WO2022057841A1 - 一种人工生物心脏瓣膜及其制备方法 - Google Patents

一种人工生物心脏瓣膜及其制备方法 Download PDF

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WO2022057841A1
WO2022057841A1 PCT/CN2021/118617 CN2021118617W WO2022057841A1 WO 2022057841 A1 WO2022057841 A1 WO 2022057841A1 CN 2021118617 W CN2021118617 W CN 2021118617W WO 2022057841 A1 WO2022057841 A1 WO 2022057841A1
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heart valve
biological
tissue
preparing
valve according
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PCT/CN2021/118617
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English (en)
French (fr)
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周仲毅
许良鹏
王云兵
郭高阳
何海洋
雷洋
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吉林启明皓月生物科技有限公司
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Publication of WO2022057841A1 publication Critical patent/WO2022057841A1/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
    • A61L27/36—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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3625—Vascular tissue, e.g. heart valves
    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • 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
    • 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
    • A61L27/52—Hydrogels or hydrocolloids
    • 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
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
    • C08F222/04—Anhydrides, e.g. cyclic anhydrides
    • 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
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
    • C08F222/36—Amides or imides
    • C08F222/38—Amides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2335/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Derivatives of such polymers

Definitions

  • the invention relates to the technical field of biomedical materials and medical devices, in particular to an artificial biological heart valve and a preparation method thereof.
  • valvular heart disease With the continuous development of the aging process and the continuous increase of the world population, the number of valvular diseases continues to rise, which seriously threatens the life of patients and affects the quality of life of patients.
  • the most effective treatment method is artificial heart valve replacement.
  • Transcatheter aortic valve implantation has completely changed the operation procedure of artificial biological heart valve replacement. It shortens the operation time, reduces the risks caused by anesthesia and blood transfusion, shortens the recovery time of patients, and also reduces the cost of treatment. Since the first interventional valve replacement was performed in 2002, more than 250,000 transcatheter replacement aortic valves have been implanted worldwide.
  • interventional valve replacement has the advantages of minimally invasive, simple operation and short postoperative recovery time
  • almost all biological tissues used in clinical interventional biological heart valves use traditional glutaraldehyde cross-linking technology.
  • Clinical data in recent decades have shown that glutaraldehyde cross-linked tissue is prone to structural deterioration of the valve during use, which will eventually lead to the failure of the biological valve.
  • a common manifestation of structural deterioration of the valve is leaflet tear, which may be caused by prolonged exposure of the leaflets to stress, resulting in a decrease in the mechanical properties of the valve tissue.
  • the currently used glutaraldehyde cross-linked tissue must always be stored in the glutaraldehyde solution during the production process, including cross-linking, sterilization, preservation and transportation, and there is a risk of leakage of the glutaraldehyde liquid.
  • the heart valve Before surgical implantation, the heart valve must be pre-flushed several times and compressed into a delivery catheter by a doctor using a special device. This procedure increases the risk of bacterial infection, prolongs the operation time, and carries the risk of incomplete irrigation and residual glutaraldehyde toxicity.
  • the wet glutaraldehyde cross-linked structure will become hard and brittle after dehydration, and the fibers will be torn and broken during crimping, which further affects its mechanical properties and durability.
  • a method of drying biological tissue using polyols has been reported in the prior art, but the obtained tissue has insufficient elasticity and toughness, and permanent deformation will occur during pressing. Therefore, biomaterials used for pre-installation of interventional heart valves must be able to resist loading stress in the dry state, and be able to return to the deployed state after rehydration without fiber damage. Based on this requirement, the prior art needs to be further improved.
  • a kind of artificial biological heart valve and preparation method thereof are provided. It has good elasticity and flexibility, will not be permanently deformed under long-term stress conditions, and can restore its original shape when placed in water.
  • valve leaflet of the artificial biological heart valve is a dry biological valve tissue filled with hydrogel; in the gap.
  • Drying means that the moisture content of the bioprosthesis is lower than the moisture content of the bioprosthesis when it is fully hydrated.
  • the in-situ copolymerization cross-linking adopted in the present invention is to realize the cross-linking of the biological tissue by introducing double bonds on the surface of the biological tissue, and a polymerization reaction occurs between the double bonds under the action of an initiator.
  • a hydrogel with a three-dimensional network is formed by the polymerization of hydrophilic monomers under the action of an initiator, so that water can be dispersed and retained in the network.
  • the hydrogel is dispersed in the voids of biological tissues, has a certain mechanical strength, can disperse and absorb stress, and enhance the mechanical strength and elasticity of the biological valve.
  • the biological valve tissue is porcine pericardium or bovine pericardium.
  • the leaflet has a moisture content of less than 30% based on the weight of the treated tissue.
  • preparation method of described artificial biological heart valve is characterized in that, comprises the following steps:
  • the biological valve tissue is immersed in a hydrophilic monomer solution for modification to obtain a modified biological tissue;
  • the biological tissue obtained after the above-mentioned d treatment is induced to polymerize by light, and then dehydrated and dried to obtain it.
  • both the hydrophilic monomer and the hydrogel monomer contain carbon-carbon double bonds.
  • step a the biological valve tissue is modified with the hydrophilic monomer, and carbon-carbon double bonds that can undergo subsequent reactions are introduced into the biological valve tissue.
