WO2024103389A1 - 醛基交联后双键聚合制备生物瓣膜材料的方法及生物瓣膜材料和应用 - Google Patents

醛基交联后双键聚合制备生物瓣膜材料的方法及生物瓣膜材料和应用 Download PDF

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WO2024103389A1
WO2024103389A1 PCT/CN2022/132870 CN2022132870W WO2024103389A1 WO 2024103389 A1 WO2024103389 A1 WO 2024103389A1 CN 2022132870 W CN2022132870 W CN 2022132870W WO 2024103389 A1 WO2024103389 A1 WO 2024103389A1
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biological
cross
double bond
carbon
glutaraldehyde
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PCT/CN2022/132870
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English (en)
French (fr)
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王云兵
郑城
杨立
李高参
罗日方
邝大军
金林赫
麻彩丽
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四川大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/24—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body
    • 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
    • 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
    • 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
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
    • C08F220/32—Esters containing oxygen in addition to the carboxy oxygen containing epoxy radicals

Definitions

  • the present application relates to the technical field of interventional materials, and in particular to a method for preparing a biological valve material by double bond polymerization after aldehyde cross-linking, the biological valve material and its application.
  • Biological heart valves are usually made from pig or cow pericardium and are used to replace functionally defective human heart valves.
  • Biological heart valves have many advantages over mechanical heart valves: patients do not need to take anticoagulants for a long time after implantation of biological heart valves, and biological heart valves can be implanted using minimally invasive surgical methods. These advantages have made biological heart valves gradually become the mainstream of the market in clinical applications.
  • Glutaraldehyde cross-linking is still the mainstream method for current bioprosthetic valve products. Therefore, further modification of bioprosthetic valves based on glutaraldehyde cross-linking to improve their cross-linking degree and stability is of great significance to scientific research and the development of related industrial fields.
  • the Chinese invention patent application document with publication number CN 114748694A disclosed a co-crosslinked biological valve material and its preparation method and application, in which the biological valve material was functionally modified by introducing functional monomers for co-crosslinking during the crosslinking treatment; in the biological valve preparation method disclosed in the Chinese invention patent application documents with publication numbers CN 114748693A, CN114748697A, CN 114748696A and CN 114748695A, while adding functional monomers for co-crosslinking, carbon-carbon double bonds were introduced from the functional monomers as a further crosslinking basis, and the modification of the biological valve material was completed through two crosslinking.
  • the present application provides a method for preparing a biological valve material by double bond polymerization after aldehyde cross-linking, a biological valve material and its application.
  • functional monomers with carbon-carbon double bonds are introduced from the active groups on the glutaraldehyde cross-linked membrane, such as residual amino groups, hydroxyl groups, carboxyl groups, etc., to provide a controllable cross-linking opportunity and range for the glutaraldehyde cross-linked membrane.
  • a method for preparing a biological valve material by double bond polymerization after aldehyde cross-linking comprising:
  • Step S110 contacting the biomaterial with an aldehyde cross-linking agent solution for cross-linking
  • Step S120 soaking the biomaterial treated in step S110 in a solution containing a first functional monomer to chemically connect the first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and an ethylene oxide group;
  • Step S200 under the action of an initiator, the carbon-carbon double bonds are polymerized to obtain a biological valve material.
  • the aldehyde-based cross-linking agent is glutaraldehyde or formaldehyde.
  • 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 that has been decellularized.
  • step S200 adding an initiator to the system treated in the previous step; or washing the biological valve material treated in the previous step and then soaking it in a solution containing an initiator.
  • the initiator is a single initiator or a mixed initiator.
  • the mixed initiator is:
  • the single initiator is any component of each mixed initiator.
  • the double bond polymerization time is 3 to 24 hours.
  • the first functional monomer is selected from at least one of allyl glycidyl ether, glycidyl methacrylate and glycidyl acrylate.
  • step S110
  • the w/w concentration of the aldehyde cross-linking agent solution is 0.1% to 5%; the cross-linking time is 0.5h to 120h.
  • step S120
  • the w/w concentration of the first functional monomer in the solution containing the first functional monomer is 1% to 10%; and the reaction time is 2 to 120 hours.
  • the solution containing the first functional monomer only contains the first functional monomer and a solvent that does not participate in the chemical reaction.
  • the solvent in the solution containing the first functional monomer is one or more of an aqueous solution of any one of methanol, ethanol, ethylene glycol, propanol, 1,2-propylene glycol, 1,3-propylene glycol, isopropanol, butanol, isobutanol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and glycerol, water, physiological saline, and pH neutral buffer.
  • the present application also provides a biological valve material prepared by the preparation method.
  • the present application also provides a biological valve material, including:
  • Step S110 contacting the biomaterial with an aldehyde crosslinking agent solution for crosslinking
  • Step S120 Soak the biomaterial treated in step S110 in a solution containing a first functional monomer to chemically connect the first carbon-carbon double bond; the first functional monomer has a first carbon-carbon double bond and an ethylene oxide group;
  • Step S200 under the action of an initiator, the carbon-carbon double bonds are polymerized to obtain a biological valve material.
  • the present application also provides a biological valve, including a stent and a valve leaflet, wherein the valve leaflet is made of the biological valve material.
  • the biological valve is a heart valve.
