接近饱和的物理渗透，以尽可能多地引入双键化的透明质酸。然后采用2.5％过硫酸铵和0.25％N,N,N',N'-四甲基乙二胺作为引发剂，在37℃下浸泡12小时，最后采用蒸馏水浸泡清洗，清除没有接枝上的双键化透明质酸，制得所述生物瓣膜材料，记为样品79。
实施例80
分别制备改性透明质酸和改性多聚赖氨酸：
称取2g分子量为10000的透明质酸钠，并使用20mlPBS溶解，再依次加入6.5ml甲基丙烯酸缩水甘油酯以及4.5ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸。
将多聚赖氨酸溶解在去离子水中，然后以摩尔比1︰1.5(甲基丙烯酸缩水甘油酯︰氨基)加入甲基丙烯酸缩水甘油酯。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸。
取新鲜采集的猪心包于4℃、100RPM转速振荡条件下蒸馏水清洗2小时，直至没有可见的粘附的非心包或非胶原组织，然后浸泡在180mM改性多聚赖氨酸水溶液中，室温浸泡24小时，确保其达到接近饱和的物理渗透，从而尽可能多地引入部分双键化的多聚赖氨酸。然后加入戊二醛溶液至质量浓度为2.5％，在37℃摇床上反应24小时。取出心包材料清洗后浸泡在30mg/ml改性透明质酸水溶液中，室温浸泡12小时，确保其达到接近饱和的物理渗透，以尽可能多地引入双键化的透明质酸。然后采用2.5％过硫酸铵和0.25％N,N,N',N'-四甲基乙二胺作为引发剂，在37℃下浸泡12小时，最后采用蒸馏水浸泡清洗，清除没有接枝上的双键化透明质酸，制得所述生物瓣膜材料，记为样品80。
实施例81
分别制备改性透明质酸和改性多聚赖氨酸：
称取2g分子量为10000的透明质酸钠，并使用20mlPBS溶解，再依次加入6.5ml甲基丙烯酸缩水甘油酯以及4.5ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸。
将多聚赖氨酸溶解在去离子水中，然后以摩尔比1︰1.5(甲基丙烯酸缩水甘油酯︰氨基)加入甲基丙烯酸缩水甘油酯。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000 的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸。
取新鲜采集的猪心包于4℃、100RPM转速振荡条件下蒸馏水清洗2小时，直至没有可见的粘附的非心包或非胶原组织，然后浸泡在180mM改性多聚赖氨酸水溶液中，室温浸泡24小时，确保其达到接近饱和的物理渗透，从而尽可能多地引入部分双键化的多聚赖氨酸。然后加入戊二醛溶液至质量浓度为2.5％，在37℃摇床上反应24小时。取出心包材料清洗后浸泡在100mg/ml改性透明质酸水溶液中，室温浸泡12小时，确保其达到接近饱和的物理渗透，以尽可能多地引入双键化的透明质酸。然后采用2.5％过硫酸铵和0.25％N,N,N',N'-四甲基乙二胺作为引发剂，在37℃下浸泡12小时，最后采用蒸馏水浸泡清洗，清除没有接枝上的双键化透明质酸，制得所述生物瓣膜材料，记为样品81。
实施例82
分别制备改性透明质酸和改性多聚赖氨酸：
称取2g分子量为10000的透明质酸钠，并使用20mlPBS溶解，再依次加入12ml甲基丙烯酸缩水甘油酯以及6ml三乙胺。在37℃摇床上放置5天。最后使用截留分子量为5000的透析袋透析5天，冷冻干燥得到双键化的透明质酸。
将多聚赖氨酸溶解在去离子水中，然后以摩尔比1︰1(甲基丙烯酸缩水甘油酯︰氨基)加入甲基丙烯酸缩水甘油酯。将混合物在37℃摇床上放置5天。最后使用截留分子量为1000的透析袋透析5天，冷冻干燥得到部分双键化的多聚赖氨酸。
