CN114748695B

CN114748695B - Method for improving calcification and anticoagulation of biological valve material by double-bond post-crosslinking

Info

Publication number: CN114748695B
Application number: CN202210273141.5A
Authority: CN
Inventors: 王云兵; 郑城; 杨立
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-11-17
Filing date: 2022-03-18
Publication date: 2022-12-23
Anticipated expiration: 2042-03-18
Also published as: CN114748693B; CN116271236A; CN114748696A; CN115645618B; CN114748696B; CN114748697A; CN114748693A; CN114748695A; CN115970060B; CN114748694A; CN115645618A; CN115970060A; CN114748697B; CN115779149B; CN115779149A

Abstract

The application discloses a method for improving calcification resistance and anticoagulation of a biological valve material by double-bond post-crosslinking, (1) soaking the biological material in a solution containing a first functional monomer for physical permeation; the first functional monomer has at least one amino group and at least one carbon-carbon double bond; (2) Adding an aldehyde crosslinking agent into the system in the step (1) for co-crosslinking; (3) Soaking the biological material treated in the step (2) in a solution containing a second functional monomer for physical permeation; the second functional monomer has at least one carbon-carbon double bond and at least one functional group B; (4) And (4) adding an initiator into the system in the step (3) to initiate double bond polymerization. According to the method, more and larger polymer cross-linked networks are formed through two times of cross-linking, so that the cross-linking degree of the biological material is improved, and the anti-calcification performance is improved; and an additional functional group is introduced while the carbon-carbon double bond is introduced for the second time, so that new characteristics can be endowed to the biological material, and the performance of the biological material is further improved.

Description

Method for improving calcification and anticoagulation of biological valve material by double-bond post-crosslinking

Technical Field

The invention relates to the technical field of intervention materials, in particular to a double-bond post-crosslinking biological valve material and preparation and application thereof.

Background

The biological heart valve is usually prepared by adopting porcine or bovine pericardium and is used for replacing the heart valve of a human body with function defect; biological heart valves have many advantages over mechanical heart valves: after the biological heart valve is implanted, a patient does not need to take anticoagulant drugs for a long time, and the biological heart valve can adopt a minimally invasive intervention operation mode, so that the advantages of the biological heart valve gradually become the main stream of the market in clinical application.

Almost all biological valve products on the current market are prepared by crosslinking glutaraldehyde, which can crosslink collagen in pericardium, but the glutaraldehyde-crosslinked biological valve has the defect of poor blood compatibility, so that the life of the valve in vivo is limited.

Disclosure of Invention

The application provides a method for improving calcification resistance and anticoagulation property of a biological valve material through double-bond post-crosslinking, and solves the problem of thrombus of a glutaraldehyde crosslinking membrane.

A method for improving calcification and anticoagulation of a biological valve material by double-bond post-crosslinking, which comprises the following steps:

(1) Soaking the biological material in a solution containing a first functional monomer for physical permeation; the first functional monomer has at least one amino group and at least one carbon-carbon double bond;

(2) Adding an aldehyde crosslinking agent into the system in the step (1) to carry out co-crosslinking;

(3) Soaking the biological material treated in the step (2) in a solution containing a second functional monomer for physical permeation; the second functional monomer has at least one carbon-carbon double bond and at least one functional group B;

(4) And (4) adding an initiator into the system in the step (3) to initiate double bond polymerization.

Optionally, the polyaldehyde crosslinking agent is glutaraldehyde or formaldehyde.

Optionally, the solvent of the solution in the step (1) is water, normal saline, pH neutral buffer solution or ethanol water solution; the concentration of the functional monomer in the solution is 10-100 mM; the soaking time is 2-20 h.

Optionally, in the step (2), the concentration of the cross-linking agent is 10-800 mM; the co-crosslinking time is 10-30 h.

Optionally, before step (3), further comprising step (2M): soaking the biological material treated in the step (2) in a solution containing a third functional monomer to eliminate residual aldehyde groups; the third functional monomer has at least one group that reacts with an aldehyde group; the group reacting with the aldehyde group is one of amino and hydrazide.

Optionally, in step (2M), the solvent of the solution is water, physiological saline, pH neutral buffer solution or an aqueous solution of ethanol; the concentration of the third functional monomer in the solution is 10-100 mM; the soaking time is 2-48 h.

Optionally, the first functional monomer and the third functional monomer are each independently selected from one of 2-methallyl amine, 3-buten-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazine, and acrylyl hydrazine.

Optionally, the first functional monomer and the third functional monomer further have at least one functional group a; the functional groups A of the first functional monomer and the third functional monomer are respectively and independently selected from at least one of hydroxyl, carboxyl, amide and sulfonic acid.

Optionally, the first functional monomer and the third functional monomer are each independently selected from one of DL-2-amino-4-pentenoic acid, 2-amino-7-ene-octanoic acid, 6-ene-heptanoic acid, 2-aminopent-4-enoic acid, 4- (1-amino-2-methyl-propyl) -hepta-1, 6-dien-4-ol, 4- (1-amino-ethyl) -hepta-1, 6-dien-4-ol, doubly bonded polylysine.

Optionally, the functional group B is one of a hydroxyl group, a carboxyl group, a choline carboxylate, a choline sulfonate, a choline phosphate, a pyrrolidone, a sulfonic acid group, a carboxylate ion, a sulfonate, a sulfoxide, an amide group, and a methoxy group.

Optionally, the second functional monomer is one of acrylamide, 2- (prop-2-enamino) acetic acid, 2-acrylamido-2-methylpropanesulfonic acid, hydroxyethyl methacrylate, 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate, N-methyl-2-acrylamide, N-isopropylacrylamide, N- (hydroxymethyl) acrylamide, N- (2-hydroxyethyl) methacrylamide, N-dimethylmethacrylamide, 3- [ N, N-dimethyl- [2- (2-methylprop-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt, 2-methacryloyloxyethylphosphate, doubly-bonded hyaluronic acid, doubly-bonded polylysine.

Optionally, in the step (3), adding a second functional monomer into the system treated in the previous step; or cleaning the biological material treated in the previous step and then soaking the biological material in a solution containing a second functional monomer.

Optionally, the solvent in the solution containing the second functional monomer is water, normal saline, pH neutral buffer solution or an aqueous solution of ethanol; the mass percentage concentration of the second functional monomer is 1-10%; the soaking time is 2-20 h.

Optionally, the initiator is a mixture of ammonium persulfate and sodium bisulfite, and the concentrations of the ammonium persulfate and the sodium bisulfite are respectively 10-100 mM; or

The initiator is a mixture of ammonium persulfate and N, N, N ', N' -tetramethyl ethylenediamine, and the mass percentage concentrations of the ammonium persulfate and the N, N, N ', N' -tetramethyl ethylenediamine are respectively 2% -5% and 0.2% -0.5%.

The application also provides a double-bond post-crosslinking biological valve material prepared by the method.

The application also provides a biological valve, which comprises a stent and a valve, wherein the valve is made of the double-bond post-crosslinking biological valve material.

Optionally, the biological valve is a heart valve.

