阅读说明：本技术 一种改性心包膜及其制备方法、人工心脏瓣膜假体 (Modified pericardium, preparation method thereof and artificial heart valve prosthesis ) 是由 张兴 应佶杉 韩日峥 郭峰 杨锐 于 2021-02-25 设计创作，主要内容包括：本发明是关于一种改性心包膜及其制备方法、人工心脏瓣膜假体,主要采用的技术方案为：所述改性心包膜的制备方法,包括如下步骤：对动物心包膜进行脱细胞处理,得到脱细胞处理后的心包膜；对脱细胞处理后的心包膜上的羧基进行封端处理,得到封端心包膜；对封端心包膜进行京尼平交联处理,得到改性心包膜。优选的,在对封端心包膜进行京尼平交联处理的步骤之前,还包括对封端心包膜进行接枝两性离子聚合物处理的步骤。本发明通过封端、交联的手段对心包膜的羧基、氨基等活性基团进行改性,减少其与血液和离子的相互作用；在心包膜表面接枝两性离子聚合物,不仅提高人工心包瓣膜假体的血液相容性,还有效抵抗钙离子的聚集沉降,实现长期抗钙化功能。(The invention relates to a modified pericardium, a preparation method thereof and a prosthetic heart valve prosthesis, which mainly adopt the technical scheme that: the preparation method of the modified pericardium comprises the following steps: carrying out acellular treatment on the animal pericardium to obtain the acellular treated pericardium; performing end capping treatment on carboxyl on the decellularized pericardium to obtain an end capped pericardium; and carrying out genipin crosslinking treatment on the end-capped pericardium to obtain the modified pericardium. Preferably, before the step of carrying out genipin cross-linking treatment on the end-capped pericardium, the method further comprises the step of carrying out grafting zwitterionic polymer treatment on the end-capped pericardium. The invention modifies carboxyl, amino and other active groups of the pericardium by means of end capping and cross linking, so as to reduce the interaction with blood and ions; the amphoteric ion polymer is grafted on the surface of the cardiac envelope, so that the blood compatibility of the artificial pericardial valve prosthesis is improved, the aggregation and sedimentation of calcium ions are effectively resisted, and the long-term anti-calcification function is realized.)

1. The preparation method of the modified pericardium is characterized by comprising the following steps:

carrying out acellular treatment on the animal pericardium to obtain the acellular treated pericardium;

performing end capping treatment on carboxyl on the decellularized pericardium to obtain an end capped pericardium;

and carrying out genipin cross-linking treatment on the end-capped pericardium to obtain the modified pericardium.

2. The preparation method of the modified pericardium according to claim 1, characterized by further comprising the step of grafting a zwitterionic polymer to the capped pericardium before the step of subjecting the capped pericardium to genipin crosslinking treatment;

preferably, the zwitterionic polymer is one or more of poly sulfobetaine, poly carboxyl betaine and poly choline phosphate.

3. The method for preparing the modified pericardium according to claim 1 or 2, wherein the step of decellularizing the animal pericardium comprises:

soaking the animal pericardium with a surfactant; cleaning the soaked animal pericardium, soaking the animal pericardium in a mixed solution of DNase and RNase, and performing oscillation treatment at a set temperature to obtain a cell-free pericardium;

preferably, the surfactant is polyethylene glycol octyl phenyl ether solution with the volume fraction of 0.5-3%; preferably, the polyethylene glycol octyl phenyl ether solution is a solution obtained by dissolving polyethylene glycol octyl phenyl ether in tris (hydroxymethyl) aminomethane buffer solution;

preferably, the time for soaking the animal pericardium by the surfactant is 12-36 hours;

preferably, after the animal pericardium is soaked by the surfactant, the animal pericardium is washed by sterile phosphate buffered saline until no foam exists;

preferably, in the mixed solution of dnase and rnase: the concentration of the DNase is 10-1000U/mL, and the concentration of the RNase is 10-100 mug/mL;

preferably, the set temperature is 37 ± 2 ℃, preferably 37 ℃;

preferably, the time of the oscillation treatment is 8-24 h.

4. The preparation method of the modified pericardium according to claim 1 or 2, wherein the step of performing end-capping treatment on the carboxyl groups on the decellularized pericardium comprises:

firstly, soaking the pericardium subjected to the cell removal treatment in a hydroxyphenylethylamine solution; then, adding ethyl [3- (dimethylamino) propyl ] carbodiimide hydrochloride and N-hydroxysuccinimide to obtain a reaction solution, and carrying out end capping reaction to obtain an end-capped pericardium;

preferably, the concentration of the hydroxyphenylethylamine solution is 0.05-0.5 mol/L;

preferably, before the step of adding ethyl [3- (dimethylamino) propyl ] carbodiimide hydrochloride and N-hydroxysuccinimide, the pericardium after the decellularization treatment is soaked in a hydroxyphenylethylamine solution for 2-6 h;

preferably, the concentration of the ethyl [3- (dimethylamino) propyl ] carbodiimide hydrochloride in the reaction solution is 1 to 100 mmol/L;

preferably, the concentration of the N-hydroxysuccinimide in the reaction solution is 1-100 mmol/L;

preferably, the end capping reaction time is 12 to 48 hours;

preferably, during the end-capping reaction, the reaction solution is oscillated at a set rotation speed; further preferably, the set rotation speed is 145-155 rpm.