  • step b the modified biological tissue is soaked in the hydrogel monomer solution, so that the hydrogel monomer is distributed in the space of the modified biological tissue, in step c, under the action of an initiator, The following two reactions occur simultaneously:
  • a polymerization reaction occurs between the carbon-carbon double bond introduced by the modified hydrophilic monomer and the double bond of the hydrogel monomer in the biological valve tissue;
  • step d a solution containing a sulfhydryl compound is placed in the treated biological tissue, and the sulfhydryl compound is used to remove the unsaturated groups and initiators remaining in the reaction.
  • the initiator-containing solution also contains dopamine and EGCG.
  • the dopamine and EGCG can generate non-covalent bonding force with the side chain of the hydrogel monomer, increase the degree of cross-linking of the hydrogel, and further improve the mechanical properties and elasticity.
  • the initiator is ammonium persulfate and sodium bisulfite, and the concentration of the initiator is 20-80 mM.
  • volume ratio of described ammonium persulfate and sodium bisulfite is 1:1.
  • the initiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and the concentration of the initiator is 0.1-1wt%.
  • the concentration of dopamine is 0.05-0.2 mg/mL.
  • the concentration of EGCG is 0.05-0.2 mg/mL.
  • the solvent is PBS buffer.
  • the preparation method of above-mentioned artificial biological heart valve comprises the following steps:
  • the biological valve tissue is immersed in a hydrophilic monomer solution for modification to obtain a modified biological tissue;
  • the biological tissue obtained after the above-mentioned d treatment is induced to polymerize by light, and then dehydrated and dried to obtain it.
  • hydrophilic monomer is methacrylic anhydride.
  • the weight ratio of the hydrophilic monomer to the biological valve tissue is 1-2:1.
  • hydrophilic monomer reacts with the biological valve tissue at room temperature for 22-26 hours.
  • hydrophilic monomer reacts with the biological valve tissue at pH 7.
  • hydrogel monomer is N-acryloylglyceramide monomer.
  • hydrophilic monomer is methacrylic anhydride
  • hydrogel monomer is N-acryloylglyceramide monomer
  • hydrogel monomer is N-acryloylglyceride monomer, and the concentration is 20-40wt%.
  • the initiator is ammonium persulfate and sodium bisulfite, and the concentration of the initiator is 20-80 mM.
  • volume ratio of described ammonium persulfate and sodium bisulfite is 1:1.
  • the initiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and the concentration of the initiator is 0.1-1wt%.
  • the concentration of dopamine is 0.05-0.2 mg/mL.
  • the concentration of EGCG is 0.05-0.2 mg/mL.
  • the solvent is PBS buffer.
  • hydrophilic monomer is methacrylic anhydride; the weight ratio of the hydrophilic monomer to the biological valve tissue is 1-2:1.
  • the above modification process is specifically as follows: soaking the biological valve tissue in deionized water, continuously adding methacrylic anhydride monomer dropwise, so that the final weight ratio of methacrylic anhydride monomer and biological valve tissue is 1:1, and adjusting The pH of the solution was around 5-9, and the reaction was allowed to stand for 24h.
  • the hydrogel monomer is N-acryloylglyceride monomer, and the concentration is 20-40wt%, preferably 30wt%.
  • the mixed solution of initiator, dopamine and EGCG is containing 20-80mM ammonium persulfate and sodium bisulfite or 0.1-1wt% 2-hydroxy-4'-(2-hydroxyethoxy)-2-methyl Propiophenone, and a mixed solution of 0.05-0.2 mg/mL dopamine and 0.05-0.2 mg/mL EGCG in PBS.
  • the mixed solution of initiator, dopamine and EGCG is containing 50mM ammonium persulfate and sodium bisulfite or 0.5wt% 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and a mixed solution of 0.1 mg/mL dopamine and 0.1 mg/mL EGCG in PBS; wherein, the volume ratio of ammonium persulfate and sodium bisulfite is 1:1.
  • the thiol compound is at least one of mercaptoethanol, cysteine and glutathione. Used to remove unsaturated groups and initiators remaining in the reaction.
  • the photo-initiated polymerization is specifically as follows: the polymerization is initiated by irradiation of ultraviolet light for 20-40 min under nitrogen protection.
  • the biological tissue is in a flattened state when the polymerization is initiated by light.
  • the dehydration is specifically as follows: the first step of dehydration is performed by soaking in a glycerol/ethanol solution, and then the second dehydration is performed in an environment with a humidity of 30-40%; the moisture content of the final obtained valve leaflet is less than 30%, based on the treated tissue. the weight of.
  • the concentration of the glycerol/ethanol solution was 50 vt%.
  • the dehydration time of the first step is not less than 24h.
  • the second step of dehydration is carried out at room temperature.
  • the biological valve is prepared by cross-linking and compounding with the hydrogel by in-situ copolymerization, and then performing moderate dehydration.
  • First by modifying double bonds on the surface of biological tissues, then introducing hydrophilic monomers into the voids of biological tissues, and then adding initiators to initiate the cross-linking reaction inside the biological tissues and polymerize the hydrophilic monomers to form a three-dimensional network structure.
  • the hydrogel is further dried and dehydrated by the glycerol/ethanol solution, which greatly improves the biocompatibility and mechanical properties of the bioprosthetic valve tissue, and can be quickly rehydrated and recovered after re-implantation in the human body. to the shape before crimping, so that it can be used for the manufacture of pre-assembled interventional biological valves;
  • the in-situ copolymerization and cross-linking method used in the present invention avoids the aldehyde residue caused by the traditional glutaraldehyde cross-linking method, enhances the anti-calcification performance and blood compatibility of the valve, and is expected to prolong the service life of the biological valve.