取新鲜采集的猪心包于4℃、100RPM转速振荡条件下蒸馏水清洗至无可见的粘附的非心包或非胶原组织，然后浸泡在500mM改性多聚赖氨酸水溶液中，室温浸泡12小时，确保其达到接近饱和的物理渗透，从而尽可能多地引入部分双键化的多聚赖氨酸。然后加入戊二醛溶液至质量浓度为2.5％，在37℃摇床上反应24小时。取出心包材料清洗后浸泡在20mg/ml改性透明质酸水溶液中，室温浸泡24小时，确保其达到接近饱和的物理渗透，以尽可能多地引入双键化的透明质酸。然后采用2％过硫酸铵和0.2％N,N,N',N'-四甲基乙二胺作为引发剂，在37℃下浸泡24小时，最后采用蒸馏水浸泡清洗，清除没有接枝上的双键化透明质酸，制得所述生物瓣膜材料，记为样品82。
实施例83
分别制备改性透明质酸和改性多聚赖氨酸：
称取2g分子量为10000的透明质酸钠，并使用20mlPBS溶解，再依次加入6ml甲基丙烯酸缩水甘油酯以及4ml三乙胺。在37℃摇床上放置7天。最后使用截留分子量为5000的透析袋透析7天，冷冻干燥得到双键化的透明质酸。
将多聚赖氨酸溶解在去离子水中，然后以摩尔比1.5︰1(甲基丙烯酸缩水甘油酯︰氨基)加入甲基丙烯酸缩水甘油酯。将混合物在37℃摇床上放置7天。最后使用截留分子量为1000的透析袋透析7天，冷冻干燥得到部分双键化的多聚赖氨酸。
取新鲜采集的猪心包于4℃、100RPM转速振荡条件下蒸馏水清洗至无可见的粘附的非心 包或非胶原组织，然后浸泡在100mM改性多聚赖氨酸水溶液中，室温浸泡48小时，确保其达到接近饱和的物理渗透，从而尽可能多地引入部分双键化的多聚赖氨酸。然后加入戊二醛溶液至质量浓度为2.5％，在37℃摇床上反应24小时。取出心包材料清洗后浸泡在60mg/ml改性透明质酸水溶液中，室温浸泡24小时，确保其达到接近饱和的物理渗透，以尽可能多地引入双键化的透明质酸。然后采用5％过硫酸铵和0.3％N,N,N',N'-四甲基乙二胺作为引发剂，在37℃下浸泡24小时，最后采用蒸馏水浸泡清洗，清除没有接枝上的双键化透明质酸，制得所述生物瓣膜材料，记为样品83。
实施例84
分别制备改性透明质酸和改性多聚赖氨酸：
称取2g分子量为10000的透明质酸钠，并使用20mlPBS溶解，再依次加入10ml甲基丙烯酸缩水甘油酯以及8ml三乙胺。在37℃摇床上放置6天。最后使用截留分子量为5000的透析袋透析6天，冷冻干燥得到双键化的透明质酸。
将多聚赖氨酸溶解在去离子水中，然后以摩尔比1︰1.5(甲基丙烯酸缩水甘油酯︰氨基)加入甲基丙烯酸缩水甘油酯。将混合物在37℃摇床上放置6天。最后使用截留分子量为1000的透析袋透析6天，冷冻干燥得到部分双键化的多聚赖氨酸。
取新鲜采集的猪心包于4℃、100RPM转速振荡条件下蒸馏水清洗至无可见的粘附的非心包或非胶原组织，然后浸泡在200mM改性多聚赖氨酸水溶液中，室温浸泡24小时，确保其达到接近饱和的物理渗透，从而尽可能多地引入部分双键化的多聚赖氨酸。然后加入戊二醛溶液至质量浓度为2.5％，在37℃摇床上反应24小时。取出心包材料清洗后浸泡在35mg/ml改性透明质酸水溶液中，室温浸泡24小时，确保其达到接近饱和的物理渗透，以尽可能多地引入双键化的透明质酸。然后采用3％过硫酸铵和0.4％N,N,N',N'-四甲基乙二胺作为引发剂，在37℃下浸泡12小时，最后采用蒸馏水浸泡清洗，清除没有接枝上的双键化透明质酸，制得所述生物瓣膜材料，记为样品84。
选取上述实施例79～81和对照例2的生物瓣膜材料进行乳酸脱氢酶相对活性测量，检测方法同实施例2，检测结果如表23。
表23
Figure PCTCN2022132374-appb-000003
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不 脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (58)

  1. 一种生物瓣膜材料的制备方法，其特征在于，包括：
    步骤S100，将生物材料依次经第一处理液和第二处理液进行处理，得到化学接枝有第一碳碳双键的预处理后的生物材料；所述第一处理液和所述第二处理液彼此相异的含有试剂A和试剂B中的其中一种，其中试剂A为带有所述第一碳碳双键的第一功能单体，试剂B为醛基交联剂；
