WO2020038293A1 - 一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法 - Google Patents

一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法 Download PDF

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WO2020038293A1
WO2020038293A1 PCT/CN2019/101014 CN2019101014W WO2020038293A1 WO 2020038293 A1 WO2020038293 A1 WO 2020038293A1 CN 2019101014 W CN2019101014 W CN 2019101014W WO 2020038293 A1 WO2020038293 A1 WO 2020038293A1
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linking
biological
tea polyphenols
combination
valves
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French (fr)
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王云兵
雷洋
訾振军
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Sichuan University
Venus Medtech Hangzhou Inc
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Sichuan University
Venus Medtech Hangzhou Inc
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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
    • 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
    • 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
    • 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
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the invention relates to the technical field of biomedical materials and medical devices, in particular to a method for treating a biological valve using a combination of enzyme cross-linking and tea polyphenols and a biological material thereof.
  • Heart valve disease is a common valvular disease. Anatomically manifested as narrowed blood passages or valvular insufficiency.
  • Heart valve disease Treatment of heart valve disease includes thoracotomy and percutaneous heart valve replacement.
  • Thoracic surgery is very traumatic, high risk, slow recovery, and requires extracorporeal circulation support, which is unacceptable to many patients.
  • Percutaneous heart valve replacement surgery has become the main trend of future valve surgery because it has less trauma to patients and low risk.
  • Biological heart valves are a class of biomedical materials used to replace heart valves in humans. Biological heart valves are generally prepared by cross-linking glutaraldehyde from porcine pericardium and bovine pericardium.
  • Glutaraldehyde cross-linking treatment has the characteristics of simple operation, low cost and high degree of collagen cross-linking, and is currently the industry's first choice for chemical cross-linking of biological heart valves.
  • glutaraldehyde cross-linked biological heart valves have problems of easy degradation and calcification, resulting in biological heart valves having a useful life of only about 10 years.
  • Glutaraldehyde can achieve stable cross-linking of collagen, but it cannot cross-link elastin, resulting in certain technical limitations.
  • the purpose of the present invention is to provide a method for treating biological valves by using a combination of enzyme cross-linking and tea polyphenols to solve the above-mentioned shortcomings of the prior art, which can effectively improve the structural stability and anti-calcification of biological materials such as biological heart valves. Performance, potentially extending its useful life.
  • a method for treating biological valves by using a combination of enzyme cross-linking and tea polyphenols specifically includes the following steps:
  • step S3 The biomaterials washed in step S2 are then modified with hydroxyphenylpropionic acid.
  • An aqueous solution having a molar concentration of hydroxyphenylpropionic acid of 1 mM to 1M is used, and a molar solution of the carbodiimide / N-hydroxysuccinimide is used. Degree is 0.01-1M;
  • step S4 The tea polyphenols are soaked in the biological material processed in step S3, and the mass concentration of the tea polyphenols used is 0.1% -10%;
  • step S5. Perform horseradish peroxidase / hydrogen peroxide-induced enzyme cross-linking on the biological material treated in step S4.
  • the mass concentration of horseradish peroxidase used is 0.1% -10%, and the mass of hydrogen peroxide used The concentration is 0.1% -10%;
  • step S1 fresh biological materials, such as pericardial tissue of pigs or cattle, are collected and stored in a low-temperature and humid state at 4 degrees Celsius.
  • step S2 the pericardial tissue is washed with distilled water under gentle vibrations and fluid pressure under oscillating conditions to remove adhered non-pericardial and non-collagenous tissues.
  • the washing achieves effective decellularization of the pericardial tissue through osmotic shock. And, preferably, the washing is continued until there is no visible adherent non-pericardial or non-collagenous tissue, and preferably, it is washed with distilled water under a shaking condition of 100 RPM at 4 degrees Celsius for 2 hours.
  • step S3 the washed biological material is modified with hydroxyphenylpropionic acid, and an aqueous solution having a molar concentration of hydroxyphenylpropionic acid of 1mM-1M is used.
  • This step needs to ensure that hydroxyphenylpropionic acid reaches a near-saturated physical penetration. Thereby, as much as possible is introduced.
  • step S4 the treated biomaterial is soaked with tea polyphenols, and the mass concentration of the used tea polyphenols is 0.1% -10%.
  • the tea polyphenols are adsorbed on the hydrophobic section of the elastin fiber by hydrogen bonding, thereby improving Structural stability of elastin.
  • step S5 the treated biological material is subjected to horseradish peroxidase / hydrogen peroxide-induced enzyme cross-linking, and the mass concentration of horseradish peroxidase used is 0.1% -10%.
  • the mass concentration of hydrogen oxide is 0.1% -10%.
  • Hydrogen peroxide is an oxidant. Under the action of oxidant, horseradish peroxidase can catalyze the phenolic hydroxyl group to achieve chemical crosslinking.
  • step S6 washing with distilled water is performed. This washing step will remove unreacted tea polyphenols, horseradish peroxidase, and hydrogen peroxide.
  • the beneficial effect of the present invention is that the method provided by the present invention can improve the structural stability and anti-calcification performance of the biological material, and potentially prolong its service life.
  • FIG. 1 is a specific flowchart of a prepared biological heart valve.
  • Figure 2 is a schematic diagram of the chemical principle of p-hydroxyphenylpropionic acid modified pericardium and horseradish peroxidase / catalase cross-linking.
  • FIG. 3 is the percentage of weight loss of enzyme degradation obtained by using Example 1.
  • FIG. FP Fresh pig pericardium.
  • GLUT glutaraldehyde treatment group.
  • EDC carbodiimide treatment group.
  • HPA / EDC / EGCG p-hydroxyphenylpropionic acid, carbodiimide, gallocatechin gallate enzyme cross-linked treatment group.
  • FIG. 4 is a result of quantitative detection of elastin obtained in Example 1.
  • FIG. GLUT glutaraldehyde treatment group.
  • EDC carbodiimide treatment group.
  • HPA / EDC / EGCG p-hydroxyphenylpropionic acid, carbodiimide, gallocatechin gallate enzyme cross-linked treatment group.
  • FIG. 5 is a test result of the amount of calcium implanted subcutaneously in the rat obtained in Example 1.
  • FIG. GLUT glutaraldehyde treatment group.
  • EDC carbodiimide treatment group.
  • HPA / EDC / EGCG p-hydroxyphenylpropionic acid, carbodiimide, gallocatechin gallate enzyme cross-linked treatment group.
  • the freshly collected pig pericardium is washed with distilled water for 2 hours under a shaking condition of 100 degrees RPM at 4 degrees Celsius. Then immerse in 50 mM hydroxyphenylpropanoic acid in water at room temperature for 4 hours. It was then soaked at room temperature for 24 hours with 10 mM carbodiimide and 10 mMN-hydroxysuccinimide. It was then soaked with 1% epigallocatechin gallate for 24 hours. It was then immersed in 1% horseradish peroxidase and 1% hydrogen peroxide at 150 RPM at 37 ° C for 24 hours.
  • the freshly collected pig pericardium is washed with distilled water for 2 hours under a shaking condition of 100 degrees RPM at 4 degrees Celsius. Then immerse in 100 mM hydroxyphenylpropanoic acid in water at room temperature for 4 hours. It was then soaked at room temperature for 24 hours with 10 mM carbodiimide and 10 mMN-hydroxysuccinimide. It was then soaked with 1% epigallocatechin gallate for 24 hours. Then it was immersed in 1% horseradish peroxidase and 1% hydrogen peroxide at 37 ° C and 150 RPM for 24 hours.
  • freshly collected pig pericardium is washed with distilled water under a shaking condition of 4 ° C and 100 RPM for 2 hours. Then immerse in 100 mM hydroxyphenylpropanoic acid in water at room temperature for 4 hours. It was then soaked at room temperature for 24 hours with 10 mM carbodiimide and 10 mMN-hydroxysuccinimide. It was then soaked with 1% epigallocatechin gallate for 24 hours. Then immerse in 5% horseradish peroxidase and 5% hydrogen peroxide at 37 ° C and 150 RPM for 24 hours.
  • pericardial (FP) group fresh pericardial (FP) group.
  • GLUT Glutaraldehyde
  • EDC carbodiimide
  • the glutaraldehyde (GLUT) treatment group the pericardium was immersed in 0.625% glutaraldehyde for 24 hours.
  • the carbodiimide (EDC) treatment group was immersed in 10 mM carbodiimide and 10 mM N-hydroxysuccinimide for 24 hours.
  • the beneficial effect of the present invention is that the method provided by the present invention can improve the stability and anti-calcification performance of the elastin of the biological material, potentially extending its service life.