  • the tissue obtained by the invention has strong mechanical properties and tear resistance in wet state, can reduce the occurrence of micro-tears, and is expected to improve the durability of the interventional biological valve;
  • the artificial biological heart valve tissue obtained in the present invention has good elasticity and the ability to resist deformation, so it can be crimped and equipped on the catheter delivery system in a dry state for a long time, and the valve tissue can be quickly rehydrated and unfolded after being released in the human body.
  • the hydrogel compounded in the biological valve tissue of the present invention can improve the blood compatibility of the glutaraldehyde cross-linked valve and reduce the immune response;
  • the present invention uses hydrogel to fill the space between fibers in the biological valve tissue. On the one hand, it can enhance the stress dispersing ability of the valve and provide excellent tear resistance and elasticity. On the other hand, the addition of hydrogel can speed up the drying of the valve. The rate of rehydration at the time of hydration, thereby facilitating the return of the valve to its pre-crimping shape after implantation.
  • Figure 1 shows the recovery of Glut-PP tissue in phosphate buffered saline solution after simulated compression
  • Figure 2 shows the recovery of pNAGA/RPC-PP tissue in phosphate-buffered saline solution after simulated crimping
  • Figure 3 shows the platelet adhesion in Glut-PP tissue
  • Figure 4 shows the platelet recovery in pNAGA/RPC-PP tissue
  • Figure 5 is a schematic diagram of the preparation process.
  • a preferred embodiment of the present invention provides a method for preparing an artificial biological heart valve, and the specific steps are as follows:
  • the polymerized tissue is thoroughly washed with deionized water and added to a solution containing mercaptoethanol to remove residual unsaturated groups and initiators from the reaction.
  • the artificial biological heart valve prepared in this example is named pNAGA/RPC-PP.
  • a preferred embodiment of the present invention provides a method for preparing an artificial biological heart valve, and the specific steps are as follows:
  • the polymerized tissue is thoroughly washed with deionized water and added to a solution containing mercaptoethanol to remove residual unsaturated groups and initiators from the reaction.
  • a preferred embodiment of the present invention provides a method for preparing an artificial biological heart valve, and the specific steps are as follows:
  • the polymerized tissue is thoroughly washed with deionized water and added to a solution containing mercaptoethanol to remove residual unsaturated groups and initiators from the reaction.
  • a preferred embodiment of the present invention provides a method for preparing an artificial biological heart valve, and the specific steps are as follows:
  • the polymerized tissue is thoroughly washed with deionized water and added to a solution containing mercaptoethanol to remove residual unsaturated groups and initiators from the reaction.
  • a control group 1 was set up, and the products prepared in Example 1 were subjected to a compression grip simulation experiment, a mechanical property test, a blood compatibility evaluation, and an anti-calcification performance test.
  • Control group 1 The porcine pericardium was immersed in 0.5vt% glutaraldehyde PBS solution for 24h cross-linking. Then soak in 5vt0% glycerol/ethanol solution for 24 hours for dehydration, and then take out the biological tissue and spread it on a plastic plate for drying. The drying temperature is room temperature and the humidity is controlled at 30-40%. The resulting valve was named Glut-PP.
  • test group samples and the control group samples were cut into squares of 3*3cm 2 respectively, curled at will, put into a polypropylene tube with an inner diameter of 5 mm and sealed, and after sterilization with ethylene oxide, the polypropylene tube was kept at 37 ° C. After 10 days in the incubator, the biological tissue was removed from the tube and completely submerged in phosphate buffered saline to observe its unfolding.
  • Figure 1 shows the recovery of the control group in a phosphate buffered saline solution after simulated pressing. It can be observed that the non-aqueous gel biological tissue cross-linked by glutaraldehyde cannot be unfolded, while in Figure 2, the samples of the test group can be fully unfolded . This indicates that the introduction of hydrogel has a good resistance to the stress generated by the pericardial tissue during the crimping and crimping process, and can significantly improve the ability of the valve to resist deformation in a dry state.
  • test group and the samples in the control group were cut into rectangular samples respectively, and the mechanical properties were tested with a universal testing machine at room temperature, and their ultimate tensile strength, shear strength and tear strength were analyzed.
  • the samples of the experimental group and the samples of the control group were cut into disk samples with a diameter of 6 mm, soaked in platelet-rich plasma, incubated at 37 °C for 1 hour, and then rinsed with phosphate buffered saline three times to remove unadhered platelets. Then the samples were fixed with 4% paraformaldehyde, dehydrated with 30%, 50%, 80%, 100% ethanol gradient, freeze-dried, sprayed with gold, and the platelet adhesion was observed by scanning electron microscope.
  • Figure 3 shows the platelet adhesion in the control group, and a large number of platelets adhered to the surface of the control rough material. On the contrary, in Fig. 4, there are almost no platelets on the surface of the materials in the test group.
  • test group The samples of the test group and the samples of the control group were cleaned and cut into 1 cm ⁇ 1 cm. Intraperitoneal injection of 3% sodium pentobarbital 0.1 into juvenile SD rats about 20 days old Anesthesia was performed, the fur on the muscles on both sides of the spine was shaved, and iodine and alcohol were routinely disinfected.
  • One sample of the test group was implanted subcutaneously on the right back, and one sample of the control group was implanted subcutaneously on the left back, and the skin incision was sutured. After 30 days, the animals were euthanized by cervical dislocation and the grafts were removed. The host tissue on the graft surface was carefully removed and rinsed with saline.