    步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
  2. 根据权利要求1所述的制备方法，其特征在于，步骤S100中，所述第一功能单体还带有活性基团，通过所述活性基团参与化学反应；所述第一处理液含有试剂A，所述活性基团可与醛基反应；或
    所述第一处理液含有试剂B，所述活性基团可与氨基反应。
  3. 根据权利要求1所述的制备方法，其特征在于，所述生物材料为动物组织，包括心包膜、瓣膜、肠膜、脑膜、肺膜、血管、皮肤或韧带的一种或多种。
  4. 根据权利要求3所述的制备方法，其特征在于，所述动物组织为新鲜的动物组织或经脱细胞处理后的生物组织。
  5. 根据权利要求1所述的制备方法，其特征在于，所述醛基交联剂为戊二醛或甲醛。
  6. 根据权利要求1所述的制备方法，其特征在于，步骤S200中：
    将引发剂加入上一步处理的体系中；或将上一步处理后的生物材料取出、直接或清洗后再浸泡于含引发剂的溶液中；
    所述引发剂为单一引发剂或混合引发剂，所述混合引发剂为：
    过硫酸铵和亚硫酸氢钠的混合物，或过硫酸铵和亚硫酸钠的混合物，或过硫酸钠和亚硫酸钠的混合物，或过硫酸钾和亚硫酸钠的混合物，或过硫酸钠和亚硫酸氢钠的混合物，或过硫酸钾和亚硫酸氢钠的混合物，所述混合物中各组分的浓度分别为1～100mM；
    或所述混合引发剂为：
    过硫酸铵和N,N,N',N'-四甲基乙二胺的混合物，或过硫酸钾和N,N,N',N'-四甲基乙二胺的混合物，或过硫酸氨和N,N,N',N'-四甲基乙二胺的混合物，或过硫酸钠和N,N,N',N'-四甲基乙二胺的混合物；混合物中过硫酸铵、过硫酸钾或过硫酸钠的质量百分浓度分别为2％～5％；四甲基乙二胺的质量百分比为0.2％～0.5％；
    所述单一引发剂为各混合引发剂中的任一组分。
  7. 根据权利要求1所述的制备方法，其特征在于，步骤S100包括：
    AS110 将生物材料与第一处理液接触进行物理渗透，所述第一处理液为含所述第一功能单体的溶液；
    AS120 将经AS110处理后的生物材料与第二处理液接触接触，进行共交联接入所述第一碳碳双键，所述第二处理液为醛基交联剂溶液。
  8. 根据权利要求7所述的制备方法，其特征在于，所述活性基团为氨基或酰肼。
  9. 根据权利要求1所述的制备方法，其特征在于，步骤S100包括：
    BS110 将生物材料与第一处理液接触进行交联，所述第一处理液为醛基交联剂溶液；
    BS120 将步骤BS110处理后的生物材料与第二处理液接触，化学反应接入所述第一碳碳双键，所述第二处理液为含所述第一功能单体的溶液。
  10. 根据权利要求9所述的制备方法，其特征在于，所述活性基团为环氧乙烷基。
  11. 根据权利要求1所述的制备方法，其特征在于，步骤S100中，采用非缩合的化学反应接入所述第一碳碳双键。
  12. 根据权利要求1所述的制备方法，其特征在于，步骤S100中，所述生物材料在经过醛基交联剂处理之前未经过任何其他试剂参与的化学反应。
  13. 根据权利要求1所述的制备方法，其特征在于，步骤S100的反应体系中通过带有活性基团的第一功能单体提供所述第一碳碳双键，且步骤S100中的反应原料仅包括所述生物材料、所述第一功能单体以及所述醛基交联剂。
  14. 根据权利要求1所述的制备方法，其特征在于，步骤S100包括：
    AS110 将生物材料浸泡于第一处理液中进行物理渗透；所述第一处理液为含所述第一功能单体的溶液；所述第一功能单体还带有活性基团；所述活性基团为氨基或酰肼；
    AS120 将步骤AS110处理后的生物材料浸泡于第二处理液中进行共交联接入第一碳碳双键，所述第二处理液为醛基交联剂溶液。
  15. 根据权利要求14所述的制备方法，其特征在于，所述第一功能单体还带有功能性基团A。
