CN114748697B

CN114748697B - Double-bond post-crosslinked biological valve material and preparation and application thereof

Info

Publication number: CN114748697B
Application number: CN202210273157.6A
Authority: CN
Inventors: 郑城; 王云兵; 雷洋; 黄雪彧
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-11-17
Filing date: 2022-03-18
Publication date: 2023-03-21
Anticipated expiration: 2042-03-18
Also published as: CN115970060B; CN115970060A; CN115779149B; CN115645618A; CN114748697A; CN114748693A; CN114748695A; CN115779149A; CN115645618B; CN114748696B; CN114748696A; CN114748693B; CN116271236A; CN114748694A; CN114748695B

Abstract

The application discloses a double-bond post-crosslinked biological valve material, and preparation and application thereof, comprising: (1) Soaking the biological material in a solution containing functional monomers for physical permeation; the functional monomer has at least one amino group, at least one carbon-carbon double bond and at least one functional group; (2) Adding an aldehyde crosslinking agent into the system in the step (1) for co-crosslinking; (3) And (3) contacting the biological material treated in the step (2) with an initiator to initiate double bond polymerization. According to the method, the functional monomer is introduced for co-crosslinking during aldehyde crosslinking, carbon-carbon double bonds are introduced during co-crosslinking, the carbon-carbon double bonds are used as the basis of secondary crosslinking, and the crosslinked biomaterial is prepared through twice crosslinking, so that the crosslinking degree of the biomaterial can be improved, and the mechanical property of the biomaterial can be improved. In the co-crosslinking process, the functional monomer can seal partial residual aldehyde groups on the biomaterial while introducing double bonds, so that the calcification and anticoagulation performances of the biomaterial are improved, and the crosslinking efficiency can be further improved.

Description

Double-bond post-crosslinked biological valve material and preparation and application thereof

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 biological valves crosslinked by glutaraldehyde have the problems of thrombus and biocompatibility.

Therefore, the development of a method for modifying the biological valve on the basis of glutaraldehyde crosslinking, particularly the development of a novel treatment method capable of improving the anticoagulation performance, will improve the overall structural stability and blood compatibility of the biological heart valve, and has great significance for scientific research and development of related industrial fields.

Disclosure of Invention

The application provides a double-bond post-crosslinked biological valve material, and preparation and application thereof, which can improve the calcification resistance, anticoagulation performance and biocompatibility of the biological material, and solve the problems of thrombus and biocompatibility of glutaraldehyde crosslinked biological valve

A preparation method of a double-bond post-crosslinking biological valve material is characterized by comprising the following steps:

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

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

(3) And (3) contacting the biological material treated in the step (2) with an initiator to initiate double bond polymerization.

Optionally, the aldehyde-based cross-linking 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, in step (3): adding an initiator 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 an initiator.

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%.

Optionally, before step (3), further comprising step (2M): soaking the biological material treated in the step (2) in a solution containing functional monomers to eliminate residual aldehyde groups; the 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 functional monomer in the solution is 10-100 mM; the soaking time is 2-48 h.

Optionally, the functional monomer in step (2M) further has at least one functional group and at least one carbon-carbon double bond; the functional monomers in the step (1) and the step (2M) are respectively and independently selected from at least one of hydroxyl, carboxyl, amide and sulfonic acid.

Optionally, the functional monomers in step (1) and step (2M) are each independently selected from one of DL-2-amino-4-pentenoic acid, 2-amino-7-ene-octanoic acid, 6-ene-heptylic 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.

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

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

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 functional monomer is introduced for co-crosslinking during aldehyde crosslinking, carbon-carbon double bonds are introduced during co-crosslinking to serve as the basis of secondary crosslinking, and the crosslinked biomaterial is prepared through secondary crosslinking, so that the crosslinking degree of the biomaterial can be improved, and the mechanical property of the biomaterial is improved.

(2) The method can introduce functional groups while introducing carbon-carbon double bonds, and can further improve the performance of the biological material, such as surface hydrophilicity, biocompatibility and the like.

(3) In the co-crosslinking process, the functional monomer can seal partial residual aldehyde groups on the biomaterial while introducing double bonds, so that the calcification and anticoagulation performances of the biomaterial are improved, and the crosslinking efficiency can be further improved.