  • the dry weight was weighed, and then digested with 6N concentrated hydrochloric acid in a water bath at 90 degrees Celsius until there were no visible solid particles, and then the quantitative analysis of calcium was carried out with an inductively coupled plasma emission spectrometer.
  • the dry biological valve tissue prepared by cross-linking and compounding with hydrogel in the present invention has good anti-calcification performance and blood compatibility, and thus the service life can be prolonged.
  • the biological tissue has good tear resistance and elastic properties, can be pressed and held in the delivery system for a long time in a dry state without structural damage, and can be quickly rehydrated to restore the original shape when immersed in a hydrochloric acid buffered saline solution. , which can be used in the manufacture of prefabricated interventional biological heart valves.

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Abstract

一种人工生物心脏瓣膜及其制备方法，制备方法如下：将异种生物组织通过原位共聚的方式交联并与水凝胶复合，然后将所得到的生物组织进行适度脱水得到干燥的生物组织；该生物组织在湿态时具有较强的机械强度和抗撕裂性能，在干燥状态下具有较好的弹性和柔韧性，在长期应力条件下不会发生永久变形，在放入水中可以恢复原有形状，因此可以在生产时以干燥状态预装在瓣膜输送系统上，降低了传统的介入生物瓣膜在生产、运输中的成本，缩短手术时间。并且避免了传统介入生物瓣膜因采用戊二醛交联而造成的醛基残留，提高了瓣膜的抗钙化性能，增强了血液相容性，有助于延长瓣膜使用寿命。

Description

一种人工生物心脏瓣膜及其制备方法 技术领域
本发明涉及生物医用材料以及医疗器械技术领域，尤其是一种人工生物心脏瓣膜及其制备方法。
 背景技术
随着老龄化进程的不断发展和世界人口的不断增加，瓣膜疾病的发病人数不断攀高，严重威胁着病人生命和影响病人的生活质量。对于药物治疗效果不佳、病程进展严重的心脏瓣膜病，目前最有效的治疗方法是人工心脏瓣膜置换术。经导管主动脉瓣植入术彻底改变了人工生物心脏瓣膜置换术的操作程序，是介入心脏病学史上的一个重大突破，使得医生可以不开胸，只需穿刺血管即可完成瓣膜置换，大大缩短了手术操作时间，降低了麻醉和输血带来的风险，缩短了患者的恢复时间，同时也降低了治疗成本。自从2002年第一例介入型瓣膜置换术实施以来，全世界已经植入了超过250000个经导管替换的主动脉瓣。
虽然介入瓣膜置换术具有微创、操作简单和病人术后恢复时间短等优点，但是目前临床上使用的介入生物心脏瓣膜所使用的生物组织几乎都是使用传统的戊二醛交联技术。近几十年的临床资料表明，戊二醛交联组织在使用过程中易发生瓣膜结构恶化，最终将导致生物瓣膜的失效。瓣膜结构恶化的一个常见表现是瓣叶撕裂，这可能是由于瓣叶长期暴露于应力下，引起瓣膜组织的机械性能下降所致。对于介入生物瓣膜来说，其耐久性问题更加严重，因为介入生物瓣膜不仅使用戊二醛处理过的生物材料，而且会经历压握卷曲和导管输送过程，这一过程会对瓣膜组织造成额外的机械损伤。此外，最新的亚临床数据表明，介入生物瓣膜在植入1至3个月后的血栓发生率高达10%-15%。因此需要改善目前临床使用的瓣膜材料的血液相容性，以降低血栓形成率。目前使用的戊二醛交联组织在生产过程中必须一直储存在戊二醛溶液中，包括交联、灭菌、保存和运输，存在戊二醛液体泄漏的风险。在手术植入之前，心脏瓣膜必须预先冲洗数次，并由医生通过专用的装置压缩安装在输送导管内。这个过程会增加细菌感染的风险，并且延长手术时间，而且存在冲洗不彻底，增加戊二醛毒性残留的风险，可以直接使用预安装的干态介入心脏瓣膜来解决这些问题。湿态的戊二醛交联组织在脱水后会变硬变脆，压握卷曲时会造成纤维撕裂和断裂，进一步影响其力学性能和耐久性。现有技术中报道过使用多元醇干燥生物组织的方法，但是得到的组织弹性和韧性不够，压握时会发生永久变形。因此，用于预安装介入心脏瓣膜的生物材料必须能够在干燥状态下抵抗加载的应力，并在复水后能够恢复到展开状态，无纤维损伤，基于此要求，现有技术还需要进一步改善。
 技术问题
