CN115970060B - Method for improving calcification resistance and anticoagulation performance of biological valve material by double bond post-crosslinking - Google Patents

Abstract

The application discloses a method for improving calcification resistance and anticoagulation performance of a biological valve material by double bond post-crosslinking, which comprises the following steps of (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 cross-linking 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) Adding an initiator into the system in the step (3) to initiate double bond polymerization; (5) And (3) dehydrating and drying the biological material treated in the step (4). According to the method, more 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 calcification resistance is improved; additional functional groups are introduced while carbon-carbon double bonds are secondarily introduced, 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 resistance and anticoagulation performance of biological valve material by double bond post-crosslinking

Technical Field

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

The application is a divisional application aiming at 202210273141.5, 2022-03-18 and the university of Sichuan of the application with the name of a method for improving calcification resistance and anticoagulation performance of biological valve materials by double bond post-crosslinking.

Background

Biological heart valves are usually prepared from porcine or bovine pericardium and are used for replacing the heart valves of the human body with defective functions; biological heart valves have many advantages over mechanical heart valves: the biological heart valve can be operated in a minimally invasive intervention mode without taking anticoagulants for a long time after being implanted, and the advantages make the biological heart valve gradually become the main stream of the market in clinical application.

The biological valve products in the current market are almost all prepared by adopting glutaraldehyde which can crosslink collagen in the pericardium, but glutaraldehyde crosslinked biological valves have the defect of poor blood compatibility, so that the service life of the biological valve products in the body is limited.

Disclosure of Invention

The application provides a method for improving calcification resistance and anticoagulation performance of a biological valve material by double bond post-crosslinking, which solves the thrombus problem of glutaraldehyde crosslinking membranes.

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

(1) Soaking 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 cross-linking 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 (3) 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, physiological saline, a pH neutral buffer or an aqueous solution of ethanol; the concentration of the functional monomer in the solution is 10-100 mM; the soaking time is 2-20 h.

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

Optionally, step (2M) is further included before step (3): 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 which reacts 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 or aqueous solution of ethanol; the concentration of the third functional monomer in the solution is 10-100 m M; 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-methylallylamine, 3-butene-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazide, and acryloyl hydrazide.

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, amido and sulfonic group.

Alternatively, 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, double-linked polylysine.

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

Optionally, the second functional monomer is one of acrylamide, 2- (prop-2-enylamino) acetic acid, 2-acrylamido-2-methylpropanesulfonic acid, hydroxyethyl methacrylate, 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate, N-methyl-2-acrylamide, N-isopropyl acrylamide, N- (hydroxymethyl) acrylamide, N- (2-hydroxyethyl) methacrylamide, N-dimethyl acrylamide, 3- [ N, N-dimethyl- [2- (2-methylpropan-2-enyloxy) ethyl ] ammonium ] propane-1-sulfonate, 2-methacryloyloxyethyl phosphorylcholine, double-linked hyaluronic acid, double-linked polylysine.

Optionally, in step (3), adding a second functional monomer into the system of the previous step 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.

Optionally, the solvent in the solution containing the second functional monomer is water, physiological saline, pH neutral buffer solution or ethanol aqueous solution; 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 concentration of the ammonium persulfate and the sodium bisulfite are respectively 10-100 mM; or (b)

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

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

The application also provides a double bond post-crosslinking biological valve material, comprising:

(1) Soaking 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 cross-linking 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) Adding an initiator into the system in the step (3) to initiate double bond polymerization;

(5) And (3) dehydrating and drying the biological material treated in the step (4).

The application also provides a biological valve, which comprises a bracket 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) The method carries out crosslinking treatment on the biological material through aldehyde group co-crosslinking and double bond polymerization secondary crosslinking, and the biological material obtained by the secondary crosslinking treatment has good crosslinking degree;

(2) According to the application, the second functional monomer is added in the double bond polymerization step for copolymerization to form more larger polymer crosslinking network, so that the crosslinking degree of the biological material can be improved and the calcification resistance can be improved;

(3) In the co-crosslinking process, the carbon-carbon double bond is introduced and part of residual aldehyde groups can be blocked;

(4) After the co-crosslinking is finished, soaking the mixture in the functional monomer solution again, eliminating residual aldehyde groups, introducing carbon-carbon double bonds through chemical reaction again, facilitating the subsequent double bond polymerization step, and further improving the crosslinking degree of the biological material.

(5) The application introduces additional functional groups while introducing carbon-carbon double bonds for the second time, which can endow the biological material with new characteristics and further improve the performance of the biological material.