5. The preparation method of the modified pericardium according to claim 1 or 2, wherein the step of subjecting the capped pericardium to genipin cross-linking treatment comprises:

soaking the end-capped pericardium in 0.05-0.3 wt% of genipin solution to perform genipin cross-linking reaction to obtain a modified pericardium;

preferably, the genipin solution is prepared by dissolving genipin in phosphate buffer solution;

preferably, the temperature of the genipin crosslinking reaction is 37 +/-2 ℃, and preferably 37 ℃;

preferably, the genipin crosslinking reaction time is 8-24 h.

6. The preparation method of the modified pericardium according to claim 2, wherein the step of subjecting the capped pericardium to the treatment of the grafted zwitterionic polymer comprises:

grafting coupling molecule: soaking the end-capped pericardium in a coupling agent solution, and incubating to obtain the end-capped pericardium grafted with coupling molecules;

photocatalytic click reaction: soaking the end-capped pericardium grafted with the coupling molecules in a photocatalytic reaction solution prepared from a zwitterionic polymer solution and a photoinitiator, and performing photocatalytic click reaction under the irradiation of ultraviolet light to obtain the end-capped pericardium grafted with the zwitterionic polymer;

preferably, in the step of grafting the coupling molecule, the coupling agent is selected from a silane coupling agent or a thiolated polyethylene glycol;

preferably, in the step of grafting the coupling molecule, the coupling agent solution is a thioglycol solution with the concentration of 1-5 mmoL/L; preferably, the thiol-polyethylene glycol solution is prepared by dissolving thiol-polyethylene glycol in sodium bicarbonate buffer solution; more preferably, the concentration of the sodium bicarbonate buffer solution is 45-50mmol/L, and more preferably, the pH value of the sodium bicarbonate buffer solution is 8.5; further preferably, the average molecular weight of the mercapto-polyethylene glycol is 2000-8000, preferably 5000;

preferably, in the step of grafting the coupling molecule: the incubation time is 24-48h, and the incubation temperature is room temperature.

7. The method for preparing the modified pericardium according to claim 6, wherein in the step of photocatalytic click reaction:

firstly, mixing a zwitterionic polymer solution and a photoinitiator to form a photocatalytic reaction solution, and then soaking the end-capped pericardium of the grafted coupling molecule in the photocatalytic reaction solution; preferably, the concentration of the zwitterionic polymer solution is 5-20 mmol/L; preferably, the photoinitiator is an ethanol solution of 2-hydroxy-4- (2-hydroxyethoxy) -2-methylacetophenone; further preferably, the concentration of the ethanol solution of the 2-hydroxy-4- (2-hydroxyethoxy) -2-methylacetophenone is 0.1 to 0.5 g/mL; preferably, the volume ratio of the photoinitiator to the zwitterionic polymer is (0.5-1.5): (95-105), preferably 1: 100; and/or

Irradiating each of the front surface and the back surface of the end-capped pericardium of the grafted coupling molecules soaked in the photocatalytic reaction solution by ultraviolet light for 20-30 minutes; preferably, the wavelength of the ultraviolet light is 300-425nm, preferably 365 nm.

8. The modified pericardium is characterized in that the modified pericardium is obtained by carrying out acellular treatment on an animal pericardium, carrying out end-capping treatment on carboxyl on the animal pericardium and carrying out genipin cross-linking treatment on the animal pericardium;

preferably, the modified pericardium is obtained by performing acellular treatment on an animal pericardium, performing end-capping treatment on carboxyl on the animal pericardium, and performing grafting zwitterionic polymer treatment and genipin crosslinking treatment; further preferably, the zwitterionic polymer is one or more of poly-sulfobetaine, poly-carboxybetaine and choline polyphosphate;

preferably, the end-capping treatment of the carboxyl group is to modify the carboxyl group into the following group:

preferably, the modified pericardium is prepared by the preparation method of any one of claims 1-7.

9. A prosthetic heart valve prosthesis, comprising a biomass heart valve; the biomass heart valve is formed by cutting a modified pericardium according to a set size; wherein, the modified pericardium is prepared by the preparation method of the modified pericardium of claims 1-7; or the modified pericardium is the modified pericardium of claim 8.

10. The prosthetic heart valve prosthesis of claim 9, further comprising a valve stent; wherein the biomass heart valve is sewn inside the valve support;

preferably, the self-expandable nickel titanium stent is selected;

preferably, the opening size of the valve stent is 21-29 mm;

preferably, the effective opening area of the artificial heart valve prosthesis is 1.0-2.3cm²The reflux ratio is less than 25%.

Technical Field

The invention relates to the field of medical materials and devices, in particular to a modified pericardium, a preparation method thereof and a prosthetic heart valve prosthesis.

Background

With the increasing aging, the incidence of heart valve diseases is increasing. More than 200000 cases are worldwide where surgical procedures involve valve replacement each year. Traditional open chest valve replacement surgery requires that circulation be established in vitro, with high surgical risk. With the development of minimally invasive interventional technology in recent years, the transcatheter interventional heart valve operation does not need to open the chest, has low operation risk and provides a new treatment scheme for the elderly patients.