本发明的目的：为了解决上述现有技术的不足提供一种人工生物心脏瓣膜及其制备方法，该人工生物心脏瓣膜在湿态时具有较强的机械强度和抗撕裂性能，在干燥状态下具有较好的弹性和柔韧性，在长期应力条件下不会发生永久变形，在放入水中可以恢复原有形状。
 技术解决方案
本发明采用的技术方案如下：
一种人工生物心脏瓣膜，人工生物心脏瓣膜的瓣叶为水凝胶填充的干燥生物瓣膜组织；瓣叶通过原位共聚与水凝胶复合，使得水凝胶填充在生物瓣膜组织中纤维间的空隙中。
干燥是指该生物瓣膜中水分含量低于生物瓣膜完全水合时候的含水量。
本发明所采用的原位共聚交联是通过在生物组织表面引入双键，在引发剂的作用下双键之间发生聚合反应，从而实现生物组织的交联。由亲水单体在引发剂的作用下聚合成的具有三维结构的网络的水凝胶，使得水分可以分散并保留于网络中。该水凝胶分散在生物组织的空隙中，具有一定的机械强度，可以分散和吸收应力，增强生物瓣膜的机械强度和弹性。
进一步地，生物瓣膜组织是猪心包或牛心包。
进一步地，基于已处理组织的重量，所述瓣叶的水分含量小于30%。
进一步的，所述的人工生物心脏瓣膜的制备方法，其特征在于，包括以下步骤：
a.将生物瓣膜组织浸泡在亲水单体溶液中进行修饰，得到修饰后生物组织；
b.将修饰后生物组织浸泡在水凝胶单体溶液中；
c.将上述经水凝胶浸泡处理后的生物组织浸泡在含引发剂的溶液中，在30-40℃下放置10-30h；
d.将上述c处理后的生物组织置入含有巯基化合物的溶液；
e.将上述d处理后得到的生物组织经光照引发聚合，然后脱水、干燥，即得。
所述亲水单体和水凝胶单体中均含有碳碳双键，步骤a中，利用亲水单体对生物瓣膜组织进行修饰，在生物瓣膜组织中引入可进行后续反应的碳碳双键，步骤b中，将修饰后的生物组织浸泡在水凝胶单体溶液中，使水凝胶单体分布于修饰后的生物组织的空隙中，步骤c中，在引发剂的作用下，同时发生以下两种反应：
（1）生物瓣膜组织中通过修饰的亲水单体引入的碳碳双键与水凝胶单体的双键之间发生聚合反应；
（2）水凝胶单体的双键之间发生聚合反应。
步骤d中，在处理后的生物组织中置入含有巯基化合物的溶液，利用巯基化合物去除反应残留的不饱和基团及引发剂。
进一步的，所述含引发剂的溶液中还包含多巴胺和EGCG。
所述多巴胺和EGCG可以与水凝胶单体的侧链之间产生非共价键的结合力，增加水凝胶的交联程度，进一步改善机械性能和弹性。
进一步的，含引发剂的溶液中，所述引发剂为过硫酸铵和亚硫酸氢钠，所述引发剂的浓度为20-80mM。
进一步的，所述过硫酸铵和亚硫酸氢钠的体积比为1:1。
进一步的，含引发剂的溶液中，所述引发剂为2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮，所述引发剂的浓度为0.1-1wt%。
进一步的，含引发剂的溶液中，多巴胺的浓度为0.05-0.2mg/mL。
进一步的，含引发剂的溶液中，EGCG的浓度为0.05-0.2mg/mL。
进一步的，含引发剂的溶液中，溶剂为PBS缓冲液。
上述的人工生物心脏瓣膜的制备方法，包括以下步骤：
a.将生物瓣膜组织浸泡在亲水单体溶液中进行修饰，得到修饰后生物组织；
b.将修饰后生物组织浸泡在水凝胶单体溶液中；
c.将上述经水凝胶浸泡处理后的生物组织浸泡在引发剂以及多巴胺和EGCG的混合溶液中，在30-40℃下放置10-30h；
d.将上述c处理后的生物组织置入含有巯基化合物的溶液；
e.将上述d处理后得到的生物组织经光照引发聚合，然后脱水、干燥，即得。
进一步地，所述亲水单体为甲基丙烯酸酐。
进一步地，所述亲水单体与生物瓣膜组织的重量比为1-2:1。
进一步地，所述亲水单体与生物瓣膜组织在室温下反应22~26h。
进一步地，所述亲水单体与生物瓣膜组织在pH为7条件下反应。
进一步地，所述水凝胶单体为N-丙烯酰甘酰胺单体。
进一步地，所述亲水单体为甲基丙烯酸酐，所述水凝胶单体为N-丙烯酰甘酰胺单体。
进一步地，所述水凝胶单体为N-丙烯酰甘酰胺单体，浓度为20-40wt%。
进一步地，所述混合溶液中，所述引发剂为过硫酸铵和亚硫酸氢钠，所述引发剂的浓度为20-80mM。
进一步地，所述过硫酸铵和亚硫酸氢钠的体积比为1:1。
进一步地，所述混合溶液中，所述引发剂为2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮，所述引发剂的浓度为0.1-1wt%。
进一步地，所述混合溶液中，多巴胺的浓度为0.05-0.2mg/mL。
进一步地，所述混合溶液中，EGCG的浓度为0.05-0.2mg/mL。
进一步地，所述混合溶液中，溶剂为PBS缓冲液。
进一步地，亲水单体为甲基丙烯酸酐；所述亲水单体与生物瓣膜组织的重量比为1-2:1。
进一步地，上述修饰过程具体为：将生物瓣膜组织浸泡在去离子水中，不断滴加甲基丙烯酸酐单体，使得甲基丙烯酸酐单体和生物瓣膜组织最终重量比为1：1，并调节溶液pH在5-9左右，静置反应24h。
进一步地，水凝胶单体为N-丙烯酰甘酰胺单体，浓度为20-40wt%，优选为30wt%。通过选择该水凝胶，能够改善生物瓣膜的血液相容性，并降低免疫反应。
进一步地，引发剂以及多巴胺和EGCG的混合溶液为含20-80mM过硫酸铵和亚硫酸氢钠或0.1-1wt% 2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮，以及0.05-0.2mg/mL多巴胺和0.05-0.2mg/mLEGCG的PBS混合溶液。