Drawings

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

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

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

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

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

FIG. 6 is a graph showing the measurement of calcium hanging amount after subcutaneous implantation of the pericarp (GA) rats in sample 1 and control group 1 of example 1;

FIG. 7 is a graph showing water contact angles of sample 2 and control 2 pericardium (GA) of example 2;

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

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

FIG. 10 is a basic schematic of example 3;

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

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

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

FIG. 14 is a chart showing alizarin red-stained sections obtained 60 days after implantation of the control sample of control group 3;

FIG. 15 is a view of alizarin red-stained sections obtained 60 days after implantation of sample 3;

FIG. 16 is a chart of alizarin red-stained sections obtained 60 days after implantation of sample 4;

FIG. 17 is a view of alizarin red-stained sections obtained 60 days after implantation of sample 5;

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

FIG. 19 is a chart of alizarin red-stained sections of sample 7 after 60 days of implantation.

Detailed Description

The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the 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 application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The biological valve products in the current market are mainly glutaraldehyde crosslinked films, and glutaraldehyde can crosslink collagen in the pericardium, but the glutaraldehyde crosslinked biological valve has unsatisfactory calcification resistance and anticoagulation performance, is easy to cause thrombus, and threatens the life quality and life of patients. On the one hand, the mechanical property, calcification resistance and anticoagulation performance of the glutaraldehyde crosslinking film are improved by improving the crosslinking means on the basis of glutaraldehyde crosslinking; on the other hand, functional groups with specific functions are introduced on the basis of improving the crosslinking means, and the properties of the biological material, such as biocompatibility and the like, are further improved.

In the improved crosslinking scheme, a functional monomer with carbon-carbon double bonds and residual amino groups is introduced before glutaraldehyde crosslinking, the functional monomer firstly physically permeates into a biological material and then is crosslinked together with an aldehyde crosslinking agent, and the amino groups of the functional monomer and amino groups on a biological film are reacted with the glutaraldehyde crosslinking agent to introduce the carbon-carbon double bonds into the biological material and simultaneously introduce functional groups; finally, the second functional monomer is initiated to copolymerize with the carbon-carbon double bond on the biological material to form a crosslinking network, and a functional group B is introduced at the same time, so that the crosslinking degree and the performance of the biological material are further improved.

Specifically, the method comprises the following steps:

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

(2) Adding an aldehyde cross-linking 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 (3) adding an initiator into the system in the step (3) to initiate double bond polymerization.

The principle of the application is as follows:

The first step: the first functional monomer is firstly physically permeated into the biological material, the introduced first functional monomer has amino and carbon-carbon double bonds, and an aldehyde cross-linking agent such as glutaraldehyde is added after the first functional monomer is permeated for co-crosslinking, and in the co-crosslinking process, the reaction at least comprises:

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

And a second step of: the second functional monomer is firstly subjected to physical permeation into the biological material which is subjected to crosslinking and is subjected to primary introduction of carbon-carbon double bonds, the carbon-carbon double bonds are further introduced, and simultaneously, the functional group B is also introduced, wherein the process of introducing the carbon-carbon double bonds in the step is physical permeation, after the second functional monomer is permeated, an initiator is added to initiate double bond copolymerization of the second functional monomer and the surface of the biological material, secondary crosslinking is carried out, and the functional group B of the second functional monomer can endow the biological material with new characteristics.

The method carries out crosslinking treatment on the biological material through aldehyde group co-crosslinking and double bond polymerization secondary crosslinking, and the biological material obtained by the secondary crosslinking treatment has good crosslinking degree; the first functional monomer can react to remove part of residual aldehyde groups in the crosslinking process; after the carbon-carbon double bond is introduced by the co-crosslinking, the carbon-carbon double bond is further introduced by the permeation of the second functional monomer, and the additional functional monomer participates in the copolymerization in the double bond polymerization process by the two-step carbon-carbon double bond introduction, so that a larger polymer crosslinking network is formed, and the crosslinking degree and the calcification resistance of the biological valve are improved; the functional group is introduced while the carbon-carbon double bond is introduced secondarily, so that the novel characteristic of the biological material can be endowed, and the performance of the biological material is further improved.

The cross-linking agent adopts a multi-aldehyde cross-linking agent used in the current mainstream cross-linking method, and optionally, the multi-aldehyde cross-linking agent can be one of glutaraldehyde and formaldehyde.

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

In a more preferred embodiment, optionally, step (2M) is further included before step (3): soaking the biological material treated in the step (2) in a solution containing a third functional monomer to eliminate residual aldehyde groups in 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 of the application has at least one amino group and at least one carbon-carbon double bond, and in one scheme, the first functional monomer can be directly used as a commercial product, and can be selected from one of 2-methylallylamine, 3-butene-1-amine, pent-4-ene-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazide and acryloyl hydrazide.

In a preferred embodiment, the third functional monomer also has at least one carbon-carbon double bond, and when the biological material is treated with the third functional monomer solution again, the residual aldehyde groups can be introduced again while the residual aldehyde groups are blocked by the reaction of the amino groups on the functional monomer with the residual aldehyde groups on the biological film.