  • the present application also provides an interventional system, including a heart valve and a catheter assembly, wherein the heart valve is folded and transported by the catheter assembly, and the heart valve includes a stent and leaflets, and the leaflets are made of the biological valve material.
  • the present invention has at least one of the following beneficial effects:
  • the method of the present application introduces double bonds as the basis for secondary cross-linking on the biological valve material cross-linked with glutaraldehyde through double bond modification, and further achieves secondary cross-linking by initiating polymerization of double bonds on the biological valve material cross-linked with glutaraldehyde, which can further increase the cross-linking degree of the biological valve material and thus improve the stability of the biological valve material.
  • the present application introduces double bonds into the glutaraldehyde cross-linked biological valve material, further initiating the polymerization of the double bonds, thereby improving the stability of the glutaraldehyde cross-linked material and further reducing the risk of calcification caused by structural degradation. Therefore, the material also has certain anti-calcification properties.
  • the functional monomers with carbon-carbon double bonds are chemically connected with the amino groups, hydroxyl groups and carboxyl groups on the surface of the glutaraldehyde crosslinking membrane through the ethylene oxide group, and the carbon-carbon double bonds are mainly connected to the surface of the biological valve material.
  • glutaraldehyde crosslinking modification of the biological valve material no other substances that can participate in the crosslinking reaction are added, which can better protect the original fiber structure of the biological material. While effectively ensuring the mechanical properties of the membrane, the orientation direction of the original fibers of the biological material can be guaranteed, avoiding the problem in the previous research that the direct addition of double-bond functional monomers during crosslinking may destroy the original fiber orientation of the biological material and increase the fiber disorder.
  • FIG1 is a process flow chart of a preferred embodiment of the present application.
  • FIG2 is a reaction principle diagram of a more preferred embodiment of the present application.
  • FIG3 is an Alizarin red staining result of control group 1 (glutaraldehyde cross-linked porcine pericardium) implanted subcutaneously in rats for 30 days;
  • FIG4 is an Alizarin red staining result of sample 1 of Example 1 after subcutaneous implantation in rats for 30 days;
  • FIG5 is an Alizarin red staining result of sample 5 of Example 5 after subcutaneous implantation in rats for 30 days;
  • FIG6 is an Alizarin red staining result of sample 7 of Example 7 after subcutaneous implantation in rats for 30 days;
  • FIG7 is a schematic diagram of the structure of the heart valve of the present application.
  • FIG8 is a schematic diagram of the structure of the intervention system of the present application.
  • Glutaraldehyde can improve the mechanical properties of the pericardium and reduce its immunogenicity to a certain extent.
  • the stability and cross-linking degree of glutaraldehyde-cross-linked biological valves are still relatively low, which will lead to the degradation of its components after implantation, causing its structure to be destroyed and structurally degraded.
  • the degradation of biological valve components will further induce mechanical damage and calcification, affecting the normal function of the valve and reducing its service life.
  • Glutaraldehyde cross-linking is still the mainstream method for current biological valve products. Therefore, further cross-linking and modification of biological valves on the basis of glutaraldehyde cross-linking to improve their cross-linking degree and stability is of great significance to scientific research and the development of related industrial fields.
  • the present application further introduces double bonds and initiates post-crosslinking on the basis of glutaraldehyde crosslinking, that is, the first carbon-carbon double bond is introduced by chemically bonding the first functional monomer (containing a first carbon-carbon double bond and an ethylene oxide group) to the glutaraldehyde crosslinked biological membrane on the basis of the glutaraldehyde crosslinked membrane.
  • the first functional monomer containing a first carbon-carbon double bond and an ethylene oxide group
  • step S200 brings the biological valve material treated in step S120 into contact with an initiator to initiate double bond polymerization.
  • the biomaterial is first subjected to a cross-linking reaction with an aldehyde cross-linking agent (S110), and then reacts with the active group of the first functional monomer to access the first carbon-carbon double bond (S120).
  • an aldehyde cross-linking agent is first added, and the aldehyde cross-linking agent first reacts with part of the amino groups of the biomaterial, and then the first functional monomer is added, and the remaining amino groups and other groups (such as hydroxyl and carboxyl groups) on the biomaterial are used to react with the active groups on the first functional group to directly access the first carbon-carbon double bond.
  • the first functional monomer also carries an oxirane group as an active group, which participates in the chemical reaction through the active group.
  • an oxirane group as an active group, which participates in the chemical reaction through the active group.
  • its hydroxyl and carboxyl groups can also react with the oxirane group to participate in the chemical reaction.
  • the first carbon-carbon double bond introduced by the chemical reaction is then polymerized under the action of an initiator to further form a cross-linked network, thereby improving the anti-coagulation, anti-calcification, elasticity and other properties of the biological valve cross-linked based on glutaraldehyde.
  • the double bond of the glutaraldehyde cross-linked biological valve material is further achieved by introducing a first carbon-carbon double bond using a first functional monomer, i.e., a double bond-forming agent.
  • the first functional monomer i.e., a double bond-forming agent, has both a first carbon-carbon double bond and an ethylene oxide group.