进一步地，引发剂以及多巴胺和EGCG的混合溶液为含50mM过硫酸铵和亚硫酸氢钠或0.5wt% 2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮，以及0.1mg/mL多巴胺和0.1mg/mLEGCG的PBS混合溶液；其中，过硫酸铵和亚硫酸氢钠的体积比为1:1。
进一步地，巯基化合物为巯基乙醇、半胱氨酸和谷胱甘肽中的至少一种。用于去除反应残留的不饱和基团及引发剂。
进一步地，光照引发聚合具体为：在氮气保护下由紫外光光照20-40min引发聚合。
进一步地，进行光照引发聚合时，生物组织处于展平状态。
进一步地，脱水具体为：使用甘油/乙醇溶液浸泡进行第一步脱水，然后在湿度30-40%的环境中进行第二次脱水；最终获得瓣叶的水分含量小于30%，基于已处理组织的重量。
进一步地，甘油/乙醇溶液的浓度为50vt%。
进一步地，第一步脱水时间不少于24h。
进一步地，第二步脱水在室温下进行。
 有益效果
综上所述，由于采用了上述技术方案，本发明的有益效果是：
1、本发明通过原位共聚的方式交联并与水凝胶复合，然后进行适度脱水制备生物瓣膜。首先通过在生物组织表面修饰双键，然后在生物组织的空隙中引入亲水单体，然后加入引发剂，引发生物组织内部的交联反应并使亲水单体聚合形成具有三维网状结构的水凝胶，进一步通过甘油/乙醇溶液对水凝胶复合生物瓣膜进行干燥脱水，从而大大提高了生物瓣膜组织的生物相容性和力学性能，并且能够在重新植入人体后快速复水，恢复到压握前的形状，从而可以用于预装介入生物瓣膜的制造；
2、本发明所使用原位共聚交联的方式避免了传统戊二醛交联方式导致的醛基残留，增强了瓣膜的抗钙化性能和血液相容性，有望延长生物瓣膜的使用寿命。本发明所得组织在湿态时具有较强的机械性能和抗撕裂性能，能够减少微撕裂的发生，有望改善介入生物瓣膜的耐久性问题；
3、本发明所得人工生物心脏瓣膜组织具有良好的弹性和抵抗变形的能力，因此可以长期以干态形式压握装备在导管输送系统上，在人体中释放后瓣膜组织可以快速复水展开，恢复原有形状并发挥瓣膜的正常功能；
4、本发明的生物瓣膜组织中复合的水凝胶可以提高戊二醛交联瓣膜的血液相容性，并降低免疫反应；
5、本发明使用水凝胶填充生物瓣膜组织中纤维间的空隙，一方面可以增强瓣膜的应力分散能力，提供优异的抗撕裂性能和弹性，另一方面水凝胶的加入可以加快干燥瓣膜水合时候的复水速度，从而便于瓣膜在植入后恢复压握前的形状。
 附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为Glut-PP组织经过模拟压握后在磷酸缓冲盐溶液中的回复情况；
图2为pNAGA/RPC-PP组织经过模拟压握后在磷酸缓冲盐溶液中的回复情况；
图3为Glut-PP组织的血小板黏附情况；
图4为pNAGA/RPC-PP组织的血小板回复情况；
图5为制备流程示意图。
 本发明的实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，即所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。通常在此处附图中描述和示出的本发明实施例的组件可以以各种不同的配置来布置和设计。
因此，以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本发明较佳实施例提供一种人工生物心脏瓣膜的制备方法，具体步骤如下：
a.首先，将新鲜的脱细胞猪心包浸泡在去离子水中，不断滴加甲基丙烯酸酐单体，使单体和瓣膜组织重量比为1.0，并调节溶液pH6左右，反应24h。
b. 将修饰单体后的猪心包洗净，随即浸泡在30wt%水凝胶单体N-丙烯酰甘酰胺单体（NAGA）溶液中。
c.将经过上述处理的猪心包浸泡在含0.5wt%2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮（Irgacure2959）引发剂，以及0.1mg/mL多巴胺和0.1mg/mLEGCG的PBS溶液中，在37℃下放置24h。
d. 聚合后的组织经去离子水彻底清洗，加入含有巯基乙醇的溶液中以去除反应残留的不饱和基团及引发剂。
e. 然后夹在两片石英玻璃中，在紫外光和氮气保护下引发聚合。光照30min后，取出生物组织并彻底清洗。
f. 然后在50vt%的甘油/乙醇溶液中浸泡24h进行脱水，然后取出生物组织平铺于塑料板上进行干燥，干燥温度为室温，湿度控制在30-40%。
本实施例中制得的人工生物心脏瓣膜命名为pNAGA/RPC-PP。
实施例2
本发明较佳实施例提供一种人工生物心脏瓣膜的制备方法，具体步骤如下：
a.首先，将新鲜的脱细胞猪心包浸泡在去离子水中，不断滴加甲基丙烯酸酐单体，使单体和瓣膜组织重量比为1.0，并调节溶液pH7左右，反应24h。
b. 将修饰单体后的猪心包洗净，随即浸泡在30wt%水凝胶单体N-丙烯酰甘酰胺单体（NAGA）溶液中。
c.将经过上述处理的猪心包浸泡在含50mM过硫酸铵和亚硫酸氢铵引发剂，以及0.1mg/mL多巴胺和0.1mg/mLEGCG的PBS溶液中，在37℃下放置24h。其中，过硫酸铵和亚硫酸氢铵的体积比为1:1。
d. 聚合后的组织经去离子水彻底清洗，加入含有巯基乙醇的溶液中以去除反应残留的不饱和基团及引发剂。
e. 然后夹在两片石英玻璃中，在紫外光和氮气保护下引发聚合。光照30min后，取出生物组织并彻底清洗。