  16. 根据权利要求15所述的制备方法，其特征在于，所述功能性基团A选自羟基、羧基、酰胺基、磺酸基、两性离子、聚乙二醇、脲基、氨基甲酸酯基、羧酸根离子、磺酸酯、亚砜、吡咯烷酮中的至少一种中的至少一种。
  17. 根据权利要求14所述的制备方法，其特征在于，步骤S100还包括：
    AS130 将经步骤AS120处理后的生物材料浸泡于含第二功能单体的溶液中进行物理渗透引入第二碳碳双键；所述第二功能单体带有第二碳碳双键。
  18. 根据权利要求17所述的制备方法，其特征在于，所述第二功能单体还带有功能性基团B。
  19. 根据权利要求18所述的制备方法，其特征在于，所述功能性基团B选自羟基、羧基、羧酸胆碱、磺酸胆碱、磷酸胆碱、吡咯烷酮、磺酸基团、羧酸根离子、磺酸酯、亚砜、酰胺基团、甲氧基中的至少一种。
  20. 根据权利要求17所述的制备方法，其特征在于，所述第二功能单体选自聚乙二醇二丙烯酸酯、1,4-丁二醇二丙烯酸酯、丙烯酸乙酯、N-甲基-2-丙烯酰胺、N-2,2-丙烯基-2-丙烯酰胺、N-乙基丙烯酰胺、N,N'-乙烯基双丙烯酰胺、(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯、双键化透明质酸、丙烯酰胺、2-(丙-2-烯酰氨基)乙酸、2-丙烯酰胺基-2-甲基丙磺酸、甲基丙烯酸羟乙酯、3-[[2-(甲基丙烯酰氧)乙基]二甲基铵]丙酸酯、N-异丙基丙烯酰胺、N-(羟甲基)丙烯酰胺、N-(2-羟基乙基)甲基丙烯酰胺、N,N-二甲基甲基丙烯酰胺、3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐、2-甲基丙烯酰氧乙基磷酸胆碱、双键化多聚赖氨酸中的至少一种。
  21. 根据权利要求17或18所述的制备方法，其特征在于，步骤AS130中，将第二功能单体加入前步处理的体系中；或将前步处理后的生物材料清洗后再浸泡于含第二功能单体的溶液中；所述含第二功能单体的溶液中仅包括第二功能单体和不参与化学反应的溶剂。
  22. 根据权利要求17或18所述的制备方法，其特征在于，所述含第二功能单体的溶液中溶剂为水、生理盐水、pH中性缓冲液或乙醇的水溶液；所述含第二功能单体的溶液中第二功能单体的质量百分浓度为1～10％；浸泡时间为2～20h。
  23. 根据权利要求14所述的制备方法，其特征在于，所述第一功能单体选自DL-2-氨基-4-戊烯酸、2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯、甲基丙烯酰肼、丙烯酰肼、双键化多聚赖氨酸、2-氨基-7-烯-辛酸、6-烯-庚氨酸、2-氨基戊-4-烯酸、4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇、4-(1-氨基-乙基)-七-1,6-二烯-4-醇中的至少一种。
  24. 根据权利要求14～18任一项权利要求所述的制备方法，其特征在于，含所述第一功能单体的溶液中仅包括第一功能单体和不参与化学反应的溶剂。
  25. 根据权利要求14～18任一项权利要求所述的制备方法，其特征在于，步骤AS110中含所述第一功能单体的溶液中溶剂为水、生理盐水、异丙醇、pH中性缓冲液或乙醇的水溶液；含所述第一功能单体的溶液中第一功能单体的浓度为10～100mM；浸泡时间为2～20h。
  26. 根据权利要求14～18任一项权利要求所述的制备方法，其特征在于，步骤AS120中，所述醛基交联剂在AS120反应体系中的终浓度为10～800mM；共交联时间为10～30h。
  27. 根据权利要求14或15所述的制备方法，其特征在于，步骤S100中：
    在步骤AS120之后还包括步骤AS120(M)：将经步骤AS120处理后的生物材料浸泡于含第三功能单体的溶液中，消除残留醛基；所述第三功能单体带有氨基或酰肼。