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Abstract

本发明公开了一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法，所述方法包括用羟基苯丙酸修饰猪或牛的心包膜，然后浸泡茶多酚，然后在辣根过氧化物酶和过氧化氢的条件下引发酶交联；羟基苯丙酸将在心包膜上引入酚羟基，外源茶多酚提供额外的酚羟基；辣根过氧化物酶和过氧化氢将实现酚羟基的化学交联；本发明提供的方法能够提升生物材料的结构稳定性及抗钙化性能，潜在地延长其使用寿命。

Description

一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法 技术领域
本发明涉及一种生物医学材料以及医疗器械技术领域，特别是一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法及其生物材料。
背景技术
心脏瓣膜疾病是一种常见的瓣膜衰退疾病。在解剖学上表现为血液通路变窄或瓣膜关闭不全。
心脏瓣膜疾病的治疗包括开胸瓣膜置换手术以及经皮心脏瓣膜置换手术。开胸手术对病人创伤大、风险高、恢复慢、需体外循环支持，很多患者无法接受。经皮心脏瓣膜置换手术因为对病人创伤小、风险低，成为未来瓣膜手术的主要趋势。
生物心脏瓣膜是指一类用于替换人体病变心脏瓣膜的生物医学材料。生物心脏瓣膜一般由猪心包膜、牛心包膜等通过戊二醛交联制备而成。
戊二醛交联处理具有操作简单，成本低以及胶原蛋白交联程度高的特点，是目前生物心脏瓣膜化学交联的行业首选。然而，戊二醛交联的生物心脏瓣膜存在容易降解以及钙化的问题，导致生物心脏瓣膜只有10年左右的有效使用年限。戊二醛可以实现胶原蛋白的稳定交联，但是不能交联弹性蛋白，导致其有一定技术局限性。
因此，通过优化生物心脏瓣膜的化学交联方法，特别是开发能够提高弹性蛋白结构稳定性的新型材料处理方法，将提升生物心脏瓣膜的整体结构稳定性以及抗钙化性能，对于科学研究以及相关产业领域的发展具有重大意义，而目前并没有很好的方法，因此需要改进。
发明内容
本发明的目的是为了解决上述现有技术的不足而提供一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法，能有效提升生物心脏瓣膜等生物材料的结构稳定性以及抗钙化性能，潜在地延长其使用寿命。
本发明的目的通过以下技术方案予以实现。
一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法，具体包括以下步骤：
S1、获取生物材料；
S2、用蒸馏水浸泡清洗；
S3、然后将步骤S2清洗后的生物材料进行羟基苯丙酸修饰，使用的羟基苯丙酸摩尔浓度为1mM-1M的水溶液，使用的碳二亚胺/N-羟基琥珀酰亚胺的摩尔溶度为0.01-1M；
S4、将步骤S3处理后的生物材料浸泡茶多酚，使用的茶多酚质量浓度为0.1％-10％；
S5、将步骤S4处理后的生物材料进行辣根过氧化物酶/过氧化氢引发的酶交联，使用的辣根过氧化物酶质量浓度为0.1％-10％，使用的过氧化氢质量浓度为0.1％-10％；
S6、最后用蒸馏水浸泡清洗。
进一步的，在步骤S1中：采集新鲜的生物材料，如猪或牛的心包组织，并于4摄氏度低温湿润状态下保存。
进一步的，在步骤S2中，采用柔和摩擦和流体压力在振荡条件之下用蒸馏水清洗心包组织，去除粘附的非心包和非胶原组织，所述清洗通过 渗压休克实现对心包组织有效脱细胞，且优选地，清洗持续到没有可见的粘附的非心包或非胶原组织，优选地，是于4摄氏度100RPM转速振荡条件之下蒸馏水清洗2小时。
进一步的，在步骤S3中，清洗后的生物材料进行羟基苯丙酸修饰，使用的羟基苯丙酸摩尔浓度为1mM-1M的水溶液，这个步骤需要确保羟基苯丙酸达到接近饱和的物理渗透，从而尽可能多地引入羟基苯丙酸。
进一步的，在步骤S4中，处理后的生物材料浸泡茶多酚，使用的茶多酚质量浓度为0.1％-10％，茶多酚通过氢键作用吸附于弹性蛋白纤维的疏水段，从而提高弹性蛋白的结构稳定性能。