Compared with the prior art, the application has at least one of the following beneficial effects:

(1) According to the method, the biological material is subjected to crosslinking treatment through aldehyde group co-crosslinking and double bond polymerization secondary crosslinking, and the biological material obtained through the secondary crosslinking treatment has good crosslinking degree;

(2) According to the method, a second functional monomer is added in the double bond polymerization step for copolymerization to form more and larger polymer cross-linked networks, so that the cross-linking degree of the biological material can be improved, and the anti-calcification performance can be improved;

(3) In the co-crosslinking process, partial residual aldehyde groups can be blocked while carbon-carbon double bonds are introduced;

(4) After the co-crosslinking is finished, the functional monomer solution is soaked again, and carbon-carbon double bonds are introduced again through chemical reaction while residual aldehyde groups are eliminated, so that the subsequent double bond polymerization step is facilitated, and the crosslinking degree of the biological material is further improved.

(5) According to the application, the carbon-carbon double bond is introduced for the second time, and the additional functional group is introduced at the same time, so that new characteristics can be endowed to the biological material, and the performance of the biological material is further improved.

Drawings

FIG. 1 is a process flow diagram of a more preferred embodiment of the present application;

FIG. 2 is a schematic diagram of the reaction of a more preferred embodiment of the present application;

FIG. 3 is a schematic diagram of a reaction according to another preferred embodiment of the present application;

FIG. 4 is an infrared spectrum of pericardium (GA) of sample 1 and control 1 of example 1;

FIG. 5 is a graph showing the results of quantifying elastin in sample 1 and control group 1 pericardium (GA) rats after subcutaneous implantation in example 1;

FIG. 6 is a schematic diagram showing the measurement of calcium content of pericardium (GA) rats subcutaneously implanted in sample 1 and control group 1 of example 1;

FIG. 7 is a schematic diagram of the water contact angle of sample 2 of example 2 with the pericardium (GA) of control 2;

FIG. 8 is a graph showing the detection of pericardium (GA) lactate dehydrogenase and the hemolysis rate of sample 2 and control 2 of example 2;

FIG. 9 is a graph showing the concentration of calcium ion in pericardium (GA) of sample 2 and control 2 of example 2;

FIG. 10 is a basic schematic diagram of embodiment 3;

FIG. 11 is a scanning electron micrograph of blood of a control of control group 3;

FIG. 12 is a scanning electron micrograph of a blood incubation experiment of sample 3;

FIG. 13 is a scanning electron micrograph of a blood incubation experiment of sample 4;

FIG. 14 is a photograph of alizarin red stained sections taken 60 days after control 3 control implantation;

FIG. 15 is a photograph of alizarin red stained sections obtained 60 days after sample 3 was implanted;

FIG. 16 is a photograph of alizarin red stained sections taken 60 days after sample 4 implantation;

FIG. 17 is a photograph of alizarin red stained sections taken 60 days after sample 5 implantation;

FIG. 18 is a photograph of alizarin red stained sections obtained 60 days after sample 6 implantation;

fig. 19 is a photograph of alizarin red stained sections obtained 60 days after sample 7 implantation.

Detailed Description

The technical solutions in the embodiments of the present application will be described clearly and completely with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making any creative effort belong to the protection scope of the present application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The biological valve products on the current market are mainly glutaraldehyde cross-linked membranes, glutaraldehyde can cross-link collagen in the pericardium, however, the biological valve cross-linked by glutaraldehyde is not ideal in calcification resistance and anticoagulation performance, is easy to cause thrombus, and threatens the life quality and life of patients. On one hand, the mechanical property, the calcification resistance and the anticoagulation property of the glutaraldehyde crosslinking membrane are improved by improving a crosslinking means on the basis of glutaraldehyde crosslinking; on the other hand, a functional group with a specific function is introduced on the basis of improving a crosslinking means, so that the performance of the biomaterial, such as biocompatibility and the like, is further improved.

According to the improved crosslinking scheme, a functional monomer with carbon-carbon double bonds and residual amino groups is introduced before glutaraldehyde crosslinking, the functional monomer physically permeates into the biological material and then is subjected to co-crosslinking with an aldehyde crosslinking agent, the amino groups of the functional monomer and the amino groups on a biological membrane react with aldehyde groups of the glutaraldehyde crosslinking agent to introduce the carbon-carbon double bonds into the biological material, and functional groups are introduced at the same time; and finally, the second functional monomer is initiated to be copolymerized with carbon-carbon double bonds on the biological material to form a cross-linking network and introduce a functional group B, so that the cross-linking degree and the performance of the biological material are further improved.

Specifically, the method comprises the following steps:

(1) Soaking the biological material in a solution containing a first functional monomer and performing reverse physical infiltration; the first functional monomer has at least one amino group and at least one carbon-carbon double bond;

The principle of the application is as follows:

the first step is as follows: the first functional monomer is firstly physically permeated into the biological material, the introduced first functional monomer has amino and carbon-carbon double bonds, an aldehyde crosslinking agent such as glutaraldehyde is added after the first functional monomer is permeated, and co-crosslinking is carried out, wherein in the co-crosslinking process, the generated reaction at least comprises the following steps:

1) Aldehyde groups at two ends of a part of cross-linking agents react with amino groups of the biological materials; 2) Aldehyde group at one end of the cross-linking agent reacts with amino of the biomaterial, and aldehyde group at the other end reacts with amino of the functional monomer; 3) One part of the cross-linking agent has aldehyde group at one end reacting with amino of the biological material and aldehyde group at the other end forming residual aldehyde group on the biological material; 4) And reacting part of residual aldehyde groups with amino groups of the functional monomers to introduce carbon-carbon double bonds into the biological material.

The second step: the second functional monomer is firstly physically permeated into the biological material which is subjected to crosslinking and is introduced with carbon-carbon double bonds for one time, the carbon-carbon double bonds are further introduced, and a functional group B is also introduced.

According to the method, the biological material is subjected to crosslinking treatment through aldehyde group co-crosslinking and double bond polymerization secondary crosslinking, and the biological material obtained through the secondary crosslinking treatment has good crosslinking degree; in the cross-linking process, the first functional monomer can react off partial residual aldehyde group; after the carbon-carbon double bond is introduced by co-crosslinking, the carbon-carbon double bond is further introduced by the permeation of a second functional monomer, and the carbon-carbon double bond introduced by two steps can have additional functional monomers to participate in the copolymerization in the double bond polymerization process to form a larger polymer crosslinking network, so that the crosslinking degree and the calcification-resistant performance of the biological valve can be improved; the carbon-carbon double bond is introduced for the second time, and simultaneously, functional groups are introduced, so that new characteristics can be endowed to the biological material, and the performance of the biological material is further improved.

The crosslinking agent of the application adopts a polyaldehyde crosslinking agent used in the current mainstream crosslinking method, and optionally, the polyaldehyde crosslinking agent can be one of glutaraldehyde and formaldehyde.

The biomaterial adopted in the application is a biomaterial which is conventional in the existing glutaraldehyde crosslinking process. The collagen content of the biological material is 60-90%. The biological material is animal tissue, the animal source is pig, cattle, horse or sheep, and comprises one or more of pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessel, skin or ligament.

In a more preferred embodiment, optionally, before the step (3), a step (2M) is further included: soaking the biological material treated in the step (2) in a solution containing a third functional monomer to eliminate residual aldehyde groups on the rest part; the third functional monomer of this step has at least one group that reacts with an aldehyde group.