  • the glutaraldehyde cross-linked biological valve material is modified by using the first functional monomer, i.e., the double-bonding reagent, and the hydroxyl group, carboxyl group and a small amount of amino group remaining after glutaraldehyde cross-linking on the glutaraldehyde cross-linked biological valve material undergo a ring-opening reaction through the ethylene oxide group in the first functional monomer, i.e., the double-bonding reagent, and then directly introduce the first carbon-carbon double bond into the glutaraldehyde cross-linked biological valve material; further, the double bonds on the glutaraldehyde cross-linked biological valve material are polymerized to achieve secondary cross-linking, and the post-cross-linking treatment of the biological valve material is completed.
  • the cross-linking degree of the biological valve material after secondary cross-linking will be further improved, and its stability, mechanical properties and anti-
  • step S120 of the present application non-condensing chemical bonding is used to connect the first carbon-carbon double bond.
  • step S110 the biomaterial is not subjected to any other chemical reaction involving any reagents before being treated with the aldehyde cross-linking agent.
  • the first carbon-carbon double bond is provided by a first functional monomer having an active group
  • the reaction raw materials in steps S110 and S120 only include the biomaterial, the first functional monomer and the aldehyde cross-linking agent.
  • step S110
  • 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 selected from one of glutaraldehyde and formaldehyde.
  • the concentration of the glutaraldehyde solution is 0.1% to 5% (w/w); and the cross-linking time can be any time between 0.5h and 120h.
  • the biomaterial used in the present application is a conventional biomaterial in the existing glutaraldehyde cross-linking process, and the collagen content of the biomaterial is 60% to 90%.
  • the biomaterial is animal tissue, the animal source is pig, cattle, 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 that has been decellularized.
  • the biological tissue is treated with a surfactant as follows:
  • the ionic surfactant is mainly used for lysing cells, and the nonionic surfactant is mainly used for removing lipid substances (such as phospholipids).
  • the ionic surfactant is at least one of sodium deoxycholate, fatty acid potassium soap, sodium dodecyl sulfate, sodium cholate, hexadecyltrimethylammonium bromide, fatty acid potassium salt, and alkyldimethylsulfonpropyl betaine.
  • the nonionic surfactant is at least one of Triton and Tween.
  • step S120
  • the double-bonding agent ie, the first functional monomer
  • the double-bonding agent is selected from at least one of allyl glycidyl ether, glycidyl methacrylate and glycidyl acrylate.
  • the concentration of the double-bonding agent in the solution containing the first functional monomer, ie, the double-bonding agent is 1% to 10% (w/w); and the reaction time of the double-bonding modification is 2 to 120 hours.
  • the solvent in the solution containing the first functional monomer, i.e., the double-bonding agent is one or more of water, physiological saline, pH neutral buffer, or an aqueous solution of methanol, ethanol, ethylene glycol, propanol, 1,2-propylene glycol, 1,3-propylene glycol, isopropanol, butanol, isobutanol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, or glycerol.
  • the biofilm material treated by S110 is taken out and washed or directly placed in a solution containing a double-bonding agent (first functional monomer).
  • step S200
  • step S120 The biological valve material treated in step S120 is washed with deionized water and then immersed in an initiator solution for treatment in step S200 or an initiator is directly added to the reaction system in step S120 to initiate a polymerization reaction, the latter being commonly known as a one-pot method.
  • the solvent in the initiator-containing solution is water, physiological saline or pH neutral buffer.
  • the concentration of the initiator can be understood as the concentration of the initiator in the solution contained in the reaction system in step S120 in the one-pot method, and can be understood as the concentration of the initiator in the solution containing the initiator in the step-by-step method.
  • the initiator is a mixture of ammonium persulfate and sodium bisulfite, or a mixture of ammonium persulfate and sodium sulfite, or a mixture of sodium persulfate and sodium sulfite, or a mixture of potassium persulfate and sodium sulfite, or a mixture of sodium persulfate and sodium bisulfite, or a mixture of potassium persulfate and sodium bisulfite, or potassium persulfate and tetramethylethylenediamine, or ammonium persulfate and tetramethylethylenediamine, or sodium persulfate and tetramethylethylenediamine; the concentration of each component in the mixture is 1 to 100 mM, respectively.
  • the double bond polymerization time is preferably 3 to 24 hours.
  • reaction processes of S110, S120 and S200 can be carried out at 0-50°C unless otherwise specified.
  • the temperature does not need to be specially controlled and can be carried out at room temperature, preferably not exceeding the temperature adapted to the human body, and is preferably carried out at 36-37°C.
  • all reactions of S110, S120 and S200 can be either static reactions or dynamic reactions unless otherwise specified.
  • the dynamic reactions can be carried out under the action of a peristaltic pump or other equipment that can circulate the solution, or can be carried out by shaking at a speed of 10rpm-150rpm.
  • the peristaltic cycle or shaking time can be continuous or intermittent.
  • dehydration and drying treatments are also included after the double bond polymerization is completed to form a dry film.
  • the biological valve material is routinely cleaned and softened, and then dehydrated and dried.
  • the cleaning solution can be one or a mixture of water, physiological saline, ethanol, isopropanol or a pH neutral buffer solution.
  • the pH can be adjusted to between 5.0 and 9.5 before and during use, or it can be left unadjusted.
  • the dehydration treatment is to expose the membrane sheet after double bond polymerization or the valve sewn from the membrane sheet to a dehydration solution.
  • the dehydration solution is a mixed solution of an alcohol solution and water, the alcohol solution accounts for 20-90% (v/v), and the alcohol reagent can be ethanol, isopropanol, or a mixture of the two.
