阅读说明：本技术 一种采用酶交联和茶多酚组合联用处理生物瓣膜的方法 (Method for treating biological valve by combining enzyme crosslinking and tea polyphenol ) 是由 王云兵 雷洋 訾振军 于 2018-08-20 设计创作，主要内容包括：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。(The invention discloses a method for treating a biological valve by combining enzyme crosslinking and tea polyphenol, which comprises the steps of modifying a porcine or bovine pericardium by hydroxy phenylpropionic acid, then soaking tea polyphenol, and then initiating enzyme crosslinking under the conditions of horseradish peroxidase and hydrogen peroxide; the hydroxyl phenylpropionic acid introduces phenolic hydroxyl on the pericardium, and the exogenous tea polyphenol provides additional phenolic hydroxyl; horseradish peroxidase and hydrogen peroxide will realize the chemical crosslinking of phenolic hydroxyl; the method provided by the invention can improve the structural stability and the anti-calcification performance of the biological material and potentially prolong the service life of the biological material.)

1. A method for treating a biological valve by combining enzyme crosslinking and tea polyphenol is characterized by comprising the following steps:

s1, obtaining the biological material;

s2, soaking and cleaning with distilled water;

s3, performing hydroxy phenylpropionic acid modification on the biological material cleaned in the step S2, wherein the molar concentration of the hydroxy phenylpropionic acid used in the biological material is 1mM-1M of aqueous solution, and the molar solubility of the carbodiimide/N-hydroxysuccinimide used in the biological material is 0.01-1M;

s4, soaking the biological material processed in the step S3 in tea polyphenol, wherein the mass concentration of the used tea polyphenol is 0.1-10%;

s5, performing horseradish peroxidase/hydrogen peroxide initiated enzyme crosslinking on the biological material treated in the step S4, wherein the mass concentration of the horseradish peroxidase is 0.1-10%, and the mass concentration of the hydrogen peroxide is 0.1-10%;

and S6, finally soaking and cleaning with distilled water.

2. The method of claim 1, wherein the tea polyphenols comprise one or more of Tannic Acid (TA), catechin (EC), gallocatechin (EGC), catechin gallate (ECG), and gallocatechin gallate (EGCG).

3. The method of claim 1, wherein the combination of enzymatic cross-linking and tea polyphenols comprises: the biological material is one or more of animal tissue including pericardium, valve, intestinal membrane, meninges, pulmonary membrane, blood vessel, skin or ligament.

4. The method of claim 1, wherein the combination of enzymatic cross-linking and tea polyphenols comprises: the biological material is not limited to percutaneous intervention biological heart valves, but also is suitable for biological materials used in open valve replacement surgery.

Technical Field

The invention relates to the technical field of biomedical materials and medical instruments, in particular to a method for treating a biological valve by combining enzyme crosslinking and tea polyphenol and a biological material thereof.

Background

Heart valve disease is a common valve failure disease. Anatomically manifested as narrowing of the blood access or incomplete valve closure.

Treatment of heart valve disease includes open chest valve replacement surgery and percutaneous heart valve replacement surgery. The thoracotomy has large trauma to patients, high risk, slow recovery and needs extracorporeal circulation support, which is unacceptable for many patients. Percutaneous heart valve replacement surgery is a main trend of valve surgery in the future because of small trauma and low risk to patients.

Biological heart valves refer to a class of biomedical materials used to replace diseased heart valves in humans. The biological heart valve is generally prepared by cross-linking porcine pericardium, bovine pericardium and the like through glutaraldehyde.

The glutaraldehyde crosslinking treatment has the characteristics of simple operation, low cost and high collagen crosslinking degree, and is the first choice in the chemical crosslinking industry of the existing biological heart valve. However, glutaraldehyde-crosslinked bioprosthetic heart valves suffer from the problems of easy degradation and calcification, resulting in a bioprosthetic heart valve that has an effective service life of only about 10 years. Glutaraldehyde can achieve stable crosslinking of collagen, but cannot crosslink elastin, resulting in certain technical limitations.