Optionally, the third functional monomer is one of 2-methylallylamine, 3-butene-1-amine, pent-4-ene-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazide and acryloyl hydrazide.

The first functional monomer and the third functional monomer of the application can be provided with 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, amido and sulfonic group.

The hydrophilic property of the biological material can be improved by introducing hydroxyl; the introduction of carboxyl groups can make the biological material take on electric neutrality; the hydrophilic property of the biological valve can be improved by introducing hydroxyl; the carboxyl is introduced to maintain the pH neutrality of the reaction system in the step (1); introducing amide groups can increase the hydrophilicity of the biological valve through hydrogen bond interaction between water molecules and the amide groups; the introduction of sulfonic acid groups can increase the hydrophilicity of the biological valve through ionic hydration between water molecules and the sulfonic acid groups.

In one version with functional monomer A, commercially available products can be used directly. Alternatively, 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 and third functional monomers can also be prepared by double bond modification, for example double bonded polylysine, outside of the commercial route as indicated previously.

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

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

The pretreated biological material is contacted with a solution containing a first functional monomer, and optionally, the contact process can be static contact or dynamic contact; when static contact is adopted, the biological material is placed in a solution containing a first functional monomer for soaking; the shaking table can vibrate during the soaking process during dynamic contact. In the process of contacting the first functional monomer, the temperature is 20-50 ℃, preferably, the final temperature of the contacting process is not required to be controlled particularly, the room temperature environment is sufficient, the temperature does not exceed the temperature suitable for human body, and the contacting is preferably carried out at 36-37 ℃.

In step (1), the concentration of the first functional monomer and the contact time of the biological material and the solution containing the first functional monomer are preferably used for ensuring that more first functional monomer permeates into the biological material, generally, the concentration of the first functional monomer is higher, the corresponding contact time can be shorter, the concentration of the first functional monomer is lower, and the corresponding contact time is adaptively prolonged.

Optionally, the solvent of the solution in the step (1) 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 usually about 50% ethanol is used; the concentration of the functional monomer in the solution is 10-100 mM.

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

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

After the functional monomer is permeated, a cross-linking agent is added into the reaction system, and the concentration of the cross-linking agent is 10-800 mM.

In the process of the co-crosslinking, the temperature is preferably 20-50 ℃, the temperature in the process of the co-crosslinking is not required to be controlled particularly, the room temperature environment is preferably not more than the temperature suitable for the human body, and the process is optionally carried out at 36-37 ℃; the reaction time of the co-crosslinking is preferably as complete as possible, and the co-crosslinking time is preferably 10-30 hours under the condition that the concentration of the crosslinking agent is 10-800 mM.

Further optionally, the concentration of the crosslinking agent in step (2) is 50 to 500mM; further, the concentration of the crosslinking agent in the step (2) is 50-150 m M, and the total crosslinking time is 20-30 h.

Alternatively, the biological material and the cross-linking agent solution can be in static contact or dynamic contact during co-crosslinking, and the dynamic contact process can be used for accelerating the cross-linking process by oscillating the reaction system while soaking.

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

Further optionally, the solvent in the solution in the step (2M) 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 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 hours.

In the step (2M), the biological material treated in the step (2) is cleaned 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 this step (2M), the soaking is carried out at 20 to 50℃and preferably at 36 to 37℃without any special control, and the room temperature environment is preferably not higher than the temperature suitable for the human body.

After the step (2) or the step (2M) is finished, the biological material is contacted with a solution containing a second functional monomer, the second functional monomer is physically permeated into the biological material, the second functional monomer is provided with a double bond and a functional group B capable of improving the performance of the biological material, and the functional group B is optionally one of hydroxyl, carboxyl, choline carboxylate, choline sulfonate, phosphorylcholine, pyrrolidone, sulfonic acid group, carboxylate ion, sulfonate, sulfoxide, amide group and methoxy.

For the second functional monomer, in one scheme, the second functional monomer is one of polyethylene glycol diacrylate, acrylamide, 2- (prop-2-enylamino) 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-methylpropan-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonate, and 2-methacryloyloxyethyl phosphorylcholine. Commercially available.

The second homoenergetic monomer can be prepared by self double bond modification besides the commercial path, and the second functional monomer is double-bonded hyaluronic acid or double-bonded polylysine.

That is, the first, second, and third functional monomers may independently select double-linked hyaluronic acid or double-linked polylysine.