Drawings

FIG. 1 is a process flow diagram of a more preferred embodiment of the preparation method 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 the reaction of 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 elastin quantification in example 1 and control group 1 pericardium (GA) rats after subcutaneous implantation;

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 graph showing 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 photograph of alizarin red stained sections taken 30 days after control 3 control implantation;

FIG. 12 is a photograph of alizarin red stained sections taken 30 days after implantation of sample No. 3;

FIG. 13 is a photograph of alizarin red stained sections taken 30 days after implantation of sample No. 4;

FIG. 14 is a photograph of alizarin red stained sections taken 30 days after implantation of sample No. 5;

FIG. 15 is a photograph of alizarin red stained sections taken 30 days after implantation of sample No. 6;

FIG. 16 is a photograph of alizarin red stained sections taken 30 days after implantation of sample No. 7;

FIG. 17 is a photograph of alizarin red stained sections taken 30 days after implantation of sample No. 8;

FIG. 18 is a scanning electron microscope image of a blood contact experiment of a control 3;

FIG. 19 is a scanning electron microscope image of a blood contact experiment of sample No. 5;

FIG. 20 is a scanning electron micrograph of sample No. 6 of a blood contact test.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the 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 in the description of the present application herein 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 market are mainly glutaraldehyde cross-linked biological membranes, and glutaraldehyde can cross-link collagen in the pericardium, but the glutaraldehyde cross-linked biological valves have the problems of thrombus and biocompatibility. The anti-calcification, anticoagulation and biocompatibility of the glutaraldehyde crosslinking membrane are improved by improving a crosslinking means on the basis of glutaraldehyde crosslinking.

According to the improved crosslinking scheme, a functional monomer with an amino group, a carbon-carbon double bond and a functional group is introduced before glutaraldehyde crosslinking, the functional monomer physically permeates into the biological material firstly and then is subjected to crosslinking with an aldehyde crosslinking agent, the amino group and the aldehyde group of the functional monomer react to simultaneously introduce the carbon-carbon double bond and the functional group into the biological material, then double bond polymerization is initiated to generate secondary crosslinking, the biological material after the secondary crosslinking has the functional group, and the biocompatibility and the like of the biological material can be further improved.

Specifically, the method comprises the following steps:

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

(2) Adding an aldehyde crosslinking agent into the solution soaked in the biological material treated in the step (1) for co-crosslinking;

The principle of the application is as follows:

the first step is as follows: the functional monomer is firstly physically permeated into the biological material, the introduced functional monomer has amino, carbon-carbon double bonds and functional groups, an aldehyde crosslinking agent (such as glutaraldehyde) is added after the functional monomer is fully 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) The aldehyde group at one end of the cross-linking agent reacts with the amino group of the biological material, and the aldehyde group at the other end reacts with the amino group 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 is that: and the biological material after finishing crosslinking and introducing the carbon-carbon double bond is contacted with a solution containing an initiator to initiate the polymerization of the carbon-carbon double bond on the biological material to generate secondary crosslinking.

According to the method, the functional monomer is introduced for co-crosslinking during aldehyde crosslinking, carbon-carbon double bonds are introduced as the basis of secondary crosslinking during co-crosslinking, and the crosslinking biological material is prepared through secondary crosslinking, so that the crosslinking degree of the biological material can be improved. The method introduces a functional group while introducing a carbon-carbon double bond, and can further improve the performance of the biological material, such as surface hydrophilicity, biocompatibility and the like.

The crosslinking agent of the application adopts an aldehyde crosslinking agent used in the current mainstream crosslinking method, and optionally, the aldehyde crosslinking agent can be selected from glutaraldehyde or formaldehyde.

The biomaterial adopted in the application is the 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 the animal source comprises one or more of pericardium, valve, intestinal membrane, meninges, pulmonary 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 functional monomers again to eliminate residual aldehyde groups on the rest part; the functional monomer of this step has at least one group that reacts with an aldehyde group. The group reacting with the aldehyde group is amino or hydrazide. Further optionally, the functional monomer in the step also has at least one carbon-carbon double bond, when the biological material is treated by the functional monomer solution again, the amino group on the functional monomer reacts with the residual aldehyde group on the biological material, the residual aldehyde group is sealed, and the carbon-carbon double bond is introduced again, so that the base number of the carbon-carbon double bond for subsequent double bond polymerization is increased, and the crosslinking degree is favorably improved.