  28. 根据权利要求27所述的制备方法，其特征在于，步骤AS120(M)中，所述含第三功能单体的溶液中溶剂为水、生理盐水、pH中性缓冲液或乙醇的水溶液；所述含第三功能单体的溶液中第三功能单体的浓度为10～100mM；浸泡时间为2～48h。
  29. 根据权利要求27所述的制备方法，其特征在于，所述第三功能基团还带有功能性基团C。
  30. 根据权利要求17或18所述的制备方法，其特征在于，步骤S100中：
    在步骤AS130之前还包括步骤AS120(M)：将经步骤AS120处理后的生物材料浸泡于含第三功能单体的溶液中，消除残留醛基；所述第三功能单体带有氨基或酰肼。
  31. 根据权利要求30所述的制备方法，其特征在于，步骤AS120(M)中，所述含第三功能单体的溶液中溶剂为水、生理盐水、pH中性缓冲液或乙醇的水溶液；所述含第三功能单体的溶液中第三功能单体的浓度为10～100mM；浸泡时间为2～48h。
  32. 根据权利要求30所述的制备方法，其特征在于，所述第三功能基团还带有功能性基团C。
  33. 根据权利要求29或32所述的制备方法，其特征在于，所述功能性基团C选自羟基、羧基、酰胺基、磺酸基、两性离子、聚乙二醇、脲基、氨基甲酸酯基、羧酸根离子、磺酸酯、亚砜、吡咯烷酮中的至少一种中的至少一种。
  34. 根据权利要求33所述的制备方法，其特征在于，所述第三功能单体选自DL-2-氨基-4-戊烯酸、2-甲基烯丙胺、3-丁烯-1-胺、戊-4-烯-1-胺、甲基丙烯酸2-氨基乙酯、甲基丙烯酰肼、丙烯酰肼、双键化多聚赖氨酸、2-氨基-7-烯-辛酸、6-烯-庚氨酸、2-氨基戊-4-烯酸、4-(1-氨基-2-甲基-丙基)-七-1,6-二烯-4-醇、4-(1-氨基-乙基)-七-1,6-二烯-4-醇中的至少一种。
  35. 根据权利要求1所述的制备方法，其特征在于，步骤S100包括：
    BS110 将生物材料与第一处理液接触进行交联，所述第一处理液为醛基交联剂溶液；
    BS120 将步骤BS110处理后的生物材料浸泡于第二处理液中，化学反应接入第一碳碳双键；所述第二处理液为含有所述第一功能单体的溶液；所述第一功能单体还带有活性基团；所述活性基团为环氧乙烷基。
  36. 根据权利要求1所述的制备方法，其特征在于，步骤S100包括：
    BS110 将生物材料与第一处理液接触进行交联，所述第一处理液为醛基交联剂溶液；
    BS120 将步骤BS110处理后的生物材料浸泡于第二处理液中，化学反应接入第一碳碳双键；所述第二处理液为含有所述第一功能单体的溶液；所述第一功能单体还带有活性基团；所述活性基团为环氧乙烷基；
    BS130 将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第二功能单体具有第二碳碳双键。
  37. 根据权利要求36所述的制备方法，其特征在于，所述第二功能单体选自聚乙二醇二丙烯酸酯、1,4-丁二醇二丙烯酸酯、乙烷-1,2-二基二丙烯酸酯、丙烯酸乙酯、N-甲基-2-丙烯酰胺、N-2,2-丙烯基-2-丙烯酰胺、N-乙基丙烯酰胺、N,N'-乙烯基双丙烯酰胺、(乙烷-1,2-二基双(氧基))双(乙烷-2,1-二基)二丙烯酸酯、N,N'-二甲基丙烯酰胺、N,N-二甲基甲基丙烯酰胺、双键化聚赖氨酸中的一种或多种。
  38. 根据权利要求36所述的制备方法，其特征在于，所述第二功能单体还带有功能性基团B。
  39. 根据权利要求38所述的制备方法，其特征在于，所述功能性基团B选自羟基、羧基、羧酸胆碱、磺酸胆碱、磷酸胆碱、吡咯烷酮、磺酸基团、羧酸根离子、磺酸酯、亚砜、酰胺基团、甲氧基中的至少一种。