An embodiment of a double bond modified hyaluronic acid comprising:

2g of sodium hyaluronate with molecular weight 10000 are weighed and dissolved by using 20ml of PBS, and then 6-12ml of glycidyl methacrylate and 4-8ml of triethylamine are added in sequence. Placing on a shaking table at 37 ℃ for 5-10 days. Finally, dialyzing for 5-7 days by using a dialysis bag with molecular weight cut-off of 5000, and freeze-drying to obtain double-bonded hyaluronic acid (which can be prepared by equal proportion amplification according to actual needs);

An embodiment of a double bond modified hyaluronic acid comprising:

Polylysine was dissolved in deionized water and then glycidyl methacrylate was added in a molar ratio of 1:1.5 to 1:5 (glycidyl methacrylate: amino). 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.

The present application re-introduces carbon-carbon double bonds after the co-crosslinking or step (2M) is completed and completes secondary crosslinking by double bond polymerization, in an alternative scheme, the second functional monomer is directly introduced after the co-crosslinking or step (2M) is completed. The scheme is commonly called a one-pot method, namely, after the co-crosslinking is finished, a second functional monomer is directly added into a reaction system of the co-crosslinking or the step (2M), after the second functional monomer permeates into the biological material, an initiator is directly added into the reaction system to initiate double bond polymerization, and the biological material is not required to be taken out for cleaning.

In another alternative, the method further comprises the step of co-crosslinking or washing the biological material after completion of step (2M). In the scheme, the biological material is taken out after the step (2M) or the co-crosslinking is carried out, the biological material is subjected to cleaning operation, residual functional monomers, crosslinking agents and the like are removed, and then the biological material is soaked in a solution containing the second functional monomers for contact to initiate double bond polymerization.

The co-crosslinked biological material 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 preferably used for ensuring that more second functional monomer permeates 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 adapted to be prolonged.

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

Alternatively, the biological material and the solution containing the second functional monomer can be contacted in a static state or a dynamic state; the contact process can be carried out at 20-50 ℃, preferably, the temperature is not required to be controlled particularly, the room temperature environment is sufficient, the temperature is not more than the temperature suitable for human body, and preferably, the contact process is carried out at 36-37 ℃.

After the second functional monomer is permeated, an initiator is added to initiate the carbon-carbon double bond to generate free radical polymerization, and secondary crosslinking is carried out.

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

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

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

The concentration of the initiator as described above, which may be understood as the concentration of ammonium sulfate and sodium bisulphite in the solution contained in the reaction system of step (2) in the one-pot method, and which may be understood as the concentration in the solution containing the initiator in the step-wise method.

Alternatively, the double bond polymerization process is carried out at 20-50 ℃, preferably, the temperature in the double bond polymerization process is not particularly controlled, the room temperature environment is sufficient, the temperature does not exceed the temperature suitable for human body, and preferably, the double bond polymerization process is carried out at 36-37 ℃. The double bond polymerization time is preferably 2 to 48 hours, more preferably 20 to 25 hours.

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

For the preparation of wet film, solvent preservation after softening treatment is sufficient, and glycerol can be used for preservation. For the requirement of preparing a dry film, the biological material is dried after the softening treatment: the drying process is one or a combination of a plurality of room temperature drying, air blast drying, vacuum drying and freeze drying. The drying time is 1 h-10 days, the room temperature drying temperature is 10-30 ℃, the forced air drying or vacuum drying temperature is 15-100 ℃, and the freeze drying temperature is-20-80 ℃.

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

step one, picking up biological valve materials, and performing conventional pretreatment operation on the biological valve materials;

Step two, soaking biological materials in an amino-double bond compound (first functional monomer) solution;

step three, glutaraldehyde (cross-linking agent) is added into the reaction system of the step two, the amino-double bond compound (first functional monomer) and the biological valve material are subjected to co-crosslinking, and carbon-carbon double bonds (free radicals) are introduced, and functional groups can be further introduced;

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

Step five, soaking the free radical polymerization monomer (second functional monomer);

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

And step seven, cleaning and glycerinum treatment are carried out on the biological material after secondary crosslinking, and the biological valve is preserved in a dry state or a wet state.

A more specific embodiment comprises the steps of:

S1, acquiring biological materials, and storing the biological materials in a low-temperature wet state at 4 ℃;

S2, washing the biological material in the step S1 with distilled water for 2 hours under the oscillation conditions of mild friction and fluid pressure at the temperature of 4 ℃ and the rotational speed of 100RPM until no visible non-pericardial or non-collagenous tissue is adhered;

S3, soaking the biological material washed in the step S2 in DL-2-amino-4-pentenoic acid aqueous solution with the molar concentration of 10-100mM for 12 hours at 37 ℃ to ensure the sufficient physical permeation of the DL-2-amino-4-pentenoic acid;

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

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.

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

And S7, adding the biological material treated in the step S6 into ammonium persulfate and sodium bisulphite initiator to initiate, wherein the molar concentration of the ammonium persulfate and the sodium bisulphite is 10-100mM.