  • the drying treatment is to expose the dehydrated membrane or valve to a softener solution for a treatment time of 20 minutes to 10 hours.
  • the main component of the softener solution is a mixed solution of one or two of glycerol and polyethylene glycol, the glycerol concentration is 10-100% (v/v), and the other components are one or more of water, ethanol, and isopropanol, accounting for 0-90% (v/v).
  • valve after drying can be sterilized by ethylene oxide sterilization or electron beam sterilization.
  • the bioprosthetic valve material prepared by the above method can be used for interventional bioprosthetic valves, such as through minimally invasive intervention; it can also be used for surgical bioprosthetic valves, such as through surgical implantation.
  • an artificial heart valve including a stent 1 and leaflets 2 connected to the stent 1.
  • the stent is cylindrical as a whole, and the side walls are a hollow grid structure.
  • the interior of the stent is a blood flow channel, and the multiple leaflets cooperate with each other to control the degree of opening and closing of the blood flow channel in the stent.
  • the corresponding materials are selected during the processing of the stent, such as nickel-titanium alloy with shape memory that can self-expand in the body, or stainless steel that is released by ball expansion, etc.
  • the stent itself can be formed by cutting tubes or weaving wires, and the leaflets can be connected to the stent by sewing, bonding or integral mold molding.
  • a positioning structure that can interact with the surrounding native tissue, such as anchor spikes, arms, etc., can be provided on the periphery of the stent.
  • a skirt or peripheral leakage prevention material can be provided on the inner and/or outer sides of the stent.
  • the leaflets, skirts, or peripheral leakage prevention materials can all be made of the bioprosthetic valve materials of the above embodiments.
  • the artificial heart valve 3 and the corresponding delivery system constitute a valve intervention system.
  • the delivery system includes a catheter assembly 4 and a handle for controlling the catheter assembly.
  • the artificial heart valve is in a radially compressed state when delivered in the body, and the catheter assembly is released from its restraints or undergoes balloon expansion and radial expansion and release in the body.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100 RPM for 2 hours, then immersed in 0.30% (w/w) glutaraldehyde solution and cross-linked at room temperature and 100 RPM for 48 hours to obtain control sample 1.
  • freshly collected porcine pericardium is cleaned with distilled water at 4°C and 100 RPM for 2 hours, immersed in a 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water and immersed in a 5% (v/v) propanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification.
  • the reaction time was 72 hours, and the solvent of the double bond modification solution was 20% (v/v) propanol aqueous solution.
  • the double bond glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bond glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 20 mM potassium persulfate and 10 mM sodium bisulfite to further induce the polymerization reaction of the double bonds on the double bond glutaraldehyde cross-linked pig pericardium, and after reacting at 37°C for 8 hours, the double bond post-cross-linked pig pericardium is obtained, which is recorded as sample 1.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water and immersed in a 6% (v/v) isopropanol aqueous solution of glycidyl acrylate at room temperature for double bond modification of the glutaraldehyde cross-linked porcine pericardium.
  • the reaction time was 72 hours, and the solvent of the double bond modification solution used was 20% (v/v) isopropanol aqueous solution.
  • the double bond glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bond glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 20 mM ammonium persulfate and 5 mM sodium bisulfite to further induce the polymerization reaction of the double bonds on the double bond glutaraldehyde cross-linked pig pericardium, and after reacting at 37°C for 8 hours, the double bond post-cross-linked pig pericardium is obtained, which is recorded as sample 2.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in an isopropanol aqueous solution containing 4% (v/v) glyceryl acrylate and 4% (v/v) allyl glycidyl ether at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium.
  • the reaction time was 72 hours, and the solvent of the double bond modification solution was 30% (v/v) ethanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 20 mM ammonium persulfate and 10 mM sodium bisulfite to further induce the polymerization reaction of the double bonds on the double bonded glutaraldehyde cross-linked pig pericardium, and the double bond cross-linked pig pericardium is obtained after reacting at 37°C for 7 hours, which is recorded as sample 3 and numbered as GAGA-PP-3.
  • Freshly collected porcine pericardium was washed with distilled water for 2 hours at 4°C with 100RPM shaking, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde-crosslinked porcine pericardium was further washed with deionized water and immersed in an isopropanol aqueous solution of 5% (v/v) glycidyl methacrylate and 2% (v/v) glycidyl acrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium.
  • the reaction time was 72 hours, and the solvent of the double bond modification solution was 35% (v/v) isopropanol aqueous solution.
  • the double bond glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bond glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 20 mM sodium persulfate and 5 mM sodium bisulfite to further induce the polymerization reaction of the double bonds on the double bond glutaraldehyde cross-linked pig pericardium, and after reacting at 37°C for 8 hours, the double bond post-cross-linked pig pericardium is obtained, which is recorded as sample 4.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde cross-linked porcine pericardium was washed with deionized water and immersed in a 4% (v/v) ethanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification of the glutaraldehyde cross-linked porcine pericardium.
  • the reaction time was 72 hours, and the solvent of the double bond modification solution was 20% (v/v) ethanol aqueous solution.