The first functional monomer has at least one amino group and at least one carbon-carbon double bond, and in one scheme, a commercially available product can be directly adopted, and optionally, the first functional monomer is one of 2-methylallyl amine, 3-butene-1-amine, pent-4-ene-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazide and acrylyl hydrazide.

The third functional monomer of the present application has at least one amino group, and in a preferred embodiment, the third functional monomer also has at least one carbon-carbon double bond, and when the biomaterial is treated with the third functional monomer solution again, the carbon-carbon double bond can be introduced again while the remaining aldehyde group is sealed by the reaction of the amino group on the functional monomer and the residual aldehyde group on the biofilm.

Optionally, the third functional monomer is one of 2-methylallyl amine, 3-buten-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazide and acrylyl hydrazide.

The first functional monomer and the third functional monomer can also have functional groups besides carbon-carbon double bonds and amino groups, and optionally, the first functional monomer and the third functional monomer also have at least one functional group A; the functional groups in the first functional monomer and the third functional monomer are respectively and independently selected from one of hydroxyl, carboxyl, amide and sulfonic acid.

The introduction of hydroxyl groups can improve the hydrophilicity of the biological material; the introduction of carboxyl groups can make the biological material exhibit electric neutrality; the introduction of hydroxyl can improve the hydrophilicity of the biological valve; carboxyl is introduced to maintain the pH neutrality of the reaction system in the step (1); the introduction of the amide group can increase the hydrophilicity of the biological valve through the hydrogen bond interaction between water molecules and the amide group; the introduction of the sulfonic acid group can increase the hydrophilicity of the biological valve through the ionic hydration between water molecules and the sulfonic acid group.

In one variant with functional monomers A, the commercially available products can be used directly. Optionally, the first functional monomer and the third functional monomer are each independently selected from one of DL-2-amino-4-pentenoic acid, 2-amino-7-ene-octanoic acid, 6-ene-heptanoic acid, 2-aminopent-4-enoic acid, 4- (1-amino-2-methyl-propyl) -hepta-1, 6-dien-4-ol, 4- (1-amino-ethyl) -hepta-1, 6-dien-4-ol.

The first functional monomer and the third functional monomer can be prepared by double bond modification, such as double-bonded polylysine, in addition to the commercially available routes shown above.

That is, the first functional monomer in step (1) and the third functional monomer in step (2M) are each independently selected from the above-described optional ranges (including commercially available and modified preparations), and may be the same or different.

The biomaterial of the present application requires conventional pretreatment prior to the introduction of the functional monomer, optionally, the pretreatment comprises conventional washing operations: obtaining biological materials, and storing the biological materials in a low-temperature wet state at 4 ℃; fresh biomaterial was washed with distilled water using gentle friction and fluid pressure at 4 ℃ with 100RPM shaking for 2 hours until no adherent non-pericardial or non-collagenous tissue was visible.

Contacting the pretreated biological material with a solution containing a first functional monomer, wherein optionally, the contacting process can be static contacting or dynamic contacting; when static contact is adopted, the biological material is soaked in a solution containing a first functional monomer; the shaking table can vibrate in the soaking process during dynamic contact. The temperature can be 20-50 ℃ during the contact with the first functional monomer, preferably, the final temperature of the contact process does not need to be controlled specially, the temperature can be room temperature, preferably, the temperature does not exceed the adaptive temperature of human body, and preferably, the temperature is 36-37 ℃.

The concentration of the first functional monomer and the contact time of the biological material with the solution containing the first functional monomer in step (1) are preferably such that more of the first functional monomer permeates into the biological material, and generally, the concentration of the first functional monomer is higher, the corresponding contact time is shorter, and the concentration of the first functional monomer is lower, and the corresponding contact time is longer.

Optionally, the solvent of the solution in the step (1) is water, normal saline, pH neutral buffer solution or ethanol water solution, wherein in the ethanol water solution, ethanol and water can be mixed according to any proportion, and the ethanol is usually about 50% ethanol; the concentration of the functional monomer in the solution is 10-100mM.

Optionally, the contact time is 2-20 h under the condition that the concentration of the first functional monomer is 10-100mM, so that the first functional monomer can fully permeate into the biological material.

Further optionally, the concentration of the first functional monomer in the solution in the step (1) is 10-30 mM, and the soaking time is 2-5 h.

And (3) after the functional monomer is permeated, adding a cross-linking agent into the reaction system, wherein the concentration of the cross-linking agent is 10-800 mM.

In the co-crosslinking process, the temperature can be within 20-50 ℃, preferably, the temperature does not need to be controlled particularly in the co-crosslinking process, the temperature can be within room temperature environment, preferably not exceeding the adaptive temperature of a human body, and optionally, the temperature is within 36-37 ℃; the reaction time of the co-crosslinking is proper to ensure that the crosslinking reaction is complete as much as possible, and optionally, the co-crosslinking time is 10 to 30 hours under the condition that the concentration of the crosslinking agent is 10 to 800mM.

Further optionally, the concentration of the cross-linking agent in step (2) is 50 to 500mM; furthermore, the concentration of the cross-linking agent in the step (2) is 50-150 mM, and the co-crosslinking time is 20-30 h.

Optionally, during co-crosslinking, the biological material and the crosslinking agent solution may be in static contact or dynamic contact, and the reaction system may be oscillated during soaking to accelerate the crosslinking process.

In the step (2M), the concentration and the soaking time of the third functional monomer are preferably more closed residual aldehyde groups, and optionally, in the step (2M), the concentration of the third functional monomer in the solution is 10 to 100mM; the soaking time is 2-48 h.

Further optionally, the solvent in the solution in the step (2M) is water, normal saline, pH neutral buffer solution or an aqueous solution of ethanol, wherein in the aqueous solution of ethanol, ethanol and water may be mixed according to any proportion, and is usually about 50% ethanol; the concentration of the third functional monomer in the solution is 20-40 mM; the soaking time is 2-4 h.

In the step (2M), the biological material treated in the step (2) is washed and then soaked in a third functional monomer solution; or directly transferring the biological material treated in the step (2) into a third functional monomer solution.

In the step (2M), the soaking temperature can be controlled at 20-50 ℃, preferably, the soaking temperature does not need to be specially controlled, the room temperature environment can be controlled, the temperature is preferably not higher than the human body adaptive temperature, and the soaking temperature is preferably 36-37 ℃.

After the step (2) or the step (2M) is finished, the biomaterial contacts a solution containing a second functional monomer, the second functional monomer physically permeates into the biomaterial, the second functional monomer has a double bond and also has a functional group B capable of improving the performance of the biomaterial, and optionally, the functional group B is one of a hydroxyl group, a carboxyl group, a choline carboxylate, a choline sulfonate, a choline phosphate, a pyrrolidone, a sulfonic acid group, a carboxylate ion, a sulfonate, a sulfoxide, an amide group and a methoxy group.

In one embodiment, the second functional monomer is one of polyethylene glycol diacrylate, acrylamide, 2- (prop-2-enamino) acetic acid, 2-acrylamido-2-methylpropanesulfonic acid, hydroxyethyl methacrylate, 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate, N-methyl-2-acrylamide, N-isopropylacrylamide, N- (hydroxymethyl) acrylamide, N- (2-hydroxyethyl) methacrylamide, N-dimethyl methacrylamide, 3- [ N, N-dimethyl- [2- (2-methylprop-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt, and 2-methacryloyloxyethyl phosphorylcholine. Are commercially available.