  40. 根据权利要求39所述的制备方法，其特征在于，所述第二功能单体选自丙烯酰胺、丙烯酸、丙烯酸钠、甲基丙烯酸、甲基丙烯酸钠、2-(丙-2-烯酰氨基)乙酸、2-丙烯酰胺基-2-甲基丙磺酸、甲基丙烯酸羟乙酯、3-[[2-(甲基丙烯酰氧)乙基]二甲基铵]丙酸酯、N-甲基-2-丙烯酰胺、N-异丙基丙烯酰胺、N-(羟甲基)丙烯酰胺、N-(2-羟基乙基)甲基丙烯酰胺、3-[N,N-二甲基-[2-(2-甲基丙-2-烯酰氧基)乙基]铵]丙烷-1-磺酸内盐、2-甲基丙烯酰氧乙基磷酸胆碱、N-(2-羟乙基)丙烯酰胺、N-(甲氧基甲基)甲基丙烯酰胺、2-丙烯酰胺-2-甲基丙磺酸、双键化透明质酸中的一种或多种。
  41. 根据权利要求36～38任一项权利要求所述的制备方法，其特征在于，步骤BS130中：所述第二功能单体通过物理渗透进入所述生物材料中；所述含第二功能单体的溶液中仅包括第二功能单体和不参与反应的溶剂。
  42. 根据权利要求36～38任一项权利要求所述的制备方法，其特征在于，所述含第二功能单体的溶液中第二功能单体的v/v浓度为0.1％-20％；浸泡时间为0.5h-120h。
  43. 根据权利要求35～38任一项权利要求所述的制备方法，其特征在于，所述第一功能单体选自烯丙基缩水甘油醚、甲基丙烯酸缩水甘油酯和丙烯酸缩水甘油酯中的至少一种。
  44. 根据权利要求35～38任一项权利要求所述的制备方法，其特征在于，步骤BS110中：
    所述醛基交联剂溶液的w/w浓度为0.1％～5％；交联时间为0.5h-120h。
  45. 根据权利要求35～38任一项权利要求所述的制备方法，其特征在于，步骤BS120中：含所述第一功能单体的溶液中仅包括第一功能单体和不参与化学反应的溶剂。
  46. 根据权利要求35～38任一项权利要求所述的制备方法，其特征在于，含所述第一功能单体的溶液中第一功能单体的w/w浓度为1％～10％；反应时间为2～120小时。
  47. 根据权利要求35～38任一项权利要求所述的制备方法，其特征在于，含所述第一功能单体的溶液中溶剂为甲醇、乙醇、乙二醇、丙醇、1,2-丙二醇、1,3-丙二醇、异丙醇、丁醇、异丁醇、1,2-丁二醇、1,3-丁二醇、1,4-丁二醇和甘油中任意一种的水溶液、水、生理盐水、pH中性缓冲液中的一种或多种。
  48. 一种生物瓣膜材料的制备方法，其特征在于，包括：
    步骤BS110 将生物材料与醛基交联剂溶液接触进行交联；
    步骤BS120 将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
    步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
  49. 一种生物瓣膜材料的制备方法，其特征在于，包括：
    步骤BS110 将生物材料与醛基交联剂溶液接触进行交联；
    步骤BS120 将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
    步骤BS130 将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第二功能单体具有第二碳碳双键；
    步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
  50. 一种生物瓣膜材料的制备方法，其特征在于，包括：
    步骤BS110 将生物材料与醛基交联剂溶液接触进行交联；
    步骤BS120 将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
    步骤BS130 将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第二功能单体具有第二碳碳双键和功能性基团B；
    步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
  51. 一种生物瓣膜材料，其特征在于，由权利要求1～50任一项权利要求所述的制备方法制备得到。
  52. 一种生物瓣膜材料，其特征在于，包括：