进一步的，在步骤S5中，处理后的生物材料进行辣根过氧化物酶/过氧化氢引发的酶交联，使用的辣根过氧化物酶质量浓度为0.1％-10％，使用的过氧化氢质量浓度为0.1％-10％，过氧化氢是一种氧化剂，辣根过氧化物酶在氧化剂的作用下，能够催化酚羟基实现化学交联。
进一步的，在步骤S6中，进行蒸馏水清洗，这个清洗步骤将清除没有反应的茶多酚、辣根过氧化物酶以及过氧化氢。
本发明的有益效果是：本发明提供的方法能够提升生物材料的结构稳定性及抗钙化性能，潜在地延长其使用寿命。
附图说明
为了进一步澄清一个或多个本发明的上述以及其他的优点和特性，通过参照附图中示出的特定实施方案，呈现一个或多个本发明更具体的描述。
图1是制备的生物心脏瓣膜的具体流程图。
图2是对羟基苯丙酸修饰心包膜和辣根过氧化物酶/过氧化氢酶交联化学原理示意图。
图3是采用实施例1所得到的酶降解重量损失百分比。FP：新鲜猪心包膜。GLUT：戊二醛处理组。EDC：碳二亚胺处理组。HPA/EDC/EGCG：对羟基苯丙酸、碳二亚胺、没食子儿茶素没食子酸酯的酶交联处理组。
图4是采用实施例1所得到的弹性蛋白定量检测结果。GLUT：戊二醛处理组。EDC：碳二亚胺处理组。HPA/EDC/EGCG：对羟基苯丙酸、碳二亚胺、没食子儿茶素没食子酸酯的酶交联处理组。
图5是采用实施例1所得到的大鼠皮下植入挂钙量检测结果。GLUT：戊二醛处理组。EDC：碳二亚胺处理组。HPA/EDC/EGCG：对羟基苯丙酸、碳二亚胺、没食子儿茶素没食子酸酯的酶交联处理组。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合具体实施例，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仅用以解释本发明，并不用于限定本发明。
实施例1
在本实施例中，新鲜采集的猪心包于4摄氏度100RPM转速振荡条件之下蒸馏水清洗2小时。然后浸泡在50mM羟基苯丙酸水溶液在室温当中4小时。然后采用10mM碳二亚胺和10mMN-羟基琥珀酰亚胺在室温浸泡24小时。然后采用1％表没食子儿茶素没食子酸酯浸泡24小时。然后采用1％辣根过氧化物酶和1％过氧化氢于37摄氏度150RPM转速振荡条 件之下浸泡24小时。
实施例2
在本实施例中，新鲜采集的猪心包于4摄氏度100RPM转速振荡条件之下蒸馏水清洗2小时。然后浸泡在100mM羟基苯丙酸水溶液在室温当中4小时。然后采用10mM碳二亚胺和10mMN-羟基琥珀酰亚胺在室温浸泡24小时。然后采用1％表没食子儿茶素没食子酸酯浸泡24小时。然后采用1％辣根过氧化物酶和1％过氧化氢于37摄氏度150RPM转速振荡条件之下浸泡24小时。
实施例3
在本实施中，新鲜采集的猪心包于4摄氏度100RPM转速振荡条件之下蒸馏水清洗2小时。然后浸泡在100mM羟基苯丙酸水溶液在室温当中4小时。然后采用10mM碳二亚胺和10mMN-羟基琥珀酰亚胺在室温浸泡24小时。然后采用1％表没食子儿茶素没食子酸酯浸泡24小时。然后采用5％辣根过氧化物酶和5％过氧化氢于37摄氏度150RPM转速振荡条件之下浸泡24小时。
处理过程当中，设置了三个对照组：(1)新鲜心包膜(FP)组。(2)戊二醛(GLUT)处理组。(3)碳二亚胺(EDC)处理组。戊二醛(GLUT)处理组即将心包膜浸泡于0.625％的戊二醛当中24小时。碳二亚胺(EDC)处理组即将心包膜浸泡于10mM碳二亚胺和10mM N-羟基琥珀酰亚胺当中24小时。
如图3所示，评估时检测的实验组(HPA/EDC/EGCG)组胶原蛋白酶、弹性蛋白酶降解的重量损失百分比较小，如图4所示，弹性蛋白含量提 高，如图5所示，大鼠皮下植入30天的挂钙量减少。
本发明的有益效果是：本发明提供的方法能够提升生物材料的弹性蛋白的稳定性及抗钙化性能，潜在地延长其使用寿命。
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 (4)