The second homoenergetic monomer can also be prepared by double bond modification in addition to the commercially available routes, and optionally, the second functional monomer is double-bonded hyaluronic acid or double-bonded polylysine.

That is, the first functional monomer, the second functional monomer and the third functional monomer may be independently selected from double-bonded hyaluronic acid or double-bonded polylysine.

One embodiment of a double bond modified hyaluronic acid, comprising:

weighing 2g of sodium hyaluronate with the molecular weight of 10000, dissolving the sodium hyaluronate by using 20ml of PBS, and sequentially adding 6-12ml of glycidyl methacrylate and 4-8ml of triethylamine. Placing on a shaker at 37 deg.C for 5-10 days. Finally dialyzing for 5-7 days by using a dialysis bag with the molecular weight cutoff of 5000, and freeze-drying to obtain double-bonded hyaluronic acid (which can be prepared in an equal-proportion amplification manner according to actual needs);

an embodiment of a double bond modified hyaluronic acid, comprising:

polylysine was dissolved in deionized water and then added to the glycidyl methacrylate in a molar ratio of 1.5 to 1 (glycidyl methacrylate: amino groups). The mixture was placed on a shaker at 37 ℃ for 5-10 days. Finally dialyzing for 5-7 days by using a dialysis bag with the molecular weight cut-off of 1000, and freeze-drying to obtain the partially double-bonded polylysine.

In the present application, the carbon-carbon double bond is introduced again after the co-crosslinking or step (2M) is completed, and the secondary crosslinking is completed by double bond polymerization, and in an alternative, the second functional monomer is introduced directly after the co-crosslinking or step (2M) is completed. The scheme is commonly called as a one-pot method, namely, a second functional monomer is directly added into a co-crosslinking reaction system or a reaction system in the step (2M) after the co-crosslinking is finished, the second functional monomer is directly added into a reaction system to initiate double bond polymerization after penetrating into the biological material, and the biological material does not need to be taken out and cleaned.

In another alternative, a step of co-crosslinking or washing the biomaterial after completion of step (2M) is included. In the scheme, the biological material is taken out after co-crosslinking or step (2M), the biological material is cleaned, residual functional monomers, crosslinking agents and the like are removed, and then the biological material is soaked in a solution containing a second functional monomer to contact, so that double bond polymerization is initiated.

The biological material after the co-crosslinking is contacted with a solution containing a second functional monomer, carbon-carbon double bonds are further introduced, the final concentration of the second functional monomer and the contact time of the biological material and the solution containing the second functional monomer are suitable for ensuring that more second functional monomers permeate into the biological material, generally, the concentration of the second functional monomer is higher, the corresponding contact time can be shorter, the concentration of the second functional monomer is lower, and the corresponding contact time is suitable for prolonging.

Optionally, the solvent in the solution containing the second functional monomer is water, normal saline, a pH neutral buffer solution or an aqueous solution of ethanol, wherein in the aqueous solution of ethanol, ethanol and water can be mixed according to any proportion, and the ethanol is usually about 50% ethanol; the mass percentage concentration of the second functional monomer is 1-10%.

Optionally, the contact time is 2-20 h under the condition that the mass percentage concentration of the second functional monomer is 1-10%. So that the second functional monomer sufficiently permeates into the biomaterial.

Further optionally, the mass percentage concentration of the second functional monomer in the solution containing the second functional monomer is 2-5%; the soaking time is 10-15 h.

Optionally, the contacting process of the biological material and the solution containing the second functional monomer can be static contacting or dynamic contacting; the contact process can be carried out at 20-50 ℃, preferably, the temperature does not need to be controlled specially, the temperature can be room temperature, and the temperature does not exceed the human body adaptive temperature, preferably, the contact process is carried out at 36-37 ℃.

And after the second functional monomer is infiltrated, adding an initiator to initiate carbon-carbon double bonds to generate free radical polymerization, and performing secondary crosslinking.

In an alternative initiation scheme, the initiator is a mixture of ammonium persulfate and sodium bisulfite; the concentrations of ammonium persulfate and sodium bisulfite in the solution are respectively 10-100 mM; further, the concentrations of ammonium persulfate and sodium bisulfite are respectively 20-40 mM.

In another alternative initiation scheme, the initiator is a mixture of ammonium persulfate and N, N, N ', N' -tetramethylethylenediamine; the mass percentage concentrations of the ammonium persulfate and the N, N, N ', N' -tetramethyl ethylenediamine in the solution are respectively 2-5% and 0.2-0.5%.

Optionally, the solvent in the initiator-containing solution is water, physiological saline or pH neutral buffer.

As the concentration of the initiator as described above, in the one-pot method, the concentration can be understood as the concentration of ammonium sulfate and sodium hydrogen sulfite in the solution contained in the reaction system of the step (2), and in the stepwise method, the concentration can be understood as the concentration in the solution containing the initiator.

Optionally, the double bond polymerization process can be carried out at 20-50 ℃, preferably, the temperature in the double bond polymerization process does not need to be controlled specially, the double bond polymerization process can be carried out in a room temperature environment, and the double bond polymerization process is preferably not more than the human body adaptive temperature, and is preferably carried out at 36-37 ℃. The polymerization time of the double bonds is preferably from 2 to 48 hours, preferably from 20 to 25 hours.

Optionally, the method further comprises a post-treatment process after the double bond polymerization is finished, wherein the post-treatment process comprises conventional cleaning, softening, drying and other operations.

For the preparation of wet films, the solvent may be stored after the softening treatment, for example, glycerol may be used for storage. For the requirement of preparing dry film, drying the biological material after softening treatment: the drying process is one or more of room temperature drying, forced air drying, vacuum drying and freeze drying. The drying time is 1 h-10 days, the room temperature drying temperature is 10-30 ℃, the blast drying or vacuum drying temperature is 15-100 ℃, and the freeze drying temperature is-20 ℃ to-80 ℃.

The process flow of the present application is described below by taking the preferred flow shown in fig. 1 as an example:

picking up a biological valve material, and performing conventional pretreatment operation on the biological valve material;

step two, soaking the biological material in an amino-double bond compound (first functional monomer) solution;

step three, adding glutaraldehyde (cross-linking agent) into the reaction system in the step two, carrying out co-crosslinking on an amino-double bond compound (first functional monomer) and the biological valve material, introducing carbon-carbon double bonds (free radicals), and further introducing functional groups;

and step four, soaking the biological material treated in the step three in an amino-double bond compound (third functional monomer) solution again.

Soaking a free radical polymerization monomer (a second functional monomer);

and step six, initiating secondary crosslinking of free radical polymerization.

And seventhly, cleaning and glycerol treating the biological material after secondary crosslinking, and storing the biological valve in a dry state or a wet state.