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

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

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

s2, washing the biological material in the step S1 with distilled water for 2 hours under the oscillation conditions of soft friction and fluid pressure at the temperature of 4 ℃ and the rotational speed of 100RPM until no visible non-pericardial or non-collagenous tissue is adhered, and simultaneously, effectively decellularizing the pericardial tissue through osmotic shock;

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

S4, dissolving polylysine in deionized water, and then adding glycidyl methacrylate in a molar ratio of 1:1.5-1:5 (glycidyl methacrylate: amino). 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 partially double-bonded polylysine;

S5, soaking the pericardium in the S2 in the partially double-bonded polylysine (the molar concentration is 100mM-500 mM) aqueous solution prepared in the S4 for 1-3 days, ensuring that the partially double-bonded polylysine reaches physical permeation close to saturation, introducing the partially double-bonded polylysine as much as possible, and then adding glutaraldehyde into the aqueous solution to the mass concentration of 2.5%.

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/or N, N, N ', N' -tetramethyl ethylenediamine, wherein the concentration of the used double-bonded hyaluronic acid is 20mg/ml-60mg/ml. Reacting at 37 ℃ for 12-24 hours;

and S7, finally, soaking and cleaning the glass fiber with distilled water, and removing the double-bonded hyaluronic acid which is not grafted.

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

The advantages of this preferred embodiment in comparison to the reported studies of hydrophilic treatment of similar pericardial polysaccharides include:

1) The methacrylic polylysine/glutaraldehyde/pericardium is adopted to jointly crosslink and simultaneously introduce a methacrylic group capable of free radical polymerization, and compared with other reported methods (firstly, pericardium is reacted with glutaraldehyde and then double bonds are introduced by residues), the method has higher double bond introducing efficiency;

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

3) Compared with most researches on surface interface hydrophilic modification by using polysaccharide, the method has the advantages that the hyaluronic acid and the pericardial material are combined in a chemical covalent combination mode, and the stability is higher.

In summary, in the scheme, polylysine and hyaluronic acid are modified by glycidyl methacrylate to obtain partially double-bonded polylysine and double-bonded hyaluronic acid, and then pericardium and partially double-bonded polylysine (with amino and double bonds) are subjected to copolymerization and cross-linking under the action of glutaraldehyde to simultaneously realize cross-linking and double-bonded modification of pericardium. Finally, copolymerization of the double-bonded glutaraldehyde valve and double-bonded hyaluronic acid free radicals is utilized to obtain the hyaluronic acid modified glutaraldehyde pericardial material.

The biological valve material prepared by the method can be used for intervention of a biological valve, for example, minimally invasive intervention; but also for surgical biological valves, for example by surgical implantation.

As one implementation form of the interventional biological valve, the interventional biological valve comprises a bracket and a valve, wherein the valve is a biological valve material prepared by the method. The valve may be secured to the stent by stitching or the like, and may generally include leaflets for controlling blood flow and a cover over the inner or outer wall of the stent as desired for function.

In a more specific embodiment, the biological valve may be a heart valve. The heart valve may be implanted by catheter intervention or surgery. As an interventional approach, its stent is typically a radially deformable mesh tube structure.

For minimally invasive interventions, the interventional system comprises a heart valve and a delivery tube through which the heart valve is delivered.

The following are further described in terms of specific examples:

Example 1:

In this example, freshly collected porcine pericardium was washed with distilled water for 2 hours at a shaking condition of 100RPM at 4℃and then immersed in 30mM DL-2-amino-4-pentenoic acid aqueous solution at 37℃for 12 hours, glutaraldehyde was then added to a concentration of 100mM, and washing was performed with distilled water after immersing for 24 hours at 37℃and 100RPM shaking condition. After cleaning, soaking in 5% aqueous solution of polyethylene glycol diacrylate for 12 hours at 37 ℃ to ensure sufficient physical penetration of the polyethylene glycol diacrylate, adding ammonium persulfate and sodium bisulfate initiator to initiate, wherein the molar concentration of the ammonium persulfate and sodium bisulfate is 40mM, and reacting for 24 hours at 37 ℃, which is marked as sample 1.

During the treatment, glutaraldehyde treatment group was set as control group 1, i.e., pericardial membrane was immersed 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 calcium-hanging amount is shown in Table 2.

TABLE 1

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

TABLE 2

	Calcium hanging amount mug/mg
		Glutaraldehyde control group 1	168.595±9.973
Examples	43.220±10.873

Infrared spectra of pericardium (GA) of sample 1 and control 1 are shown in fig. 4; the results of elastin quantification after subcutaneous implantation in pericardial (GA) rats for sample 1 and control group 1 are schematically shown in figure 5; the schematic of calcium hanging amount detection after subcutaneous implantation of pericardial (GA) rats for sample 1 and control 1 is shown in fig. 6.