  • ammonium persulfate and sodium bisulfite were added to initiate the polymerization reaction of the double bonds on the double-bonded glutaraldehyde cross-linked pig pericardium, wherein the concentration of ammonium persulfate was 20 mM and the concentration of sodium bisulfite was 5 mM; after adding the initiator and reacting at 37°C for 8 hours, the double-bond cross-linked pig pericardium was obtained, which was recorded as sample 5.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water and immersed in a 4% (v/v) isobutanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification of the glutaraldehyde cross-linked porcine pericardium.
  • the reaction time was 72 hours, and the solvent of the double bond modification solution was 15% (v/v) isobutanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 20 mM ammonium persulfate and 5 mM sodium bisulfite to further induce the polymerization reaction of the double bonds on the double bonded glutaraldehyde cross-linked pig pericardium. After reacting at 37°C for 8 hours, the double bond cross-linked pig pericardium is obtained, which is recorded as sample 6.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water and immersed in a 4% (v/v) isopropanol aqueous solution of glycidyl acrylate at room temperature for double bond modification of the glutaraldehyde cross-linked porcine pericardium.
  • the reaction time was 48 hours, and the solvent of the double bond modification solution was 20% (v/v) methanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 20 mM ammonium persulfate and 6.5 mM sodium sulfite to further induce the polymerization reaction of the double bonds on the double bonded glutaraldehyde cross-linked pig pericardium. After reacting at 37°C for 10 hours, the double bond cross-linked pig pericardium is obtained, which is recorded as sample 7.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water and immersed in a 4% (v/v) ethylene glycol aqueous solution of glycidyl methacrylate at room temperature for double bond modification.
  • the reaction time was 72 hours, and the solvent of the double bond modification solution was 20% (v/v) ethylene glycol aqueous solution.
  • the double bond glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bond glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 40 mM ammonium persulfate and 15 mM sodium bisulfite to further induce the polymerization reaction of the double bonds on the double bond glutaraldehyde cross-linked pig pericardium. After reacting at 37°C for 7 hours, the double bond post-cross-linked pig pericardium is obtained, which is recorded as sample 8.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water and immersed in a 7% (v/v) propanol aqueous solution of glycidyl acrylate at room temperature for double bond modification of the glutaraldehyde cross-linked porcine pericardium.
  • the reaction time was 60 hours, and the solvent of the double bond modification solution was 40% (v/v) propanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 30 mM sodium persulfate and 10 mM sodium bisulfite to further induce the polymerization reaction of the double bonds on the double bonded glutaraldehyde cross-linked pig pericardium. After reacting at 37°C for 8 hours, the double bond cross-linked pig pericardium is obtained, which is recorded as sample 9.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde-crosslinked porcine pericardium was immersed in an isopropanol aqueous solution containing 6% (v/v) glycidyl methacrylate and 3% (v/v) glycidyl acrylate at room temperature for double bond modification of the glutaraldehyde-crosslinked porcine pericardium.
  • the reaction time was 84 hours, and the solvent of the double bond modification solution used was 50% (v/v) ethanol aqueous solution.
  • the double bonded glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bonded glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 40 mM ammonium persulfate and 10 mM sodium sulfite to further induce the polymerization reaction of the double bonds on the double bonded glutaraldehyde cross-linked pig pericardium, and after reacting at 37°C for 12 hours, the double bond post-cross-linked pig pericardium is obtained, which is recorded as sample 10.
  • the thermal stability and cross-linking degree of the biological valve materials were characterized by measuring the thermal shrinkage temperature of the biological valve materials; the stability of the biological valve materials was characterized by an enzyme degradation experiment; and the calcification degree (anti-calcification performance) of the samples was characterized by a rat subcutaneous implantation experiment.
  • the bioprosthetic valve material was cut into circular sheets with a diameter of 0.6 cm, dried and placed in a crucible, and the thermal shrinkage temperature of the bioprosthetic valve material was measured at a heating rate of 10°C/min in the range of 40-120°C on a differential scanning calorimeter.
  • the thermal stability and cross-linking degree of the bioprosthetic valve material were characterized by measuring the thermal shrinkage temperature; the higher the thermal shrinkage temperature, the higher the corresponding thermal stability and cross-linking degree.
  • the thermal shrinkage temperature of Example 1, Example 2, Example 9, Example 10 and Control Group 1 was measured and it was found that: as shown in Table 1, the thermal shrinkage temperatures of Example 1, Example 2, Example 9, and Example 10 were all higher than that of Control Group 1 (glutaraldehyde cross-linked porcine pericardium), that is, the thermal stability and cross-linking degree of Example 1, Example 2, Example 9, and Example 10 were all higher than that of Control Group 1 (glutaraldehyde cross-linked porcine pericardium).
  • the results of the thermal shrinkage temperature measurement experiment show that the method of preparing biological valve materials by double bond post-cross-linking of the present application can improve the thermal stability and cross-linking degree of biological valves.
  • Samples 3, 6, 10 and control group 1 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 at minus 80°C overnight and then transferred to a vacuum freeze dryer for freeze drying for 48 hours. The weight of each sample was weighed on a 1/100,000 balance and recorded as the initial weight (W0) and returned to the 48-well plate.
  • Control group 1 (glutaraldehyde cross-linked pig pericardium) 7.45 ⁇ 1.33
  • Sample 3 5.31 ⁇ 0.30
  • Sample 4 4.47 ⁇ 1.05
  • Sample 6 5.12 ⁇ 0.97
  • control group 1 (glutaraldehyde cross-linked porcine pericardium), sample 3, sample 4, sample 6, and sample 10 were subjected to enzyme degradation experiments to characterize the cross-linking efficiency of each group of samples.