A more specific embodiment, comprising the steps of:

s1, obtaining a biological material, and storing the biological material in a low-temperature wet state at 4 ℃;

s2, washing the biological material in the step S1 for 2 hours by using distilled water under the conditions of soft friction and fluid pressure, and oscillating at the rotating speed of 100RPM at 4 ℃ until no visible adhered non-pericardial or non-collagenous tissue exists;

s3, soaking the biological material cleaned in the step S2 in a DL-2-amino-4-pentenoic acid aqueous solution with the molar concentration of 10-100mM for 12 hours at 37 ℃ to ensure the full physical permeation of DL-2-amino-4-pentenoic acid;

and S4, adding glutaraldehyde into the solution soaked by the biological material treated in the step S3 for copolymerization, wherein the molar concentration of the glutaraldehyde in a solution system is 10-500mM, and reacting for 24 hours at 37 ℃.

And S5, soaking and cleaning the biological material treated in the step S4 by using distilled water, and removing unreacted DL-2-amino-4-pentenoic acid and glutaraldehyde.

And S6, soaking the biological material treated in the step S5 in a 5% aqueous solution of polyethylene glycol diacrylate, and soaking for 12 hours at 37 ℃ to ensure sufficient physical permeation of the polyethylene glycol diacrylate.

And S7, adding the biological material treated in the step S6 into an ammonium persulfate and sodium bisulfite initiator for initiation, wherein the molar concentration of the ammonium persulfate and the sodium bisulfite is 10-100mM.

A schematic chemical diagram of this embodiment is shown in fig. 2.

In this embodiment, in step S3, the method of introducing the radical polymerizable allyl group by co-crosslinking DL-2-amino-4-pentenoic acid/glutaraldehyde/pericardium has higher efficiency of introducing the radical polymerizable group than similar research reported in the literature, and the present solution can further improve the degree of crosslinking of the pericardium while introducing the allyl group.

In another more specific embodiment, the method comprises the steps of: s1, obtaining a biological material, and storing the biological material in a low-temperature wet state at 4 ℃;

s2, washing the biological material in the step S1 for 2 hours by using distilled water under the conditions of soft friction and fluid pressure oscillation at the temperature of 4 ℃ and the rotating speed of 100RPM until no visible adhered non-pericardial or non-collagen tissue exists, and meanwhile, realizing effective decellularization on the pericardial tissue through osmotic shock;

s3, weighing 2g of sodium hyaluronate with the molecular weight of 10000, dissolving the sodium hyaluronate with the molecular weight of 20ml of PBS, and sequentially adding 6-12ml of glycidyl methacrylate and 4-8ml of triethylamine. Placing on a shaker at 37 deg.C for 5-10 days. Finally dialyzing for 5-7 days by using a dialysis bag with the molecular weight cutoff of 5000, and freeze-drying to obtain double-bonded hyaluronic acid (which can be prepared in an equal-scale amplification manner according to actual needs);

s4, dissolving polylysine in deionized water, and then adding the glycidyl methacrylate in a molar ratio of 1.5-1. The mixture was placed on a shaker at 37 ℃ for 5-10 days. Finally dialyzing for 5-7 days by using a dialysis bag with the molecular weight cutoff of 1000, and freeze-drying to obtain the partially double-bonded polylysine;

s5, soaking the pericardium in the S2 in the aqueous solution of the partially double-bonded polylysine (with the molar concentration of 100mM-500 mM) prepared in the S4 for 1-3 days to ensure that the solution reaches physical permeation close to saturation so as to introduce the partially double-bonded polylysine as much as possible, and then adding glutaraldehyde to the aqueous solution until the mass concentration of the glutaraldehyde is 2.5%.

And S6, carrying out free radical copolymerization reaction on the biological material treated in the step S5 and the double-bonded hyaluronic acid prepared in the step S3 under the initiation of ammonium persulfate and/N, N, N ', N' -tetramethylethylenediamine, wherein the concentration of the double-bonded hyaluronic acid is 20-60 mg/ml. Reacting at 37 deg.c for 12-24 hr;

s7, soaking and cleaning with distilled water, and removing double-bonded hyaluronic acid without grafting.

In this embodiment, a schematic diagram of the modification of hyaluronic acid and polylysine and a schematic diagram of the principle of double-bonded polylysine-modified pericardium and double-bonded hyaluronic acid radical polymerization are shown in fig. 3.

In contrast to the hydrophilic treatment studies that have been reported for similar pericardial polysaccharides, the advantages of this preferred embodiment include:

1) The methacrylic acid-acidified polylysine/glutaraldehyde/pericardium is adopted for crosslinking together and simultaneously introducing the free radical polymerizable methacrylic group, and compared with other reported methods (the pericardium is reacted with glutaraldehyde first, and then double bonds are introduced by using residues), the method has higher efficiency of introducing the double bonds;

2) The research strategy is to adopt double crosslinking, including glutaraldehyde crosslinking and free radical polymerization crosslinking, and the material crosslinking degree is higher;

3) Compared with the research of mostly using polysaccharide to carry out hydrophilic modification on the surface interface, the method has the advantages that the combination mode of hyaluronic acid and the pericardium material is chemical covalent combination, and the stability is higher.

In conclusion, according to the scheme, polylysine and hyaluronic acid are respectively modified by glycidyl methacrylate to obtain partially double-bonded polylysine and double-bonded hyaluronic acid, and then the pericardium and the partially double-bonded polylysine (simultaneously provided with amino and double bonds) are subjected to copolymerization crosslinking under the action of glutaraldehyde to simultaneously realize the crosslinking and double-bonding modification of the pericardium. And finally, copolymerizing the double-bonded glutaraldehyde valve and the double-bonded hyaluronic acid free radical to obtain the hyaluronic acid modified glutaraldehyde pericardial material.

The biological valve material prepared by the method can be used for interventional biological valves, such as minimally invasive intervention; it may also be used for surgical biological valves, for example by surgical implantation.

The valve is a biological valve material prepared by the method. The valve may be secured to the stent by means of stitching or the like, and may generally include leaflets for controlling blood flow and a covering membrane applied to the inner or outer wall of the stent, depending on functional needs.

In a more specific embodiment, the biological valve can be a heart valve. The heart valve may be implanted by catheter intervention or surgery. The stent is generally a radially deformable mesh tube structure as an intervention mode.

When the minimally invasive catheter is used for minimally invasive intervention, the interventional system comprises a heart valve and a delivery pipe, and the heart valve is delivered through the delivery pipe.

The following is further illustrated by the specific examples:

example 1:

in this example, freshly collected pig hearts were washed with distilled water at 4 ℃ under 100RPM shaking for 2 hours, then soaked in 30mM DL-2-amino-4-pentenoic acid aqueous solution at 37 ℃ for 12 hours, then glutaraldehyde was added to give a concentration of 100mM, and washed with distilled water after soaking at 37 ℃ under 100RPM shaking for 24 hours. After cleaning, the sample is soaked in a 5% aqueous solution of polyethylene glycol diacrylate, the mixture is soaked for 12 hours at 37 ℃ to ensure sufficient physical permeation of the polyethylene glycol diacrylate, ammonium persulfate and a sodium bisulfite initiator are added for initiation, the molar concentrations of the ammonium persulfate and the sodium bisulfite are both 40mM, and the mixture reacts for 24 hours at 37 ℃ and is marked as sample 1.

In the treatment process, the glutaraldehyde treatment group was set as the control group 1, i.e., the pericardium was soaked in 0.625% glutaraldehyde for 24 hours.

The results of analysis of the relative activities of lactate dehydrogenase in example 1 and glutaraldehyde control 1 are shown in Table 1, and the amount of calcium attachment is shown in Table 2.