Currently, the interventional heart valves used clinically consist of a valve stent and a biomass valve, which has similar mechanical strength and hemodynamic properties to the native valve. And the biomass valve has higher biocompatibility and can support cell adhesion, proliferation and differentiation. However, calcification and biological enzymolysis can lead to a shorter life span of the biomass valve due to the immunogenic response caused by the xenogenic tissue.

Currently, commercial biomass valves are all sourced from glutaraldehyde-fixed xenogeneic tissues. The immunogenicity and biological enzymolysis of the biomass valve can be obviously reduced by using glutaraldehyde for fixation, and the inflammatory reaction after implantation can be reduced. However, the inventors of the present invention found that: since the foreign tissue fixed by glutaraldehyde does not block carboxyl and other groups with affinity for calcium ions in the cross-linking process, calcium ions are easy to concentrate and deposit at the carboxyl, and finally calcification of the valve is caused. Although other cross-linking agents are reported, they mainly include carbodiimides, epoxy compounds, etc.; however, these crosslinking methods give poor tissue stability.

Disclosure of Invention

In view of the above, the present invention provides a modified pericardium, a preparation method thereof, and a heart valve prosthesis, and mainly aims to block and modify active groups (such as carboxyl and amino groups) on an animal pericardium to prevent calcium ions from binding with the animal pericardium, so that the modified pericardium and the heart valve prosthesis have calcification-resistant performance.

In order to achieve the purpose, the invention mainly provides the following technical scheme:

in one aspect, an embodiment of the present invention provides a method for preparing a modified pericardium, including the following steps:

carrying out acellular treatment on the animal pericardium to obtain the acellular treated pericardium;

performing end capping treatment on carboxyl on the decellularized pericardium to obtain an end capped pericardium;

and carrying out genipin cross-linking treatment on the end-capped pericardium to obtain the modified pericardium.

Preferably, before the step of carrying out genipin cross-linking treatment on the blocked pericardium, the method further comprises the step of carrying out grafting zwitterionic polymer treatment on the blocked pericardium; preferably, the zwitterionic polymer is one or more of poly sulfobetaine, poly carboxyl betaine and poly choline phosphate.

Preferably, the step of subjecting the animal pericardium to a decellularization process comprises: soaking the animal pericardium with a surfactant; and cleaning the soaked animal pericardium, soaking the animal pericardium in a mixed solution of DNase and RNase, and performing oscillation treatment at a set temperature to obtain the pericardium subjected to cell removal treatment. Preferably, the surfactant is polyethylene glycol octyl phenyl ether solution with the volume fraction of 0.5-3%; preferably, the solution of octyl phenyl ether of polyethylene glycol is a solution obtained by dissolving octyl phenyl ether of polyethylene glycol in tris (hydroxymethyl) aminomethane buffer. Preferably, the time for soaking the animal pericardium by the surfactant is 12-36 hours. Preferably, after the animal pericardium is soaked by the surfactant, the animal pericardium is washed by sterile phosphate buffered saline until no foam exists. Preferably, in the mixed solution of dnase and rnase: the concentration of the DNase is 10-1000U/mL, and the concentration of the RNase is 10-100 mug/mL. Preferably, the set temperature is 37 ± 2 ℃, preferably 37 ℃. Preferably, the time of the oscillation treatment is 8-24 h.

Preferably, the step of performing a capping treatment on the carboxyl groups on the decellularized pericardium includes: firstly, soaking the pericardium subjected to the cell removal treatment in a hydroxyphenylethylamine solution; then, ethyl [3- (dimethylamino) propyl ] carbodiimide hydrochloride and N-hydroxysuccinimide are added to the obtained mixture to obtain a reaction solution, and end capping reaction is carried out to obtain the end-capped pericardium. Preferably, the concentration of the hydroxyphenylethylamine solution is 0.05-0.5 mol/L. Preferably, the pericardium after the decellularization treatment is soaked in a hydroxyl phenethylamine solution for 2 to 6 hours before the step of adding ethyl [3- (dimethylamino) propyl ] carbodiimide hydrochloride and N-hydroxyl succinimide. Preferably, the concentration of the ethyl [3- (dimethylamino) propyl ] carbodiimide hydrochloride in the reaction solution is 1 to 100 mmol/L. Preferably, the concentration of the N-hydroxysuccinimide in the reaction solution is 1 to 100 mmol/L. Preferably, the capping reaction time is 12 to 48 hours. Preferably, during the end-capping reaction, the reaction solution is oscillated at a set rotation speed; further preferably, the set rotation speed is 145-155 rpm.

Preferably, the step of subjecting the capped pericardium to genipin cross-linking treatment comprises: soaking the end-capped pericardium in 0.05-0.3% genipin solution to perform genipin crosslinking reaction to obtain a modified pericardium; preferably, the genipin solution is prepared by dissolving genipin in phosphate buffer solution; preferably, the temperature of the genipin crosslinking reaction is 37 +/-2 ℃, and preferably 37 ℃; preferably, the genipin crosslinking reaction time is 8-24 h.