  • the enzyme degradation weight loss rate of each group of samples was calculated after treating the control group 1 (glutaraldehyde cross-linked porcine pericardium), sample 3, sample 4, sample 6, and sample 10 with collagenase I as shown in Table 2.
  • the enzyme degradation weight loss rates of sample 3, sample 4, sample 6, and sample 10 were all lower than those of the control group (glutaraldehyde cross-linked porcine pericardium), which indicates that the stability of sample 3, sample 4, sample 6, and sample 10 was higher than that of the control group (glutaraldehyde cross-linked porcine pericardium), that is, sample 3, sample 4, sample 6, and sample 10 were higher in stability.
  • the results of the enzyme degradation experiment show that the method for preparing biological valve materials by double bond post-cross-linking of the present application can improve the stability of biological valves.
  • the sample bioprosthetic valve material was cut into sheets of 0.8 ⁇ 0.8cm2 , sterilized and implanted into rats' subcutaneous tissues, and then removed after 30 days. Each sample was divided into two parts. One part was freeze-dried and weighed after removing the capsule, and the calcium content per gram of the sample was determined after digestion with 6M hydrochloric acid; the other part of the sample was fixed with paraformaldehyde tissue fixative. After fixation, it was taken out and trimmed with a scalpel and transferred to a dehydration box. The material sample was dehydrated with gradient ethanol. After dehydration, the material sample was transferred to an embedding machine for embedding with melted paraffin, and then transferred to a -20°C refrigerator for cooling and trimming.
  • 5 ⁇ m thick slices were cut from the trimmed wax block on a slicer, transferred from the spreader to a glass slide, and dewaxed and rehydrated.
  • the slices were stained with alizarin red dye for 3 minutes, washed with water, dried, and then permeabilized with xylene for 5 minutes.
  • the slices were sealed with neutral gum and the staining result images were collected on a pathological slice scanner.
  • Control group 1 (glutaraldehyde cross-linked pig pericardium) 74.9 ⁇ 12.3 Sample 1 15.1 ⁇ 4.7 Sample 5 8.4 ⁇ 4.6 Sample 7 12.7 ⁇ 5.1
  • Samples 1, 5, 7 and control group 1 were implanted into the subcutaneous tissue of rats for 30 days and then removed and fixed with paraformaldehyde tissue fixative. After fixation, the samples were removed and trimmed with a scalpel and then transferred to a dehydration box. The material samples were dehydrated in a gradient manner using 50%, 75%, 85%, 95% (v/v) and anhydrous 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 for cooling and trimming. 3-5 ⁇ m thick slices were cut from the trimmed wax block on a microtome, transferred from the slide spreader to a glass slide, and dewaxed and rehydrated. The slices were stained with alizarin red dye for 3 minutes, washed with water, dried, and then permeabilized with xylene for 5 minutes. The slices were sealed with neutral gum and the staining result images were collected on a pathology slice scanner.
  • control group 1 (glutaraldehyde cross-linked pig pericardium), sample 1, sample 5, and sample 7 were directly observed for the degree of calcification of each group of samples by alizarin red staining after 30 days of subcutaneous implantation in rats.
  • the alizarin red staining results of the sample slices implanted in rats for 30 days are shown in Figures 3 to 6, wherein the darker the color of the sample after alizarin red staining, the higher the degree of calcification.
  • the alizarin red staining images of the slices of sample 1 are obviously lighter and lighter, which directly indicates that the degree of calcification of sample 1, sample 5, and sample 7 is lower than that of the control group 1, that is, sample 1, sample 5, and sample 7 have a stronger anti-calcification effect than that of the control group 1.
  • the alizarin red staining results of the biological valve material implanted in rats for 30 days show that the method of preparing biological valve materials by double bond post-crosslinking of the present application can improve the anti-calcification performance of biological valves.
  • Freshly collected porcine pericardium was washed with distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde cross-linked porcine pericardium was washed with deionized water and immersed in a 4% (v/v) ethanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification of the glutaraldehyde cross-linked porcine pericardium.
  • the reaction time was 72 hours, and the solvent of the double bond modification solution was 20% (v/v) ethanol aqueous solution.
  • ammonium persulfate and sodium bisulfite are added to initiate the polymerization reaction of the double bonds on the double-bonded glutaraldehyde cross-linked pig pericardium, wherein the concentration of ammonium persulfate is 20mM and the concentration of sodium bisulfite is 5mM; after adding the initiator and reacting at 37°C for 8 hours, the double-bond post-cross-linked pig pericardium is obtained.
  • the pig pericardium material cross-linked after double bond copolymerization was placed in a 70% ethanol aqueous solution and soaked for 20 minutes, and then placed in a drying solution (80% glycerol, 2% water, 18% ethanol) and soaked for 1.5 hours at room temperature. The excess glycerol on the surface of the pig pericardium material was removed, and the material was sterilized with ethylene oxide, and recorded as sample 31.
  • Fresh porcine pericardium was placed in a PS solution containing 0.5% sodium deoxycholate (surfactant) by mass, shaken for 4 hours at room temperature, and then washed three times with a 0.9% sodium chloride aqueous solution (ie, normal saline).