f. 然后在50vt%的甘油/乙醇溶液中浸泡24h进行脱水，然后取出生物组织平铺于塑料板上进行干燥，干燥温度为室温，湿度控制在30-40%。
实施例3
本发明较佳实施例提供一种人工生物心脏瓣膜的制备方法，具体步骤如下：
a.首先，将新鲜的脱细胞猪心包浸泡在去离子水中，不断滴加甲基丙烯酸酐单体，使单体和瓣膜组织重量比为1.0，并调节溶液pH6左右，反应24h。
b. 将修饰单体后的猪心包洗净，随即浸泡在30wt%水凝胶单体N-丙烯酰甘酰胺单体（NAGA）溶液中。
c.将经过上述处理的猪心包浸泡在含0.5wt%2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮（Irgacure2959）引发剂的PBS溶液中，在37℃下放置24h。
d. 聚合后的组织经去离子水彻底清洗，加入含有巯基乙醇的溶液中以去除反应残留的不饱和基团及引发剂。
e. 然后夹在两片石英玻璃中，在紫外光和氮气保护下引发聚合。光照30min后，取出生物组织并彻底清洗。
f. 然后在50vt%的甘油/乙醇溶液中浸泡24h进行脱水，然后取出生物组织平铺于塑料板上进行干燥，干燥温度为室温，湿度控制在30-40%。
实施例4
本发明较佳实施例提供一种人工生物心脏瓣膜的制备方法，具体步骤如下：
a.首先，将新鲜的脱细胞猪心包浸泡在去离子水中，不断滴加甲基丙烯酸酐单体，使单体和瓣膜组织重量比为1.0，并调节溶液pH7左右，反应24h。
b. 将修饰单体后的猪心包洗净，随即浸泡在30wt%水凝胶单体N-丙烯酰甘酰胺单体（NAGA）溶液中。
c.将经过上述处理的猪心包浸泡在含50mM过硫酸铵和亚硫酸氢铵引发剂的PBS溶液中，在37℃下放置24h。其中，过硫酸铵和亚硫酸氢铵的体积比为1:1。
d. 聚合后的组织经去离子水彻底清洗，加入含有巯基乙醇的溶液中以去除反应残留的不饱和基团及引发剂。
e. 然后夹在两片石英玻璃中，在紫外光和氮气保护下引发聚合。光照30min后，取出生物组织并彻底清洗。
f. 然后在50vt%的甘油/乙醇溶液中浸泡24h进行脱水，然后取出生物组织平铺于塑料板上进行干燥，干燥温度为室温，湿度控制在30-40%。
实验例
设置对照组1，与实施例1制得的产品分别进行压握模拟实验 、机械性能测试、血液相容性评价、抗钙化性能测试。
对照组1：将猪心包浸泡在0.5vt%的戊二醛PBS溶液中交联24h。然后在5vt0%的甘油/乙醇溶液中浸泡24小时进行脱水，然后取出生物组织平铺于塑料板上进行干燥，干燥温度为室温，湿度控制在30-40%。制得的瓣膜命名为Glut-PP。
（1）压握模拟实验
分别将将试验组样品与对照组样品裁剪成3*3cm 2的正方形，随意卷曲，放入内径5毫米的聚丙烯管中密封，用环氧乙烷灭菌后，将聚丙烯管在37℃培养箱中放置10天，然后从管中取出生物组织，并完全浸没在磷酸缓冲盐缓冲溶液中，观察其展开情况。
图1为对照组经过模拟压握后在磷酸缓冲盐溶液中的回复情况，可以观察到戊二醛交联的不含水凝胶生物组织无法展开，而在图2中，试验组样品能完全展开。这表明水凝胶的引入对于心包组织在压握卷曲过程中产生的应力有较好的抵抗作用，可以明显提高瓣膜在干燥状态下抵抗变形的能力。
（2）机械性能测试
将试验组样品与对照组样品分别切成长方形样品，在室温下用万能试验机进行机械性能测试，并分析其极限抗拉强度、抗剪切强度和抗撕裂强度。
表1 机械强度对比表
  极限抗拉强度（MPa） 抗剪切强度（N） 抗撕裂强度（N/mm）
对照组1 8±1 1.2±0.4 32±5
实施例1 13±2 1.9±0.4 40±4
由表1可以发现， 试验组的极限抗拉强度、抗剪切强度和抗撕裂强度均优于对照组。这表明水凝胶的引入对于提高戊二醛交联组织的机械强度有显著作用。
（3）血液相容性评价
将试验组样品与对照组样品分别切成直径6毫米的圆片样品，浸泡于富血小板血浆中，在37℃下孵育1小时，然后用磷酸缓冲盐冲洗3次，去除未黏附的血小板。随后用4%的多聚甲醛固定样品，并用30%、50%、80%、100%的乙醇梯度脱水、冻干、喷金，进行扫描电镜观察血小板黏附情况。
图3为对照组的血小板黏附情况，对照粗材料表面粘附有大量的血小板。相反，在图4中，试验组材料表面几乎没有血小板，这些结果说明通过原位共聚交联并复合水凝胶的生物组织具有良好的抗血小板黏附的能力。
（4）抗钙化性能测试
将试验组样品和对照组样品清洗好并裁剪成1cm×1cm的。向20天左右幼年SD大鼠大鼠腹腔注射3%戊巴比妥钠0.1 mL进行麻醉，剃除脊柱两旁肌肉上的皮毛，碘酒和酒精常规消毒。右侧背部皮下植入试验组样品1个，左侧背部皮下植入对照组样品1个，缝合皮肤切口。30天后，采用颈椎脱臼法对动物进行安乐死，取出移植物。小心除去移植物表面的宿主组织，生理盐水冲洗干净。冷冻干燥后称量干重，之后采用6N浓盐酸在90摄氏度水浴锅中消解直到无可见固体颗粒，之后采用电感耦合等离子体发射光谱仪进行钙元素的定量分析。
对实施例1、对照组1制得的材料分别进行抗钙化性能测试，结果如下表2所示。由表2可知，实施例组的挂钙量减少。
表2 挂钙量含量表
  挂钙量 µg/mg
对照组1 32.15 ± 1.56
实施例1 2.31 ± 0.52
综上，本发明通过原位共聚的方式交联并与水凝胶复合制备得到的干燥的生物瓣膜组织，该生物组织具有良好的抗钙化性能和血液相容性，使用寿命能因此得以延长。同时该生物组织具有良好的抗撕裂性能和弹性性能，能够在干燥状态下长时间压握于输送系统而不产生结构损伤，并在浸泡于盐酸缓冲盐溶液时快速复水以恢复原有形状，可以用于预装式介入生物心脏瓣膜的制造。