Preferably, the step of subjecting the end-capped pericardium to the grafted zwitterionic polymer treatment comprises the following steps:

grafting coupling molecule: soaking the end-capped pericardium in a coupling agent solution, and incubating to obtain the end-capped pericardium grafted with coupling molecules;

photocatalytic click reaction: soaking the end-capped pericardium grafted with the coupling molecules in a photocatalytic reaction solution prepared from a zwitterionic polymer solution and a photoinitiator, and performing photocatalytic click reaction under the irradiation of ultraviolet light to obtain the end-capped pericardium grafted with the zwitterionic polymer;

preferably, in the step of grafting the coupling molecule, the coupling agent is selected from a silane coupling agent or a thiolated polyethylene glycol;

preferably, in the step of grafting the coupling molecule, the coupling agent solution is a thioglycol solution with the concentration of 1-5 mmoL/L; preferably, the thiol-polyethylene glycol solution is prepared by dissolving thiol-polyethylene glycol in sodium bicarbonate buffer solution; more preferably, the concentration of the sodium bicarbonate buffer solution is 45-50mmol/L, and more preferably, the pH value of the sodium bicarbonate buffer solution is 8.5; further preferably, the average molecular weight of the mercapto-polyethylene glycol is 2000-8000, preferably 5000;

preferably, in the step of grafting the coupling molecule: the incubation time is 24-48h, and the incubation temperature is room temperature.

Preferably, in the step of photocatalytic click reaction: firstly, mixing a zwitterionic polymer solution and a photoinitiator to form a photocatalytic reaction solution, and then soaking the end-capped pericardium of the grafted coupling molecule in the photocatalytic reaction solution; preferably, the concentration of the zwitterionic polymer solution is 5-20 mmol/L; preferably, the photoinitiator is an ethanol solution of 2-hydroxy-4- (2-hydroxyethoxy) -2-methylacetophenone; further preferably, the concentration of the ethanol solution of the 2-hydroxy-4- (2-hydroxyethoxy) -2-methylacetophenone is 0.1 to 0.5 g/mL; preferably, the volume ratio of the photoinitiator to the zwitterionic polymer is (0.5-1.5): (95-105), preferably 1: 100.

Preferably, in the step of photocatalytic click reaction: irradiating each of the front surface and the back surface of the end-capped pericardium of the grafted coupling molecules soaked in the photocatalytic reaction solution by ultraviolet light for 20-30 minutes; preferably, the wavelength of the ultraviolet light is 300-425nm, preferably 365 nm.

On the other hand, the embodiment of the invention also provides a modified pericardium, which is obtained by carrying out acellular treatment on an animal pericardium, carrying out end-capping treatment on carboxyl on the animal pericardium and carrying out genipin cross-linking treatment on the animal pericardium;

preferably, the modified pericardium is a pericardium obtained by performing acellular treatment on an animal pericardium, performing end-capping treatment on carboxyl on the animal pericardium, performing graft zwitterionic polymer treatment and genipin crosslinking treatment; further preferably, the zwitterionic polymer is one or more of poly-sulfobetaine, poly-carboxybetaine and choline polyphosphate;

preferably, the end-capping treatment of the carboxyl group is to modify the carboxyl group into the following group:

preferably, the modified pericardium is prepared by the preparation method of any one of the above methods.

In yet another aspect, embodiments of the present invention provide a prosthetic heart valve prosthesis, wherein the prosthetic heart valve prosthesis comprises a biomass heart valve; the biomass heart valve is formed by cutting a modified pericardium according to a set size; the modified pericardium is prepared by the preparation method of any one of the modified pericardium; or the modified pericardium is the modified pericardium.

Preferably, the prosthetic heart valve prosthesis further comprises a valve stent; wherein the biomass heart valve is sewn inside the valve support; preferably, the valve stent is a self-expanding nickel titanium stent; preferably, the opening size of the valve stent is 21-29 mm; preferably, the effective opening area of the artificial heart valve prosthesis is 1.0-2.3cm²The reflux ratio is less than 25%.

Compared with the prior art, the modified pericardium, the preparation method thereof and the artificial heart valve prosthesis have at least the following beneficial effects:

on the one hand, according to the modified pericardium and the preparation method thereof provided by the embodiment of the invention, the animal pericardium is subjected to decellularization treatment, carboxyl end capping treatment and genipin crosslinking treatment in sequence, so that the active groups such as carboxyl, amino and the like on the pericardium are subjected to end capping and modification (it is to be noted that the carboxyl and amino on the pericardium are mainly reacted, and if some active groups such as double bonds and hydroxyl groups are present, the reaction may occur, but is not the main reaction needed), and the interaction between the active groups and blood and ions (such as calcium ions) is reduced. Therefore, when the modified pericardium prepared by the embodiment of the invention is used as a biomass valve on a prosthetic heart valve prosthesis, calcium ions can be prevented from being combined with the modified pericardium, so that the biomass valve has anti-calcification performance, and the service life of a valve device can be prolonged.

Further, according to the modified pericardium and the preparation method thereof provided by the embodiment of the invention, on the basis, the treatment of grafting zwitterions is performed, so that the adhesion of blood platelets in blood can be effectively prevented, and the aggregation and permeation of calcium ions in the modified pericardium can be avoided. Therefore, when the modified pericardium prepared by the embodiment of the invention is used as a biomass valve on a prosthetic heart valve prosthesis, the blood compatibility of the prosthetic heart valve prosthesis can be improved, the aggregation and sedimentation of calcium ions can be effectively resisted, and the long-term anti-calcification function is realized.