  • a PS solution containing 0.5% sodium deoxycholate (surfactant) by mass, shaken for 4 hours at room temperature, and then washed three times with a 0.9% sodium chloride aqueous solution (ie, normal saline).
  • the cleaned porcine pericardium was cleaned in distilled water at 4°C and 100RPM for 2 hours, immersed in 0.30% (w/w) glutaraldehyde solution at room temperature, and immersed at room temperature for 48 hours to perform glutaraldehyde cross-linking treatment on the biological valve to obtain glutaraldehyde cross-linked porcine pericardium.
  • the glutaraldehyde cross-linked porcine pericardium was further washed with deionized water and immersed in a 5% (v/v) propanol aqueous solution of glycidyl methacrylate at room temperature for double bond modification.
  • the reaction time was 72 hours, and the solvent of the double bond modification solution was 20% (v/v) propanol aqueous solution.
  • the double bond glutaraldehyde cross-linked pig pericardium is washed with deionized water; then the double bond glutaraldehyde cross-linked pig pericardium is immersed in a mixture of 20 mM potassium persulfate and 10 mM sodium bisulfite to further induce the polymerization reaction of the double bonds on the double bond glutaraldehyde cross-linked pig pericardium, and after reacting at 37°C for 8 hours, the double bond post-cross-linked pig pericardium is obtained, which is recorded as sample 34.

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Abstract

一种醛基交联后双键聚合制备生物瓣膜材料的方法及生物瓣膜材料和应用，制备方法包括：步骤S110，将生物材料与醛基交联剂溶液接触进行交联；步骤S120，将步骤S110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。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能力。

Description

醛基交联后双键聚合制备生物瓣膜材料的方法及生物瓣膜材料和应用 技术领域
本申请涉及介入材料技术领域，具体涉及一种醛基交联后双键聚合制备生物瓣膜材料的方法及生物瓣膜材料和应用。
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。另一方面，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。
本申请的申请人长期致力于生物心脏瓣膜的研究，例如在前期的研究中，公开号为CN 114748694A的中国发明专利申请文献公开了一种共交联生物瓣膜材料及其制备方法和应用，在交联处理同时通过引入功能单体共交联对生物瓣膜材料进行功能修饰处理；公开号为CN 114748693A、CN114748697A、CN 114748696A和CN 114748695A的中国发明专利申请文献公开的生物瓣膜制备方法中，在加入功能单体共交联的同时由功能单体引入碳碳双键，作为进一步的交联基础，通过两次交联完成生物瓣膜材料的改性。
在如前所述的研究中，不管是通过戊二醛交联的同时引入功能单体进行共交联改性，还是共交联过程中还引入碳碳双键作为进一步交联的基础，都是对戊二醛交联过程中引入新的改性物质参与交联反应。
发明内容
本申请提供一种醛基交联后双键聚合制备生物瓣膜材料的方法及生物瓣膜材料和应用，在戊二醛交联后，由戊二醛交联膜片上的活性基团例如残余氨基、羟基、羧基等引入带有碳碳双键的功能单体，为戊二醛交联膜片重新提供一个可控的交联机会与范围。
一种醛基交联后双键聚合制备生物瓣膜材料的方法，包括：
步骤S110将生物材料与醛基交联剂溶液接触进行交联；
步骤S120将步骤S110处理后的生物材料浸泡于含第一功能单体的溶液中，化学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。
可选的，所述醛基交联剂为戊二醛或甲醛。
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。
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瓣膜材料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。
可选的，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，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，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S200，所述双键聚合的时间为3～24h。
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S110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S120中：
所述含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，所述含第一功能单体的溶液中仅包含第一功能单体和不参与化学反应的溶剂。
可选的，所述含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，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S110 将生物材料与醛基交联剂溶液接触进行交联；
步骤S120 将步骤S110处理后的生物材料浸泡于含第一功能单体的溶液中，化学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，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系统，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)本申请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。
(3)相对于申请人前期研究的戊二醛改性过程中通过加入功能单体进行共交联引入碳碳双键的改性方法，本申请的生物瓣膜材料改性过程中，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，在戊二醛交联改性生物瓣膜材料的过程中没有其他可参与交联反应的物质加入，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，避免前期研究中交联同时直接加入双键功能单体可能破坏生物材料原始纤维取向及增加纤维混乱度的问题。
附图说明
图1为本申请的一种较优选实施方式的工艺流程图；
图2为本申请的一种较优选实施方案的反应原理图；
图3为对照组1(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4为实施例1的样品1在大鼠皮下植入30天后的茜素红染色结果图；
图5为实施例5的样品5在大鼠皮下植入30天后的茜素红染色结果图；
图6为实施例7的样品7在大鼠皮下植入30天后的茜素红染色结果图；
图7为本申请心脏瓣膜的结构示意图；
图8为本申请介入系统的结构示意图。
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。
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键合引入第一碳碳双键，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。
一种实施方式中，具体包括(参见图1)：
S110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；
S120将步骤S110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S120处理后的生物瓣膜材料与引发剂接触，引发双键聚合。
本申请中，所述生物材料先与醛基交联剂进行交联反应(S110)，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(S120)。制备过程中，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，所述第一功能单体还带有为环氧乙烷基作为活性基团，通过该活性基团参与所述化学反应，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。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2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。
在戊二醛交联后再引入碳碳双键，碳碳双键主要接入生物瓣膜材料的表面，在戊二醛交联改性生物瓣膜材料的过程中没有其他可参与交联反应的物质加入，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，避免前期研究中交联同时直接加入双键功能单体可能破坏生物材料原始纤维取向及增加纤维混乱度的问题。
可选的，本申请的步骤S120中采用非缩合的化学键合接入所述第一碳碳双键。
可选的，步骤S110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S120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S110和S120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。
步骤S110中：