Example 2

Preparation of modified hyaluronic acid: 2g of sodium hyaluronate having a molecular weight of 10000 was weighed and dissolved in 20ml of PBS, followed by the addition of 6.5ml of glycidyl methacrylate and 4.5ml of triethylamine. Placed on a shaker at 37℃for 7 days. Finally, dialyzing for 7 days by using a dialysis bag with molecular weight cut-off 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 (1:1.5). 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 cut-off of 1000, and freeze-drying to obtain partially double-bonded polylysine;

In this example, freshly collected porcine pericardium was washed with distilled water at a rotational speed of 100RPM at 4℃for 2 hours, then immersed in a 180mM modified polylysine aqueous solution 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 pericardium material was taken out and washed, then immersed in a 50mg/ml modified hyaluronic acid aqueous solution at room temperature for 12 hours, then immersed in 2.5% ammonium persulfate and 0.25% N, N' -tetramethyl ethylenediamine at 37℃for 12 hours, and finally washed with distilled water, which was designated as sample 2.

The sample 2 prepared in example 2 and the sample of control group 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: the freshly collected pig pericardium is washed with distilled water for 2 hours under the shaking condition of 100RPM at 4 ℃, then is soaked in glutaraldehyde solution with the mass concentration of 0.625 percent for 24 hours, and is taken out and soaked in glutaraldehyde solution with the mass fraction of 0.2 percent for preservation after the reaction is completed.

(1) Water contact angle test

The control and example 1 materials were cut into 1 x 1cm square pieces, placed in the middle of two glass sheets, flattened, and subjected to water contact angle testing after vacuum freeze drying.

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

(3) Hemolysis rate test

Fresh rabbit blood was collected, centrifuged at 1500rpm for 15min, the supernatant was discarded and erythrocytes were taken. Control and example 1 samples were placed in a 2ml centrifuge tube, and red blood cells (9/1, PBS/RBC) were diluted with PBS and incubated at 37℃for 1 hour. Red blood cells diluted 10-fold with PBS and deionized water were negative and positive controls. The supernatant was transferred to a 96-well plate by centrifugation at 3000rpm for 5 min. Absorbance at 545nm was recorded with a microplate reader and the haemolysis rate was calculated.

(4) Calcification test

Incisions were made on the backs of 45-50g male SD rats, and subcutaneous tissues were separated with a blunt instrument to create a cavity, the samples of the control group and example 1 were placed in the cavity, then the skin was sutured, the samples were taken out after 30 days, freeze-dried and weighed, digested with 1ml of 6M hydrochloric acid at 100℃and then the digested solution was diluted to 10ml with deionized water, and inductively coupled plasma atomic emission spectrometry was performed 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 Table 3

	Water contact angle (°)
		Glutaraldehyde control group 2	84.29
Example 2	55.26

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

TABLE 4 Table 4

	Lactate dehydrogenase activity	Hemolysis rate (%)
			Glutaraldehyde control group 2	0.41	1.54
Example 2	0.24	0.38

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

TABLE 5

It can be seen from a combination of tables 1, 2 and 3 that the biological material was treated by the method of example 2, the water contact angle of the biological material was decreased, the lactate dehydrogenase activity was decreased, and the calcium ion content was decreased.

As shown in fig. 7, the experiment was a water contact angle test, the control group was glutaraldehyde-treated, the experiment group was hydrophilic-treated, and the experiment group was reduced in water contact angle.

As shown in FIG. 8, the experiment was performed by measuring the activity and the hemolysis rate of lactate dehydrogenase, the control group was glutaraldehyde-treated, the experimental group was hydrophilically-treated, and the activity and the hemolysis rate of lactate dehydrogenase were both reduced.

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

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

Control group 3

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

Example 3

Freshly collected pig pericardium is washed with distilled water for 2 hours at 4 ℃ under the shaking condition of 100RPM,

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 soaked for 24 hours at 37℃with shaking at 100 RPM.

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

The pig pericardium was soaked in 30mM 2-aminopent-4-enoic acid aqueous solution for 2 hours

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

Ammonium persulfate and sodium bisulphite initiators were added to initiate, the molar concentrations of ammonium persulfate and sodium bisulphite were 30mM, and the reaction was carried out at 37℃for 24 hours. In order to facilitate distinguishing between the samples prepared in the examples, the sample obtained in this example is designated sample 3.

Example 4

Freshly collected pig pericardium is washed with distilled water for 2 hours at 4 ℃ under the shaking condition of 100RPM,

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

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

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

After washing, the mixture was immersed in a 3wt% aqueous solution of 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate at 37℃for 12 hours to ensure sufficient physical penetration of the 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate.