On the other hand, the embodiment of the invention also provides a prosthetic heart valve prosthesis, wherein the biomass valve in the prosthetic heart valve prosthesis is formed by cutting and sewing the modified pericardium according to a set size, so that the prosthetic heart valve prosthesis provided by the embodiment of the invention has the beneficial effects. In addition, the artificial heart valve prosthesis provided by the embodiment of the invention has excellent hydrodynamic performance, and the effective opening area is 1.0-2.3cm²Ratio of backflow<25％。

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a topography picture of a fluid scour calcification simulation experiment in vitro of a pericardium after a decellularization treatment for 14 days;

FIG. 2 is a topographical picture of the modified pericardium prepared in example 1 after a fluid erosion calcification simulation experiment for 14 days in vitro;

FIG. 3 is a topographical picture of the modified pericardium prepared in example 2 after a fluid erosion calcification simulation experiment for 14 days in vitro;

FIG. 4 is a surface topography picture of a pericardium after decellularization treatment after a platelet adhesion experiment;

FIG. 5 is a surface topography picture of the modified pericardium prepared in example 2 after a platelet adhesion experiment;

FIG. 6 is a schematic view of a prosthetic heart valve prosthesis prepared in example 3;

FIG. 7 is a graph of the hydrodynamic test of the prosthetic heart valve prosthesis prepared in example 3;

FIG. 8 is a graph of the hydrodynamic test of the prosthetic heart valve prosthesis prepared in example 4;

FIG. 9 is a graph of the hydrodynamic test of the prosthetic heart valve prosthesis prepared in example 5;

fig. 10 is a graph showing the hydrodynamic test of the prosthetic heart valve prosthesis prepared in example 6.

Detailed Description

To further explain the technical means and effects of the present invention adopted to achieve the predetermined object, the following detailed description of the embodiments, structures, features and effects according to the present invention will be made with reference to the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Genipin is obtained by enzymolysis of geniposide separated from fructus Gardeniae with b-glucosidase. Genipin exhibits high cross-linking properties in animal tissue through linkage with primary amines; and because no aldehyde group is involved in the crosslinking process, the crosslinked product has higher biocompatibility.

The zwitterionic polymer is a high molecular material which is electrically neutral as a whole and simultaneously contains anionic and cationic groups on the same monomer side chain, and widely exists in nature, and comprises penetrating fluid, cell membranes, proteins and the like. Amphoteric ion is grafted in the cardiac envelope, so that the adhesion of blood platelet and the electrostatic adsorption of calcium ion can be effectively resisted.

The invention aims at the problems of short service life and easy calcification of the existing biomass valve. The animal pericardium (the animal pericardium is a film which surrounds the outside of the heart of an animal, the animal pericardium can be a pig pericardium or a bovine pericardium and the like) is used as a raw material, the carboxyl end capping technology and genipin cross-linking are utilized to treat the pericardium, so that the problems of cytotoxicity and ion aggregation caused by free aldehyde groups existing in the conventional biomass artificial valve are solved, and the amphoteric ion polymer is grafted on the surface of the pericardium, so that the problems of aggregation and permeation of calcium ions in the pericardium are solved. And the calcification-resisting performance of the artificial valve is characterized in vitro, and the treated valve is sewn by an interventional valve device.

The specific scheme of the invention is as follows:

in one aspect, an embodiment of the present invention provides a method for preparing a modified pericardium, including the following steps:

and (3) cell removal treatment: and (3) carrying out cell removal treatment on the animal pericardium to obtain the cell-removed pericardium.

The method comprises the following steps: the animal pericardium is soaked in 0.5-3% (v/v) volume fraction of octyl phenyl polyglycol ether solution (the term "soaking" as used in this invention means that the solution can submerge the pericardium; preferably, 0.5-3mL solution is soaked in per square centimeter of pericardium) until the pericardium is ruptured, and the soaking time is 12-36 hours. Then cleaning the animal pericardium (thoroughly cleaning with sterile Phosphate Buffered Saline (PBS), rinsing until no foam exists), soaking in a mixed solution of DNase and RNase (the concentration of DNase is 10-1000U/mL, the concentration of RNase is 10-100 mu g/mL), and performing oscillation treatment at 37 +/-2 ℃ and preferably 37 ℃ (overnight oscillation and oscillation time is 8-24h) to remove organelles on the animal pericardium and substances such as DNA and RNA in cells, so as to reduce the immunogenicity of the implanted device and remove easily calcified components such as phospholipid.

The polyethylene glycol octyl phenyl ether solution is prepared by dissolving polyethylene glycol octyl phenyl ether in Tris (hydroxymethyl) aminomethane buffer solution (10mmol/L Tris, pH 8.0).

End-capping treatment: and performing end capping treatment on carboxyl on the decellularized pericardium to obtain an end capped pericardium.

The method comprises the following steps: firstly, adding the decellularized pericardium into a hydroxyphenylethylamine solution (the concentration of the hydroxyphenylethylamine solution is 0.05-0.5mol/L), soaking for 2-6 hours (so as to enable the hydroxyphenylethylamine to be fully soaked into the decellularized pericardium), then adding N-hydroxysuccinimide (NHS) and ethyl [3- (dimethylamino) propyl ] carbodiimide hydrochloride (EDC) into the decellularized pericardium to obtain a reaction solution (the final concentration of the NHS and the EDC in the reaction solution is 1-100mmol/L), and oscillating at 150RPM for 12-48 hours to perform end capping reaction. Wherein, the chemical reaction formula is shown as the following formula, wherein R represents collagen molecules in the pericardium.