Therefore, by optimizing the chemical crosslinking method of the biological heart valve, especially developing a novel material processing method capable of improving the structural stability of elastin, the overall structural stability and calcification resistance of the biological heart valve will be improved, which is of great significance to scientific research and development of related industrial fields, and there is no good method at present, so improvement is needed.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provide a method for treating a biological valve by combining enzyme crosslinking and tea polyphenol, which can effectively improve the structural stability and anti-calcification performance of biological materials such as biological heart valves and the like and potentially prolong the service life of the biological heart valves.

The purpose of the invention is realized by the following technical scheme.

A method for treating a biological valve by combining enzyme crosslinking and tea polyphenol comprises the following steps:

s1, obtaining the biological material;

s2, soaking and cleaning with distilled water;

s3, performing hydroxy phenylpropionic acid modification on the biological material cleaned in the step S2, wherein the molar concentration of the hydroxy phenylpropionic acid used in the biological material is 1mM-1M of aqueous solution, and the molar solubility of the carbodiimide/N-hydroxysuccinimide used in the biological material is 0.01-1M;

s4, soaking the biological material processed in the step S3 in tea polyphenol, wherein the mass concentration of the used tea polyphenol is 0.1-10%;

s5, performing horseradish peroxidase/hydrogen peroxide initiated enzyme crosslinking on the biological material treated in the step S4, wherein the mass concentration of the horseradish peroxidase is 0.1-10%, and the mass concentration of the hydrogen peroxide is 0.1-10%;

and S6, finally soaking and cleaning with distilled water.

Further, in step S1: fresh biological material, such as porcine or bovine pericardial tissue, is collected and stored in a low temperature humidified state at 4 degrees celsius.

Further, in step S2, the pericardial tissue is washed with distilled water under shaking conditions using gentle friction and fluid pressure to remove the adherent non-pericardial and non-collagenous tissues, the washing is performed by osmotic shock to effectively decellularize the pericardial tissue, and preferably, the washing is continued until there is no visible adherent non-pericardial or non-collagenous tissue, preferably, the washing with distilled water under shaking conditions at 4 degrees celsius and 100RPM for 2 hours.

Further, in step S3, the washed biological material is modified with hydroxy phenylpropionic acid, and an aqueous solution with a molar concentration of 1mM-1M of hydroxy phenylpropionic acid is used, and this step is required to ensure that the hydroxy phenylpropionic acid reaches a physical permeation close to saturation, so as to introduce as much hydroxy phenylpropionic acid as possible.

Further, in step S4, the processed biological material is soaked in tea polyphenol, the mass concentration of the tea polyphenol is 0.1% -10%, and the tea polyphenol is adsorbed on the hydrophobic section of the elastin fiber through hydrogen bond action, so as to improve the structural stability of elastin.

Further, in step S5, the processed biomaterial is subjected to horseradish peroxidase/hydrogen peroxide-initiated enzyme crosslinking, the mass concentration of the horseradish peroxidase used is 0.1% to 10%, the mass concentration of the hydrogen peroxide used is 0.1% to 10%, the hydrogen peroxide is an oxidizing agent, and the horseradish peroxidase can catalyze phenolic hydroxyl groups to realize chemical crosslinking under the action of the oxidizing agent.

Further, in step S6, a distilled water washing step is performed, and this washing step removes unreacted tea polyphenol, horseradish peroxidase and hydrogen peroxide.

The invention has the beneficial effects that: the method provided by the invention can improve the structural stability and the anti-calcification performance of the biological material and potentially prolong the service life of the biological material.

Drawings

To further clarify the above and other advantages and features of one or more of the present inventions, a more particular description of one or more of the present inventions will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.

FIG. 1 is a detailed flow diagram of a prepared biological heart valve;

FIG. 2 is a schematic diagram of the chemical principle of para-hydroxy-phenylpropionic acid modified pericardium and horseradish peroxidase (HRP)/hydrogen peroxide (H2O2) enzyme crosslinking.

FIG. 3 is the percent weight loss for enzymatic degradation obtained using example 1.

FIG. 4 shows the results of quantitative measurement of insoluble elastin in example 1.

FIG. 5 shows the results of measurement of the amount of calcium suspended in a rat subcutaneously implanted in the rat obtained in example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.