Ammonium persulfate and sodium bisulphite initiators were added to initiate, the molar concentrations of ammonium persulfate and sodium bisulphite were 30mM, and the reaction was carried out at 37℃for 24 hours. In order to facilitate distinguishing between the samples prepared in the examples, the sample obtained in this example is designated sample 4.

Control 3, sample 3 and sample 4 of control 3 were subjected to a blood contact experiment:

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

The blood contact test sem images of control 3, sample 3 and sample 4 are shown in fig. 11, 12 and 13. More blood cells adhered and aggregated were observed on the sem image of control 3 after incubation with rabbit blood, while less blood cells adhered to sample 3 and sample 4, with only a few blood cells adhered to the surface in a dispersed manner. The results show that the sample 3 and the sample 4 can inhibit the adhesion of blood cells to a certain extent, so that the risk of coagulation is reduced, and the anticoagulation effect is achieved.

Example 5

Freshly collected pig pericardium is washed with distilled water for 2 hours at 4 ℃ under the shaking condition of 100RPM,

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

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

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

After washing, the mixture was immersed in a 5wt% aqueous solution of N-isopropylacrylamide at 37℃for 12 hours to ensure sufficient physical penetration of N-isopropylacrylamide.

Ammonium persulfate and sodium bisulphite initiators were added to initiate, the molar concentrations of ammonium persulfate and sodium bisulphite were 30mM, and the reaction was carried out at 37℃for 24 hours. In order to facilitate distinguishing between the samples prepared in the examples, the sample obtained in this example is designated sample 5.

Enzyme degradation experiment

Sample 3, sample 4, sample 5 and control group 3 were cut into circular sheets of 1cm diameter, each group being provided with 6 parallel samples. The round sheet samples were placed in 48-well plates, frozen overnight at minus 80 ℃ and then transferred to a vacuum freeze dryer for 48 hours. The weight of each sample was weighed on a ten-thousandth balance and recorded as the initial weight (W0) and returned to the 48-well plate. 0.5mL of collagenase I in PBS was added to each well of the 48-well plate using a pipette 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 for incubation for 24 hours. After the incubation is finished, the solution in the pore plate is discarded, and deionized water is sucked by a rubber head dropper to repeatedly blow biological valve samples in the pore plate. After 3 repeated washings, the mixture was frozen overnight at minus 80 ℃ and then transferred to a vacuum freeze dryer for freeze-drying for 48 hours. The weight of each sample after degradation with collagenase solution was weighed on a ten-thousandth balance and recorded as the final weight (Wt). The weight loss rate of enzyme degradation is calculated as follows:

the results are shown in Table 6, and it is apparent from the results in Table 6 that the preparation method of the present application can significantly improve the crosslinking degree of the biomaterial.

TABLE 6 weight loss rate of collagenase degradation

Sample numbering	Collagenase degradation weight loss rate
		Control group 3	8.6％
Sample 3	2.9％
		Sample 4	1.7％
Sample 5	2.4％

The enzymatic degradation experiments were performed on sample 3, sample 4, sample 5 and control group 3 to characterize the degree of crosslinking of each group of samples, and the enzymatic degradation weight loss rates of each group of samples were calculated after treating sample 3, sample 4, sample 5 and control group 3 with collagenase i as shown in the above table. The enzyme degradation weight loss rates of the sample 3, the sample 4 and the sample 5 are lower than those of the control group 3, which shows that the enzyme degradation stability of the sample 3, the sample 4 and the sample 5 is higher than that of the control group 3, namely the crosslinking degree of the sample 3, the sample 4 and the sample 5 is higher. The enzyme degradation experimental result shows that the method for preparing the biological valve material can improve the crosslinking degree of the biological valve material.

Example 6

Freshly collected pig pericardium is washed with distilled water for 2 hours at 4 ℃ under the shaking condition of 100RPM,

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

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

N-isopropyl acrylamide was added to a final concentration of 5wt% and immersed at 37℃for 12 hours to ensure sufficient physical permeation of N-isopropyl acrylamide.

Ammonium persulfate and sodium bisulphite initiators were added to initiate, the molar concentrations of ammonium persulfate and sodium bisulphite were 30mM, and the reaction was carried out at 37℃for 24 hours. In order to facilitate distinguishing between the samples prepared in the examples, the sample obtained in this example is designated sample 6.

Example 7

Freshly collected pig pericardium is washed with distilled water for 2 hours at 4 ℃ under the shaking condition of 100RPM,

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 soaked for 24 hours at 37℃with shaking at 100 RPM.

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

The pig pericardium was soaked in 30mM 2-aminopent-4-enoic acid aqueous solution for 2 hours

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

Ammonium persulfate and sodium bisulphite initiators were added to initiate, the molar concentrations of ammonium persulfate and sodium bisulphite were 30mM, and the reaction was carried out at 37℃for 24 hours.