TABLE 1

	Relative lactate dehydrogenase Activity
		Glutaraldehyde control 1	0.410±0.072
Examples	0.100±0.019

TABLE 2

	The amount of calcium is mu g/mg
		Glutaraldehyde control 1	168.595±9.973
Examples	43.220±10.873

The infrared spectrograms of the pericardium (GA) of the sample 1 and the control 1 sample are shown in fig. 4; the results of the quantification of elastin after subcutaneous implantation of pericardium (GA) rats for sample 1 and control 1 sample are shown in fig. 5; a schematic diagram of the detection of calcium entrapment after subcutaneous implantation in pericardium (GA) rats for sample 1 and control 1 is shown in FIG. 6.

Example 2

Preparation of modified hyaluronic acid: 2g of sodium hyaluronate with a molecular weight of 10000 are weighed and dissolved with 20ml of PBS, and 6.5ml of glycidyl methacrylate and 4.5ml of triethylamine are added in sequence. The mixture was placed on a shaker at 37 ℃ for 7 days. Finally dialyzing for 7 days by using a dialysis bag with the molecular weight cutoff of 5000, and freeze-drying to obtain double-bonded hyaluronic acid;

preparation of modified polylysine: polylysine was dissolved in deionized water, and then glycidyl methacrylate (glycidyl methacrylate: amino group) was added in a molar ratio of (1. The mixture was placed on a shaker at 37 ℃ for 7 days. Finally dialyzing for 7 days by using a dialysis bag with the molecular weight cutoff of 1000, and freeze-drying to obtain the partially double-bonded polylysine;

in this example, freshly collected pig pericardium was washed with distilled water at 4 ℃ under 100RPM shaking for 2 hours, then soaked in an aqueous solution of 180mM modified polylysine at room temperature for 12 hours, then glutaraldehyde solution was added to a mass concentration of 2.5%, and reacted on a shaker at 37 ℃ for 24 hours, the pericardial material was taken out, washed, then soaked in an aqueous solution of 50mg/ml modified hyaluronan at room temperature for 12 hours, then soaked with 2.5% ammonium persulfate and 0.25% N, N' -tetramethylethylenediamine at 37 ℃ for 12 hours, and finally washed with distilled water, as sample 2.

The sample 2 prepared in example 2 and the control 2 were subjected to a water contact angle test, a lactate dehydrogenase activity test, a hemolysis rate test, and a calcification test, respectively.

Control group 2: washing a freshly collected pig heart bag with distilled water for 2 hours at the temperature of 4 ℃ under the condition of 100RPM rotation speed oscillation, then soaking the pig heart bag in a glutaraldehyde solution with the mass concentration of 0.625% for 24 hours, taking out the pig heart bag after the reaction is finished, and soaking the pig heart bag in a glutaraldehyde solution with the mass fraction of 0.2% for storage.

(1) Water contact Angle test

The control group and the material of example 1 were cut into square pieces of 1 × 1cm, flattened between two glass sheets, vacuum freeze-dried and subjected to water contact angle test.

(2) Lactate dehydrogenase activity assay: fresh rabbit blood is collected and centrifuged at 1500rpm for 15min to obtain platelet rich plasma. Control and example 1 material were cut into 10mm diameter discs and washed 3 times with PBS, placed in 48 well plates, and 100 μ L of platelet rich plasma was added and soaked at 37 ℃ for 1h. 100 mul of platelet rich plasma was selected as a positive control for quantitative detection. After incubation, wash 3 times with PBS. The relative amount of platelet adhesion was determined using a lactate dehydrogenase assay kit. The absorbance at 490nm of each group was recorded with a microplate reader, and the relative lactate dehydrogenase activity of each group was calculated, and the relative number of platelets was expressed as the relative lactate dehydrogenase activity.

(3) Hemolysis rate test

Collecting fresh rabbit blood, centrifuging at 1500rpm for 15min, removing supernatant, and collecting erythrocyte. The control and example 1 samples were placed in 2ml centrifuge tubes and red blood cells (9/1, PBS/RBC) diluted with PBS were added and incubated at 37 ℃ for 1 hour. Red blood cells diluted 10-fold in PBS and deionized water set negative and positive controls. The supernatant was transferred to a 96-well plate by centrifugation at 3000rpm for 5 min. The absorbance at 545nm was recorded with a microplate reader and the hemolysis rate was calculated.

(4) Calcification testing

An incision was made in the back of 45-50g male SD rats, and subcutaneous tissue was separated with a blunt instrument to create a cavity, and the control group and the sample of example 1 were placed in the cavity, followed by suturing the skin, taking out the sample after 30 days, freeze-drying and weighing, digesting at 100 ℃ with 1ml of 6M hydrochloric acid, and then diluting the digested solution to 10ml with deionized water, and performing inductively coupled plasma atomic emission spectrometry to determine the calcium concentration.

The final water contact angle results for example 2 and glutaraldehyde control 2 are shown in table 3.

TABLE 3

	Water contact Angle (°)
		Glutaraldehyde control 2	84.29
Example 2	55.26

The final lactate dehydrogenase activity and hemolysis results of the examples and the glutaraldehyde control are shown in Table 4.

TABLE 4

	Lactate dehydrogenase Activity	Hemolysis ratio (%)
			Glutaraldehyde control 2	0.41	1.54
Example 2	0.24	0.38

The final calcium ion concentration results for the examples and the glutaraldehyde control are shown in table 5.

TABLE 5

	Calcium ion concentration (μ g/mg)
		Glutaraldehyde control 2	188.39
Example 2	36.95

By combining tables 1, 2 and 3, it can be found that the water contact angle of the biomaterial is reduced, the lactate dehydrogenase activity is reduced, and the calcium ion content is reduced after the biomaterial is treated by the method of example 2.

As shown in fig. 7, the test is a water contact angle test, the control group is a glutaraldehyde-treated group, the test group is a hydrophilic-treated group, and the water contact angle of the test group is decreased.

As shown in fig. 8, the experiment was performed by detecting the lactate dehydrogenase activity and the hemolysis rate, the control group was glutaraldehyde-treated, the experiment group was hydrophilic-treated, and the lactate dehydrogenase activity and the hemolysis rate were decreased in the experiment group.

As shown in fig. 9, the test is calcium ion concentration detection, the control group is glutaraldehyde test group, the test group is hydrophilic treatment group, and the calcium ion content of the test group is reduced.

The method provided by the embodiment can improve the hydrophilic performance, blood compatibility and calcification resistance of the biological material, and potentially prolong the service life of the biological material.

Control group 3

Freshly collected pig hearts were washed with distilled water at 4 ℃ under 100RPM shaking conditions for 2 hours, then soaked in 100mM glutaraldehyde solution and crosslinked at room temperature under 100RPM shaking conditions for 24 hours to obtain a control.

Example 3

Washing freshly collected pig hearts with distilled water at 4 ℃ under 100RPM oscillation for 2 hours,

then immersed in 30mM 2-aminopent-4-enoic acid aqueous solution at 37 ℃ for 2 hours,

glutaraldehyde was then added to a final concentration of 100mM, and the mixture was soaked at 37 ℃ for 24 hours with shaking at 100 RPM.

Taking out the porcine pericardium, and cleaning with distilled water.