Here, in the presence of EDC/NHS, hydroxyphenylethylamine binds to carboxyl groups on the surface and inside of the pericardium, thereby reducing valve calcification caused by the binding of carboxyl groups to calcium ions in the blood.

Crosslinking treatment of genipin: and carrying out genipin cross-linking treatment on the end-capped pericardium to obtain the modified pericardium.

The method comprises the following steps: soaking the end-capped pericardium in 0.05-0.3 wt% genipin solution, and performing crosslinking reaction at 37 + -2 deg.C (preferably 37 deg.C) for 8-24 hr. The chemical reaction formula is shown as the following formula, wherein R represents a collagen molecule in the pericardium.

Preferably, before the step of performing genipin cross-linking treatment on the capped pericardium, the method further comprises a step of performing graft zwitterionic polymer treatment on the capped pericardium, specifically as follows:

graft zwitterionic Polymer: and (3) carrying out grafting zwitterionic polymer treatment on the end-capped pericardium.

In this step: firstly, end-capped pericardium is grafted with coupling molecules, and then the zwitterionic polymer is combined with the coupling molecules through a click chemistry method to form long-chain molecules. The zwitterionic polymer includes, but is not limited to, poly sulfobetaine (pSBMA), poly carboxybetaine (pCBMA), poly choline phosphate (pMPC), etc., and the coupling molecule may be a silane coupling agent, a thiolated polyethylene glycol molecule, etc.

Preferably, the steps are specifically:

grafting coupling molecule: and soaking the end-capped pericardium in a coupling agent solution, and incubating to obtain the end-capped pericardium grafted with coupling molecules.

After incubation, the capped pericardium, grafted with coupling molecules, was washed thoroughly in phosphate buffer to remove residual reagents.

Photocatalytic click reaction: soaking the end-capped pericardium grafted with the coupling molecules in a photocatalytic reaction solution prepared from a zwitterionic polymer solution and a photoinitiator, and carrying out photocatalytic click reaction under the irradiation of ultraviolet light to obtain the end-capped pericardium grafted with the zwitterionic polymer.

The zwitterionic polymer solution is prepared by dissolving a zwitterionic polymer in deionized water.

The photocatalytic click reaction comprises the following specific steps: firstly, mixing a zwitterionic polymer solution and a photoinitiator to obtain a photocatalytic reaction solution, and then soaking the end-capped pericardium of the grafted coupling molecule in the photocatalytic reaction solution. The concentration of the zwitterionic polymer solution is 5-20 mmol/L. The photoinitiator is an ethanol solution of 2-hydroxy-4- (2-hydroxyethoxy) -2-methylacetophenone. The concentration of the ethanol solution of the 2-hydroxy-4- (2-hydroxyethoxy) -2-methylacetophenone is 0.1 to 0.5 g/mL. The volume ratio of the photoinitiator to the zwitterionic polymer is (0.5-1.5): (95-105), preferably 1: 100. Irradiating each of the front surface and the back surface of the end-capped pericardium of the grafted coupling molecules soaked in the photocatalytic reaction solution by ultraviolet light for 20-30 minutes; wherein the wavelength of the ultraviolet light is 365 nm.

The specific procedure for grafting the zwitterionic polymer is illustrated here by the example of the coupling molecule being mercaptopolyethylene glycol (MW 5000): thiol polyethylene glycol (MW5000) was dissolved in 50mmol/L sodium bicarbonate buffer (pH 8.5) to give a coupling agent solution (wherein the concentration of thiol polyethylene glycol was 1 to 5 mmol/L). The capped pericardium was soaked in the coupling agent solution and incubated at room temperature for 24-48 hours. After the reaction, the resulting thiolated capped pericardium was washed well in phosphate buffer to remove residual reagents. The chemical reaction formula is shown as the following formula (wherein the black bars in the chemical reaction formula represent collagen molecules on the pericardium):

mixing a photoinitiator and a zwitterionic polymer solution according to a volume ratio of (0.5-1.5): (95-105), preferably in a ratio of 1:100, to obtain a photocatalytic reaction solution. The thiolated blocked pericardium is soaked in a photocatalytic reaction solution, and ultraviolet light with the wavelength of 300-425nm is irradiated on each side (front side and back side) of the thiolated blocked pericardium for 20-30 minutes to fully react.

The chemical reaction formula is shown as the following formula:

in summary, the preparation method of the modified pericardium provided by the embodiment of the present invention mainly adopts two schemes to modify the animal pericardium. The first scheme is as follows: and sequentially carrying out decellularization treatment, end-capping treatment and genipin crosslinking treatment on the animal pericardium to obtain the modified pericardium. The second scheme is as follows: and sequentially carrying out decellularization treatment, end-capping treatment, grafting of a zwitterionic polymer and genipin crosslinking treatment on the animal pericardium to obtain the modified pericardium. Here, it should be noted that: the order of processing of each step in the two schemes is critical and ensures that the capping treatment, genipin cross-linking, and grafting of the zwitterionic polymer are successful.