    步骤S100，将生物材料依次经第一处理液和第二处理液进行处理，得到化学接枝有第一碳碳双键的预处理后的生物材料；所述第一处理液和所述第二处理液彼此相异的含有试剂A和试剂B中的其中一种，其中试剂A为带有所述第一碳碳双键的第一功能单体，试剂B为醛基交联剂；
    步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
  53. 一种生物瓣膜材料，其特征在于，包括：
    步骤BS110 将生物材料与醛基交联剂溶液接触进行交联；
    步骤BS120 将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
    步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
  54. 一种生物瓣膜材料，其特征在于，包括：
    步骤BS110 将生物材料与醛基交联剂溶液接触进行交联；
    步骤BS120 将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
    步骤BS130 将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第二功能单体具有第二碳碳双键；
    步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
  55. 一种生物瓣膜材料，其特征在于，包括：
    步骤BS110 将生物材料与醛基交联剂溶液接触进行交联；
    步骤BS120 将步骤BS110处理后的生物材料浸泡于含第一功能单体的溶液中，反应接入第一碳碳双键；所述第一功能单体具有第一碳碳双键和环氧乙烷基；
    步骤BS130 将步骤BS120处理后的生物材料浸泡于含第二功能单体的溶液中，所述第二功能单体具有第二碳碳双键和功能性基团B；
    步骤S200，在引发剂的作用下使碳碳双键进行聚合反应，得到生物瓣膜材料。
  56. 一种生物瓣膜，包括支架和瓣叶，其特征在于，所述瓣叶为权利要求51～55任一项权利要求所述的生物瓣膜材料。
  57. 根据权利要求56所述的生物瓣膜，其特征在于，所述生物瓣膜为心脏瓣膜。
  58. 一种介入***，包括心脏瓣膜和导管组件，所述心脏瓣膜折叠后由导管组件输送，其特征在于，心脏瓣膜包括支架和瓣叶，所述瓣叶为权利要求51～55任一项权利要求所述的生物瓣膜材料。
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CN113101412A (zh) * 2021-03-02 2021-07-13 四川大学华西医院 一种长效稳定的抗凝生物瓣膜材料及其制备方法
CN113476659A (zh) * 2021-05-31 2021-10-08 四川大学 一种人工生物瓣膜处理方法
CN114748695A (zh) * 2021-11-17 2022-07-15 四川大学 一种双键后交联提高生物瓣膜材料抗钙化及抗凝血性的方法
CN114748697A (zh) * 2021-11-17 2022-07-15 四川大学 一种双键后交联生物瓣膜材料及其制备和应用
CN114748696A (zh) * 2021-11-17 2022-07-15 四川大学 一种共交联后双键交联生物瓣膜材料及其制备方法和应用
CN114748694A (zh) * 2021-11-17 2022-07-15 四川大学 一种共交联生物瓣膜材料及其制备方法和应用
CN114748693A (zh) * 2021-11-17 2022-07-15 四川大学 一种共交联联合双键交联制备生物瓣膜材料的方法及生物瓣膜材料

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