Soaking pig heart bag in 30mM 2-amino-pent-4-enoic acid water solution for 2 hr

After washing, the mixture was immersed in a 5wt% aqueous solution of 3- [ N, N-dimethyl- [2- (2-methylprop-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt and immersed at 37 ℃ for 12 hours to ensure sufficient physical permeation.

Initiating by adding ammonium persulfate and sodium bisulfite initiator, wherein the molar concentration of the ammonium persulfate and the sodium bisulfite are both 30mM, and reacting for 24 hours at 37 ℃. To facilitate the differentiation of the samples prepared in the examples, the sample obtained in this example was designated as sample 3.

Example 4

then immersed in a 20mM aqueous solution of 2-aminopent-4-enoic acid at 37 ℃ for 2 hours,

glutaraldehyde was then added to a final concentration of 100mM, and the mixture was immersed at 37 ℃ for 24 hours with shaking at 100 RPM.

Taking out the porcine pericardium, and cleaning with distilled water.

After cleaning, the solution was immersed in a 3wt% aqueous solution of 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate, and immersed at 37 ℃ for 12 hours, thereby ensuring sufficient physical permeation of 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate.

Initiating by adding ammonium persulfate and sodium bisulfite initiator, wherein the molar concentration of the ammonium persulfate and the sodium bisulfite are both 30mM, and reacting for 24 hours at 37 ℃. To facilitate the differentiation of the samples prepared in the examples, the sample obtained in this example was designated as sample 4.

Control 3, sample 3 and sample 4 of control group 3 were subjected to blood contact experiments:

control 3, sample 3 and sample 4, which had uniform surface and thickness, were cut into sheets of 1cm in diameter, washed with physiological saline, drained and placed in a 24-well plate, 300. Mu.L of rabbit blood was added to each well and incubated at 37 ℃ for 1 hour with shaking at 70 bpm. After the incubation was completed, the rabbit blood was discarded and 500. Mu.L of physiological saline was added to each well to wash off the non-adherent blood with gentle shaking on a shaker. At the end of the washing, the samples were transferred to a 2.5% (w/w) glutaraldehyde solution for fixation for 4 hours. The fixed samples were dehydrated with gradient ethanol (25%, 50%, 75% and 100%, v/v) for 20 min each gradient. The dried samples were fixed on a test table with conductive adhesive and subjected to gold spraying treatment, and images of blood adhesion on each group of samples were observed and photographed on a field emission scanning electron microscope.

Scanning electron micrographs of the blood contact experiments of control 3, sample 3 and sample 4 are shown in fig. 11, 12 and 13. More blood cell adhesion and aggregation were observed on the scanning electron micrograph of control 3 after incubation with rabbit blood, while less blood cells adhered to the surface of samples 3 and 4, and only a few blood cells adhered dispersedly to the surface. The results show that samples 3 and 4 can inhibit the adhesion of blood cells to a certain extent, thereby reducing the risk of blood coagulation and having an anticoagulation effect.

Example 5

Washing freshly collected pig hearts with distilled water at 4 ℃ under the condition of 100RPM rotation speed oscillation for 2 hours,

then immersed in a 20mM 2-aminoethyl methacrylate aqueous solution at 37 ℃ for 2 hours,

Taking out the porcine pericardium, and cleaning with distilled water.

After cleaning, the cloth is soaked in 5wt% N-isopropyl acrylamide water solution and soaked for 12 hours at 37 ℃ to ensure that the N-isopropyl acrylamide fully and physically permeates.

Initiating by adding ammonium persulfate and sodium bisulfite initiator, wherein the molar concentration of the ammonium persulfate and the sodium bisulfite are both 30mM, and reacting for 24 hours at 37 ℃. To facilitate the differentiation of the samples prepared in the examples, the sample obtained in this example was designated as sample 5.

Enzyme degradation experiments

Sample 3, sample 4, sample 5 and control 3 were cut into circular sheets of 1cm diameter, with 6 replicates per set. These circular sheet samples were placed in 48-well plates, frozen overnight at minus 80 ℃ and then transferred to a vacuum lyophilizer for lyophilization for 48 hours. The weight of each sample was weighed as the initial weight (W0) on a one-hundred-thousandth balance and placed back into the 48-well plate. 0.5mL of collagenase I in PBS was added to each well of the 48-well plate using a pipette gun and the biological valve sample was completely immersed in collagenase in PBS (100U/mL), and the 48-well plate was transferred to a 37 ℃ incubator and incubated for 24 hours. And after the incubation is finished, removing the solution in the pore plate, and sucking deionized water by using a rubber head dropper to repeatedly blow and beat the biological valve sample in the pore plate. After repeated purging 3 times, the mixture was frozen overnight at minus 80 ℃ and then transferred to a vacuum freeze dryer for 48 hours. The weight of each sample after degradation by collagenase solution was weighed on a one-hundred-thousand balance and recorded as the final weight (Wt). The weight loss rate of enzyme degradation is calculated by the following formula:

as shown in table 6, it is understood from the results in table 6 that the preparation method of the present application can significantly improve the degree of crosslinking of the biomaterial.

TABLE 6 collagenase degradation weight loss ratio

The samples 3, 4, 5 and 3 were subjected to enzyme degradation experiments to characterize the degree of cross-linking of the samples in each group, and the weight loss rate of enzyme degradation of the samples in each group was calculated after treating the samples 3, 4, 5 and 3 with collagenase i as shown in the table above. The enzyme degradation weight loss rates of the samples 3, 4 and 5 are all lower than that of the control group 3, which indicates that the enzyme degradation stabilities of the samples 3, 4 and 5 are all higher than that of the control group 3, i.e. the cross-linking degrees of the samples 3, 4 and 5 are higher. The enzyme degradation experiment result shows that the method for preparing the biological valve material can improve the crosslinking degree of the biological valve material.

Example 6

Adding N-isopropyl acrylamide to make the final concentration of the N-isopropyl acrylamide to be 5wt%, and soaking the N-isopropyl acrylamide for 12 hours at the temperature of 37 ℃ to ensure that the N-isopropyl acrylamide fully and physically permeates.

Initiating by adding ammonium persulfate and sodium bisulfite initiator, wherein the molar concentration of the ammonium persulfate and the sodium bisulfite are both 30mM, and reacting for 24 hours at 37 ℃. To facilitate the differentiation of the samples prepared in the examples, the sample obtained in this example was designated as sample 6.

Example 7

Taking out the porcine pericardium, and cleaning with distilled water.

Soaking pig heart bag in 30mM 2-amino-pent-4-enoic acid water solution for 2 hr

Initiating by adding ammonium persulfate and sodium bisulfite initiator, wherein the molar concentration of the ammonium persulfate and the sodium bisulfite is 30mM, and reacting for 24 hours at 37 ℃.

After the reaction is finished, washing the porcine pericardium with distilled water, soaking the porcine pericardium with glycerol, and dehydrating to obtain a dry film, wherein a sample obtained in the embodiment is marked as sample 7.