当然，以上只是本发明的典型实例，除此之外，本发明还可以有其它多种具体实施方式，凡采用等同替换或等效变换形成的技术方案，均落在本发明要求保护的范围之内。

Claims (25)

  1. 一种人工生物心脏瓣膜，其特征在于，所述人工生物心脏瓣膜的瓣叶为水凝胶填充的干燥生物瓣膜组织；所述瓣叶通过原位共聚与水凝胶复合，使得水凝胶填充在所述生物瓣膜组织中纤维间的空隙中。
  2. 根据权利要求1所述的人工生物心脏瓣膜，其特征在于，所述生物瓣膜组织是猪心包或牛心包。
  3. 根据权利要求1中所述的人工生物心脏瓣膜，其特征在于，基于已处理组织的重量，所述瓣叶的水分含量小于30%。
  4. 权利要求1-3中任一项中所述的人工生物心脏瓣膜的制备方法，其特征在于，包括以下步骤：
    a.将生物瓣膜组织浸泡在亲水单体溶液中进行修饰，得到修饰后生物组织；
    b.将修饰后生物组织浸泡在水凝胶单体溶液中；
    c.将上述经水凝胶浸泡处理后的生物组织浸泡在含引发剂的溶液中，在30-40℃下放置10-30h；
    d.将上述c处理后的生物组织置入含有巯基化合物的溶液；
    e.将上述d处理后得到的生物组织经光照引发聚合，然后脱水、干燥，即得。
  5. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述含引发剂的溶液中还包含多巴胺和EGCG。
  6. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述亲水单体为甲基丙烯酸酐。
  7. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述亲水单体与生物瓣膜组织的重量比为1-2:1。
  8. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述亲水单体与生物瓣膜组织在室温下反应22~26h。
  9. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述亲水单体与生物瓣膜组织在pH为7条件下反应。
  10. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述水凝胶单体为N-丙烯酰甘酰胺单体。
  11. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述亲水单体为甲基丙烯酸酐，所述水凝胶单体为N-丙烯酰甘酰胺单体。
  12. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述水凝胶单体为N-丙烯酰甘酰胺单体，浓度为20-40wt%。
  13. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，含引发剂的溶液中，所述引发剂为过硫酸铵和亚硫酸氢钠，所述引发剂的浓度为20-80mM。
  14. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述过硫酸铵和亚硫酸氢钠的体积比为1:1。
  15. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，含引发剂的溶液中，所述引发剂为2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮，所述引发剂的浓度为0.1-1wt%。
  16. 根据权利要求5所述的人工生物心脏瓣膜的制备方法，其特征在于，含引发剂的溶液中，多巴胺的浓度为0.05-0.2mg/mL。
  17. 根据权利要求5所述的人工生物心脏瓣膜的制备方法，其特征在于，含引发剂的溶液中，EGCG的浓度为0.05-0.2mg/mL。
  18. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，含引发剂的溶液中，溶剂为PBS缓冲液。
  19. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述巯基化合物为巯基乙醇、半胱氨酸和谷胱甘肽中的至少一种。
  20. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述光照引发聚合具体为：在氮气保护下由紫外光光照20-40min引发聚合。
  21. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，进行光照引发聚合时，生物组织处于展平状态。
  22. 根据权利要求4所述的人工生物心脏瓣膜的制备方法，其特征在于，所述脱水过程具体为：使用甘油/乙醇溶液浸泡进行第一步脱水，然后在湿度30-40%的环境中进行第二次脱水。
  23. 根据权利要求21所述的人工生物心脏瓣膜的制备方法，其特征在于，甘油/乙醇溶液的浓度为50vt%。
  24. 根据权利要求21所述的人工生物心脏瓣膜的制备方法，其特征在于，第一步脱水时间不少于24h。
  25. 根据权利要求21所述的人工生物心脏瓣膜的制备方法，其特征在于，第二步脱水在室温下进行。
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