After the reaction was completed, the pig pericardium was washed with distilled water, then soaked with glycerol, and dehydrated to obtain a dry film, and the sample obtained in this example was designated as sample 7.

Example 8

Preparation of modified hyaluronic acid: 2g of sodium hyaluronate having a molecular weight of 10000 was weighed and dissolved in 20ml of PBS, followed by the addition of 6.5ml of glycidyl methacrylate and 4.5ml of triethylamine. Placed on a shaker at 37℃for 7 days. Finally, dialyzing for 7 days by using a dialysis bag with molecular weight cut-off 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 (1:1.5). 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 cut-off of 1000, and freeze-drying to obtain partially double-bonded polylysine;

In this example, freshly collected pig pericardium was washed with distilled water at 4℃and a rotational speed of 100RPM for 2 hours, then immersed in a 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 shaking table at 37℃for 24 hours, the pericardium material was taken out, washed, then immersed in a 50mg/ml aqueous solution of modified hyaluronic acid at room temperature for 12 hours, then immersed in 2.5% ammonium persulfate and 0.25% N, N' -tetramethyl ethylenediamine 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 calcification resistance of the biological material can be improved, and the service life of the biological material is potentially prolonged.

Alizarin red staining experiment

Samples of material (sample 3, sample 4, sample 5, sample 6, sample 7 and control sample 3) taken 30 days after subcutaneous implantation of the mice were washed with PBS. After the washing, the tissue was fixed with 4% (w/v) paraformaldehyde PBS at room temperature for 24 hours. And after the fixation is finished, taking out the tissue, repairing and leveling the tissue by using a surgical knife, and transferring the tissue into a dehydration box. The material samples were gradient dehydrated with 50%, 75%, 85%, 95% (v/v) and absolute ethanol. After dehydration, the material sample is transferred to an embedding machine for embedding by melted paraffin, and then transferred to a refrigerator at the temperature of-20 ℃ for cooling and shape trimming. Cut 3-5 μm thick sections from the trimmed wax blocks on a microtome, transferred from the slide to a slide and dewaxed and rehydrated. The slices were stained with alizarin red dye for 3 minutes, washed with water, dried and permeabilized with xylene for 5 minutes. After the sections are sealed by neutral resin, the images of the dyeing results are collected on a pathological section scanner.

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

The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.

Claims (22)

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

(1) Soaking 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 cross-linking 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) Adding an initiator into the system in the step (3) to initiate double bond polymerization;

(5) And (3) dehydrating and drying the biological material treated in the step (4).

2. The method of claim 1, wherein the dehydration drying comprises treatment with glycerol.

3. The method of claim 1, wherein the aldehyde-based cross-linking agent is glutaraldehyde or formaldehyde.

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

5. The method of claim 1, further comprising, prior to 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 which reacts with the aldehyde group is one of amino and hydrazide.

6. The method of claim 5, wherein in step (2M), the solvent of the solution is water, physiological saline, pH neutral buffer or 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.

7. The method of claim 1, wherein the first functional monomer is selected from one of 2-methylallylamine, 3-butene-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazide, and acryloyl hydrazide.

8. The method according to claim 5, wherein the third functional monomer is selected from one of 2-methylallylamine, 3-butene-1-amine, pent-4-en-1-amine, 2-aminoethyl methacrylate, methacryloyl hydrazide, and acryloyl hydrazide.

9. 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, amido and sulfonic acid.

10. The method of claim 5, 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, amido and sulfonic acid groups.

11. The method according to 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-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, double-linked polylysine.

12. The method according to claim 5, wherein the third functional monomer is selected from the group consisting of DL-2-amino-4-pentenoic acid, 2-amino-7-ene-octanoic acid, 6-ene-heptanoic acid, 2-aminopentan-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, double-linked polylysine.

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

14. The method of claim 1, wherein the second functional monomer is one of acrylamide, 2- (prop-2-enylamino) 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-methylpropan-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonate, 2-methacryloyloxyethyl choline phosphate, doubly linked hyaluronic acid, double linked polylysine.

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

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

17. The method according to claim 1, wherein the initiator is a mixture of ammonium persulfate and sodium bisulfite, and the concentration of each of the ammonium persulfate and sodium bisulfite is 10 to 100mM; or (b)

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

18. A double bond post-crosslinked biological valve material prepared by the method of any one of claims 1 to 17.

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

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

21. A double bond post-crosslinked biological valve material, comprising:

(1) Soaking 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 cross-linking 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) Adding an initiator into the system in the step (3) to initiate double bond polymerization;

(5) And (3) dehydrating and drying the biological material treated in the step (4).

22. A biological valve comprising a stent and a valve, wherein the valve is the double bond post-crosslinked biological valve material of claim 21.