Example 8

in this example, freshly collected pig hearts were washed with distilled water at 4 ℃ under 100RPM shaking for 2 hours, then soaked in 60mM (calculated as lysine) aqueous solution of modified polylysine at room temperature for 12 hours, then glutaraldehyde solution was added to a mass concentration of 250mM, reacted on a shaker at 37 ℃ for 24 hours, the pericardial material was washed out and then soaked in 50mg/ml aqueous solution of modified hyaluronic acid at room temperature for 12 hours, then soaked with 2.5% ammonium persulfate and 0.25% N, N' -tetramethylethylenediamine at 37 ℃ for 12 hours, and finally washed with distilled water, which was designated as sample 8. Compared with the control sample 2, the water contact angle of the sample 8 is reduced, the lactate dehydrogenase activity is reduced, the calcium ion content is reduced, the hydrophilic performance, the blood compatibility and the anti-calcification capability of the biological material can be improved, and the service life of the biological material can be potentially prolonged.

Alizarin Red staining experiment

Samples of the material (sample 3, sample 4, sample 5, sample 6, sample 7 and control 3) taken 30 days after subcutaneous implantation in mice were washed with PBS. After washing, the tissue was fixed in 4% (w/v) paraformaldehyde PBS tissue fixative for 24 hours at room temperature. After the fixation is finished, the operation knife is taken out for repairing and leveling and then is transferred into the dehydration box. The material samples were gradient dehydrated with 50%, 75%, 85%, 95% (v/v) and absolute ethanol. And after dehydration, transferring the material sample to an embedding machine for embedding by using melted paraffin, and then transferring to a refrigerator with the temperature of 20 ℃ below zero for cooling and shape trimming. Sections of 3-5 μm thickness were cut from the trimmed wax block on a microtome, transferred from the spreader onto glass slides and dewaxed and rehydrated. The section is dyed by alizarin red dye solution for 3 minutes, and is permeated by dimethylbenzene for 5 minutes after being washed and dried. The sections were mounted on neutral gum and stained images were collected on a pathological section scanner.

Samples 3, 4, 5, 6, 7 and 3, which were implanted into the rat subcutaneously for 30 days, were stained by alizarin red staining test to characterize the degree of calcification of each group of samples. The results of alizarin red staining of the sample sections 30 days after implantation into the rat skin are shown in fig. 14-19, where the darker the color of the alizarin red stained sample indicates the higher degree of calcification. The alizarin red staining results of the sections of sample 3, sample 4, sample 5, sample 6 and sample 7 were significantly lighter in color than the alizarin red staining results of the section of control 3 (fig. 14), which indicates that the calcification degree of sample 3, sample 4, sample 5, sample 6 and sample 7 is lower than that of control 3, i.e., sample 3, sample 4, sample 5, sample 6 and sample 7 have a certain anti-calcification effect compared to control 3. The alizarin red staining results of the sample 3, the sample 4, the sample 5, the sample 6 and the sample 7 which are implanted into the rat skin for 30 days show that the method for preparing the biological valve material can improve the calcification resistance of the biological valve.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for improving calcification and anticoagulation of a biological valve material by double-bond post-crosslinking is characterized by comprising the following steps:

2. The method of claim 1, wherein the aldehyde-based crosslinking agent is glutaraldehyde or formaldehyde.

3. The method according to claim 1, wherein the solvent of the solution in step (1) is water, physiological saline, a pH neutral buffer or an aqueous solution of ethanol; the concentration of the functional monomer in the solution is 10 to 100mM; the soaking time is 2 to 20h; in the step (2), the concentration of the cross-linking agent is 10 to 800mM; the co-crosslinking time is 10 to 30 hours.

4. The method of claim 1, further comprising, before step (3), step (2M): soaking the biological material treated in the step (2) in a solution containing a third functional monomer to eliminate residual aldehyde groups; the third functional monomer has at least one group that reacts with an aldehyde group; the group reacting with the aldehyde group is one of amino and hydrazide.

5. The method according to claim 4, wherein in step (2M), the solvent of the solution is water, physiological saline, pH neutral buffer or an aqueous solution of ethanol; the concentration of the third functional monomer in the solution is 10 to 100mM; the soaking time is 2 to 48 hours.

6. The method of claim 1, wherein the first functional monomer is selected from the group consisting of 2-methylallyl amine, 3-buten-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazide, and acrylyl hydrazide.

7. The method of claim 4, wherein the third functional monomer is selected from one of 2-methylallyl amine, 3-buten-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazide, and acrylyl hydrazide.

8. The method of claim 1, wherein the first functional monomer further has at least one functional group a; the functional group A of the first functional monomer is at least one selected from hydroxyl, carboxyl, amide and sulfonic acid.

9. The method of claim 4, wherein the third functional monomer further has at least one functional group A; the functional group A of the third functional monomer is at least one selected from hydroxyl, carboxyl, amide and sulfonic acid.

10. The method of claim 1, wherein the first functional monomer is selected from one of DL-2-amino-4-pentenoic acid, 2-amino-7-ene-octanoic acid, 6-ene-heptinic acid, 2-aminopent-4-enoic acid, 4- (1-amino-2-methyl-propyl) -hepta-1, 6-dien-4-ol, 4- (1-amino-ethyl) -hepta-1, 6-dien-4-ol, and doubly bonded polylysine.

11. The method of claim 4, wherein the third functional monomer is selected from one of DL-2-amino-4-pentenoic acid, 2-amino-7-ene-octanoic acid, 6-ene-heptinic acid, 2-aminopent-4-enoic acid, 4- (1-amino-2-methyl-propyl) -hepta-1, 6-dien-4-ol, 4- (1-amino-ethyl) -hepta-1, 6-dien-4-ol, doubly bonded polylysine.

12. The method of claim 1, wherein the functional group B is one of a hydroxyl group, a carboxyl group, a choline carboxylate, a choline sulfonate, a choline phosphate, a pyrrolidone, a sulfonic acid group, a carboxylate ion, a sulfonate, a sulfoxide, an amide group, and a methoxy group.

13. The method of claim 1, wherein the second functional monomer is one of acrylamide, 2- (prop-2-enamino) acetic acid, 2-acrylamido-2-methylpropanesulfonic acid, hydroxyethyl methacrylate, 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate, N-methyl-2-acrylamide, N-isopropylacrylamide, N- (hydroxymethyl) acrylamide, N- (2-hydroxyethyl) methacrylamide, N-dimethyl methacrylamide, 3- [ N, N-dimethyl- [2- (2-methylprop-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt, 2-methacryloyloxyethylphosphocholine, doubly-bound hyaluronic acid, doubly-bound polylysine.

14. The method according to claim 1, wherein in the step (3), a second functional monomer is added to the system of the previous treatment; or cleaning the biological material treated in the previous step and then soaking the biological material in a solution containing a second functional monomer.

15. The method according to claim 1, wherein the solvent in the second functional monomer-containing solution is water, physiological saline, a pH neutral buffer, or an aqueous solution of ethanol; the mass percentage concentration of the second functional monomer is 1 to 10 percent; the soaking time is 2 to 20 hours.

16. The method according to claim 1, wherein the initiator is a mixture of ammonium persulfate and sodium bisulfite, the concentrations of the ammonium persulfate and the sodium bisulfite are respectively 10 to 100mM; or

17. A double-bond post-crosslinked biological valve material, which is prepared by the method as claimed in any one of claims 1 to 16.

18. A biological valve comprising a stent and a valve, wherein the valve is the double bond post-crosslinked biological valve material of claim 17.

19. The biological valve of claim 18, wherein the biological valve is a heart valve.