  1. 一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法，其特征在于,具体包括以下步骤：
    S1、获取生物材料；
    S2、用蒸馏水浸泡清洗；
    S3、然后将步骤S2清洗后的生物材料进行羟基苯丙酸修饰，使用的羟基苯丙酸摩尔浓度为1mM-1M的水溶液，使用的碳二亚胺/N-羟基琥珀酰亚胺的摩尔溶度为0.01-1M；
    S4、将步骤S3处理后的生物材料浸泡茶多酚，使用的茶多酚质量浓度为0.1％-10％；
    S5、将步骤S4处理后的生物材料进行辣根过氧化物酶/过氧化氢引发的酶交联，使用的辣根过氧化物酶质量浓度为0.1％-10％，使用的过氧化氢质量浓度为0.1％-10％；
    S6、最后用蒸馏水浸泡清洗。
  2. 根据权利要求1所述的一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法，其特征在于:所述茶多酚包括单宁酸、儿茶素、没食子儿茶素、儿茶素没食子酸酯和没食子儿茶素没食子酸酯的一种或多种。
  3. 根据权利要求1所述的一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法，其特征在于：所述生物材料为动物组织包括心包膜、瓣膜、肠膜、脑膜、肺膜、血管、皮肤或韧带的一种或多种。
  4. 根据权利要求1所述的一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法，其特征在于：所述生物材料不限于经皮介入生物心脏瓣膜，也适用于开胸瓣膜置换手术所用的生物材料。
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