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，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。
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。
步骤S120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中的至少一种。
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。
步骤S200中：
步骤S120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S120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S120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。
本申请中，S110、S120和S200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。
本申请中，S110、S120和S200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。
本申请中，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7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8，采用导管介入时，人工心脏瓣膜3与相应的输送系统组成瓣膜介入系统，输送系统包括导管组件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：
对照组1
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1。
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，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1。
实施例2
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2。
实施例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，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3，编号为GAGA-PP-3。
实施例4
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，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，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，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，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8。
实施例9
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9。
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，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10。
对实施例1～10及对照组1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1各组样品的热收缩温度
样品 热收缩温度(℃)
对照组1(戊二醛交联猪心包) 84.7
实施例1 88.9
实施例2 89.3
实施例9 91.5
实施例10 92.0
对实施例1、实施例2、实施例9、实施例10和对照组1(戊二醛交联猪心包)进行热收缩温度测定发现：如表1所示，实施例1、实施例2、实施例9、实施例10的热收缩温度均高于对照组1(戊二醛交联猪心包)，即实施例1、实施例2、实施例9、实施例10的热稳定性和交联度均高于对照组1(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
将样品3、样品6、样品10和对照组1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 PCTCN2022132870-appb-000001
结果如表2所示：
表2各组样品的酶降解失重率
样品 酶降解失重率(％)
对照组1(戊二醛交联猪心包) 7.45±1.33
样品3 5.31±0.30
样品4 4.47±1.05
样品6 5.12±0.97
样品10 3.06±0.59
对对照组1(戊二醛交联猪心包)、样品3、样品4、样品6、样品10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1(戊二醛交联猪心包)、样品3、样品4、样品6和样品10后计算各组样品的酶降解失重率如表2所示。样品3、样品4、样品6、样品10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3、样品4、样品6、样品10的稳定性均高于对照组(戊二醛交联猪心包)，即样品3、样品4、样品6、样品10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
将样品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3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1(戊二醛交联猪心包) 74.9±12.3
样品1 15.1±4.7
样品5 8.4±4.6
样品7 12.7±5.1
通过对植入到大鼠皮下30天后的样品1、样品5、样品7和对照组1(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3所示，样品1、样品5、样品7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。
茜素红染色实验：
将样品1、样品5、样品7和对照组1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天后的对照组1(戊二醛交联猪心包)、样品1、样品5、样品7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3～6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1(戊二醛交联猪心包)切片的茜素红染色结果(图3)，样品1(图4)、样品5(图5)、样品7(图6)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1、样品5、样品7的钙化程度低于对照组1，即样品1、样品5、样品7相比于对照组1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。
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，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31。
实施例12
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PS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。
清洗后的猪心包于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34。
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申请专利范围的限制。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 (19)

  1. 一种醛基交联后双键聚合制备生物瓣膜材料的方法，其特征在于，包括：
    步骤S110 将生物材料与醛基交联剂溶液接触进行交联；
    步骤S120 将步骤S110处理后的生物材料浸泡于含第一功能单体的溶液中，化学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。
  2. 根据权利要求1所述的方法，其特征在于，所述醛基交联剂为戊二醛或甲醛。
  3. 根据权利要求1所述的方法，其特征在于，所述生物材料为动物组织，所述动物组织选自心包膜、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、直接或经清洗后再浸泡于含引发剂的溶液中。
  6. 根据权利要求1所述的方法，其特征在于，所述引发剂为单一引发剂或混合引发剂。
  7. 根据权利要求6所述的方法，其特征在于，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，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。
  8. 根据权利要求7所述的方法，其特征在于，所述单一引发剂为各混合引发剂中的任一组分。
  9. 根据权利要求1所述的方法，其特征在于，步骤S200中，所述双键聚合的时间为3～24h。
  10. 根据权利要求1所述的方法，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。
  11. 根据权利要求1所述的方法，其特征在于，步骤S110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。
  12. 根据权利要求1所述的方法，其特征在于，步骤S120中：
    所述含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。
  13. 根据权利要求1所述的方法，其特征在于，所述含第一功能单体的溶液中仅包含第一功能单体和不参与化学反应的溶剂。
  14. 根据权利要求1所述的方法，其特征在于，所述含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。
  15. 一种生物瓣膜材料，其特征在于，由权利要求1～14任一项权利要求所述的方法制备得到。
  16. 一种生物瓣膜材料，其特征在于，包括：
    步骤S110 将生物材料与醛基交联剂溶液接触进行交联；
    步骤S120 将步骤S110处理后的生物材料浸泡于含第一功能单体的溶液中，化学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。
  17. 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15或16所述的生物瓣膜材料。
  18. 根据权利要求17所述的生物瓣膜，其特征在于，所述生物瓣膜为心脏瓣膜。
  19. 一种介入系统，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15或16所述的生物瓣膜材料。
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