On the other hand, the embodiment of the invention provides a modified pericardium, which is mainly prepared by the preparation method of the modified pericardium.

The pericardium prepared by the preparation method of the modified pericardium or the modified pericardium provided by the preparation method is mainly used as a biomass valve.

In yet another aspect, embodiments of the present invention provide a prosthetic heart valve prosthesis, wherein the prosthetic heart valve prosthesis comprises a biomass heart valve and a valve stent. Specifically, the modified pericardium is cut into a specific size, and the modified pericardium is sewn inside the valve stent by using a medical suture line with the aid of medical polyester fabric. After sewing, the three valve leaflets can be well jointed in a natural state.

The invention is further illustrated by the following specific examples:

example 1

In this embodiment, a fresh and healthy bovine pericardium is used as a raw material, and the pericardium is modified to obtain a modified pericardium. The modified pericardium may be used as an anticalcific biomass valve. The specific modification steps are as follows:

and (3) cell removal treatment: dissolving polyethylene glycol octyl phenyl ether in Tris (hydroxymethyl) aminomethane buffer (10mmol/L Tris, pH 8.0) to obtain polyethylene glycol octyl phenyl ether solution as a surfactant, wherein the volume fraction of the polyethylene glycol octyl phenyl ether solution is 1% (v/v); the bovine pericardium is soaked by the surfactant for 24 hours. The bovine pericardium soaked with surfactant was then thoroughly washed with sterile Phosphate Buffered Saline (PBS) and rinsed until free of foam. Finally, bovine pericardium was soaked with a mixed solution of DNase and RNase (DNase concentration 100U/mL and RNase concentration 20. mu.g/mL), and the resulting solution was shaken at 37 ℃ for 12 hours to obtain a decellularized pericardium.

End-capping treatment: the pericardium after the cell removal treatment was soaked with 0.2mol/L hydroxyphenylethylamine (tyramine) solution for 4 hours. Then, N-hydroxysuccinimide (NHS) and ethyl [3- (dimethylamino) propyl ] carbodiimide hydrochloride (EDC) were added thereto to obtain a reaction solution (the final concentration of NHS and EDC in the reaction solution was 10mmol/L), and the reaction solution was constantly shaken at 150rpm for 24 hours to obtain a capped pericardium.

Crosslinking treatment of genipin: dissolving genipin in phosphate buffer solution to obtain 0.1 wt% genipin solution; and (3) putting the sealed pericardium into the genipin solution (the sealed pericardium is immersed in the genipin solution), and reacting at the temperature of 37 ℃ for 12 hours to obtain the modified pericardium.

The modified pericardium prepared in this example and the pericardium after the simple decellularization treatment were subjected to in vitro fluid scour calcification test. Wherein, the appearance picture of the pericardium after the pure decellularization treatment after the in vitro 14 days fluid scouring calcification simulation experiment is shown in figure 1. The morphological picture of the modified pericardium prepared in this example after the in vitro 14-day fluid erosion calcification simulation experiment is shown in fig. 2.

As is apparent from fig. 2, the modified pericardium prepared in this example has better calcification resistance.

Example 2

In this embodiment, a fresh and healthy bovine pericardium is used as a raw material, and the pericardium is modified to obtain a modified pericardium. The modified pericardium may be used as an anticalcific biomass valve. The specific modification steps are as follows:

and (3) cell removal treatment: this step corresponds to the step of the decellularization treatment in example 1.

End-capping treatment: this step corresponds to the step of the end-capping treatment in example 1.

Graft zwitterionic Polymer: (1) grafting coupling molecule: sulfhydryl polyethylene glycol (MW5000) and 50mmol/L sodium bicarbonate buffer (pH 8.5) are prepared into a coupling agent solution, wherein the concentration of the sulfhydryl polyethylene glycol in the coupling agent solution is 3 mmol/L. The capped pericardium was soaked in the coupling agent solution and incubated at room temperature for 24 hours. After incubation, the capped pericardium (thiolated capped pericardium) grafted with the coupling molecule was washed thoroughly in phosphate buffer to remove residual reagents.

(2) Photocatalytic click reaction: mixing a photoinitiator with a zwitterionic polymer solution with the concentration of 12mol/L according to the volume ratio of 1:100 to obtain a photocatalytic reaction solution (wherein the photoinitiator is an ethanol solution of 2-hydroxy-4- (2-hydroxyethoxy) -2-methylacetophenone, and the concentration of the 2-hydroxy-4- (2-hydroxyethoxy) -2-methylacetophenone is 0.3 g/mL). And soaking the thiolated blocked pericardium in a photocatalytic reaction solution, and irradiating ultraviolet light with the wavelength of 365nm on each surface (front and back surfaces) of the thiolated blocked pericardium for 30 minutes to fully react to obtain the blocked pericardium grafted with the zwitterionic polymer.

Crosslinking treatment of genipin: dissolving genipin in phosphate buffer solution to obtain 0.1 wt% genipin solution; and (3) putting the end-capped pericardium grafted with the zwitterionic polymer into the genipin solution (the end-capped pericardium grafted with the zwitterionic polymer is immersed in the genipin solution), and reacting at the temperature of 37 ℃ for 12 hours to obtain the modified pericardium.

The modified pericardium prepared in this example and the pericardium after the simple decellularization treatment were subjected to in vitro fluid scour calcification test. Wherein, the appearance picture of the pericardium after the pure decellularization treatment after the in vitro 14 days fluid scouring calcification simulation experiment is shown in figure 1. The morphological picture of the modified pericardium prepared in this example after the in vitro 14-day fluid erosion calcification simulation experiment is shown in fig. 3. As is apparent from fig. 3, the modified pericardium prepared in this example has better calcification resistance.

Platelet adhesion tests were performed on the modified pericardium prepared in this example and on the pericardium that was simply decellularized. FIG. 4 is a surface topography picture of a pericardium after decellularization treatment after a platelet adhesion experiment; fig. 5 is a surface topography picture of the modified pericardium prepared in example 2 after a platelet adhesion experiment. As can be seen from fig. 4 and 5: compared with the pericardium after pure acellular treatment, the modified pericardium prepared by the embodiment has no platelet adhesion phenomenon basically.

Example 3

The embodiment of the invention prepares a prosthetic heart valve prosthesis, which comprises the following specific steps:

preparing a modified pericardium: this example the procedure for preparing a modified pericardium was identical to that of example 1.

Suturing of the interventional valve: and cutting the prepared modified pericardium into a specific size. The modified pericardium cut into a specific size is sewn inside the valve stent with the inner diameter of 21mm by using a medical suture line with the aid of medical polyester fabric. After sewing, the three valve leaflets can be well attached in a natural state, and the obtained artificial heart valve prosthesis is shown in fig. 6.

Performing fluid dynamics evaluation on the artificial heart valve prosthesis of the embodiment; the evaluation results are shown in fig. 7, and the results show that: the artificial heart valve prosthesis prepared by the embodiment has good fluid dynamic performance, and the effective opening area is more than 1.02cm²The reflux ratio is less than 3.2 percent and reaches the ISO-5840-3 standard.

Example 4

The embodiment of the invention prepares a prosthetic heart valve prosthesis, which comprises the following specific steps:

preparing a modified pericardium: this example the procedure for preparing a modified pericardium was identical to that of example 1.

Suturing of the interventional valve: and cutting the prepared modified pericardium into a specific size. The modified pericardium cut into a specific size is sewn inside the valve stent with the inner diameter of 25mm by using a medical suture line with the aid of medical polyester fabric. After sewing, the three valve leaflets can be well jointed in a natural state, and the artificial heart valve prosthesis is obtained.

Performing fluid dynamics evaluation on the artificial heart valve prosthesis of the embodiment; the evaluation results are shown in fig. 8, and the results show that: the artificial heart valve prosthesis prepared by the embodiment has good fluid dynamic performance, and the effective opening area is more than 1.73cm²The reflux ratio is less than 6.2 percent and reaches the ISO-5840-3 standard.

Example 5

The embodiment of the invention prepares a prosthetic heart valve prosthesis, which comprises the following specific steps:

preparing a modified pericardium: this example the procedure for preparing a modified pericardium was identical to that of example 1.

Suturing of the interventional valve: and cutting the prepared modified pericardium into a specific size. The modified pericardium cut into a specific size is sewn inside the valve stent with the inner diameter of 29mm by using a medical suture line with the aid of medical polyester fabric. After sewing, the three valve leaflets can be well jointed in a natural state, and the artificial heart valve prosthesis is obtained.

Performing fluid dynamics evaluation on the artificial heart valve prosthesis of the embodiment; the evaluation results are shown in fig. 9, and the results show that: the artificial heart valve prosthesis prepared by the embodiment has good fluid dynamic performance, and the effective opening area is more than 1.21cm²The reflux ratio is less than 11.8 percent and reaches the ISO-5840-3 standard.

Example 6

The embodiment of the invention prepares a prosthetic heart valve prosthesis, which comprises the following specific steps:

preparing a modified pericardium: this example corresponds to the procedure of example 2 for the preparation of a modified pericardium.

Suturing of the interventional valve: and cutting the prepared modified pericardium into a specific size. The modified pericardium cut into a specific size is sewn inside the valve stent with the inner diameter of 21mm by using a medical suture line with the aid of medical polyester fabric. After sewing, the three valve leaflets can be well jointed in a natural state, and the artificial heart valve prosthesis is obtained.

Performing fluid dynamics evaluation on the artificial heart valve prosthesis of the embodiment; the evaluation results are shown in fig. 10, and the results show that: the artificial heart valve prosthesis prepared by the embodiment has good fluid dynamic performance, and the effective opening area is more than 1.20cm²The reflux ratio is less than 4.1 percent and reaches the ISO-5840-3 standard.

In summary, the modified pericardium, the preparation method thereof and the artificial heart valve prosthesis provided by the embodiment of the invention modify active groups such as carboxyl, amino and the like on the pericardium by means of end capping and cross linking, so that the interaction between the modified pericardium and blood and ions is reduced; the amphoteric ion polymer is grafted on the surface of the cardiac envelope, so that the blood compatibility of the artificial pericardial valve prosthesis can be improved, the aggregation and sedimentation of calcium ions can be effectively resisted, and the long-term calcification resisting function is realized.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are still within the scope of the technical solution of the present invention.