The functional monomer in step (1) may further have a functional group in addition to the carbon-carbon double bond and the amino group, and the functional monomer in step (2M) may further have a functional group in addition to the carbon-carbon double bond and the amino group, and optionally, the functional groups in step (1) and step (2) are each independently selected from at least one of a hydroxyl group, a carboxyl group, an amide group, and a sulfonic acid group. Namely, the functional group of the functional monomer in the step (1) is at least one of hydroxyl, carboxyl, amide and sulfonic acid; the functional group of the functional monomer in the step (2M) is at least one of hydroxyl, carboxyl, amido and sulfonic group; may be the same or different.

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.

With respect to the functional monomers used in step (1) and step (2M), in one embodiment, commercially available products can be directly used, and optionally, the functional monomers in step (1) and step (2M) are each independently selected from one of DL-2-amino-4-pentenoic acid, 2-amino-7-en-octanoic acid, 6-en-heptylic acid, 2-aminopent-4-enoic acid, 4- (1-amino-2-methyl-propyl) -hepta-1, 6-dien-4-ol, and 4- (1-amino-ethyl) -hepta-1, 6-dien-4-ol.

In another embodiment, the functional monomers required in step (1) and step (2M) can also be prepared by double bond modification, such as double-bond modified polylysine.

That is, the functional monomers in step (1) and 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.

An 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-scale amplification manner according to actual needs);

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

polylysine was dissolved in deionized water and then diluted with 1:1.5-1:5 (glycidyl methacrylate: amino group) glycidyl methacrylate was added in a molar ratio. 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.

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 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 functional monomers; the shaking table can vibrate in the soaking process during dynamic contact. The temperature can be 20-50 ℃ in the contact process with the functional monomer, preferably, the final temperature of the contact process does not need to be controlled specially, the temperature can be room temperature, the temperature is preferably not higher than the adaptive temperature of a human body, and the temperature is preferably 36-37 ℃.

The concentration of the functional monomer and the contact time of the biological material and the solution containing the functional monomer in the step (1) are preferably used for ensuring that more functional monomers permeate into the biological material, generally, the concentration of the functional monomer is higher, the corresponding contact time can be shorter, the concentration of the functional monomer is lower, and the corresponding contact time is suitable for prolonging.

Optionally, the solvent of the solution in the step (1) is water, normal saline or 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 concentration of the functional monomer in the solution is 10-100mM.

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

Further optionally, the concentration of the functional monomer in the solution in the step (1) is 20-30 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 be complete as much as possible, and optionally, the co-crosslinking time is 10 to 30 hours under the condition that the final concentration of the crosslinking agent is 10 to 800mM.

Further optionally, the final concentration of the cross-linking agent is 10-500 mM; 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, the contact process may be static contact or dynamic contact, and the reaction system may be oscillated while soaking in the dynamic contact process to accelerate the crosslinking process.

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

Further optionally, in the step (2M), the solvent of the solution is water, normal saline, pH neutral buffer solution or ethanol aqueous solution, wherein in the ethanol aqueous solution, ethanol and water may be mixed according to any proportion, and is usually about 50% ethanol; the concentration of the functional monomer in the solution is 30-50 mM; the soaking time is 3-8 h.

In the step (2M), the biological material treated in the step (2) is cleaned and then soaked in a functional monomer solution; or directly transferring the biological material treated in the step (2) into a 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 co-crosslinking is completed, carbon-carbon double bonds are introduced into the biological material, and further, the polymerization of the carbon-carbon double bonds is initiated to complete the secondary crosslinking.

In an alternative embodiment, the double bond polymerization is initiated directly after the co-crosslinking is completed. The scheme is commonly called a one-pot method, namely, the process of directly adding the initiator into a reaction system after the completion of the co-crosslinking without taking out and cleaning the biological material is adopted.

In another alternative, the method further comprises the step of washing the biomaterial after the completion of the co-crosslinking. In the scheme, the biological material is taken out after co-crosslinking, is cleaned, and is soaked in a solution containing an initiator, and residual functional monomers, a crosslinking agent and the like are removed.

In the preferred scheme of additionally arranging the step (2M), an initiator is directly added into the system soaked in the step (2M); or cleaning the biological material soaked in the step (2M) and then soaking the biological material in a solution containing an initiator.

In an alternative initiation scheme, 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; 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 are respectively 2% -5% and 0.2% -0.5%.

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

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

Optionally, the double bond polymerization process can be carried out at 20-50 ℃, preferably, the temperature does not need to be controlled particularly, the double bond polymerization process can be carried out in a room temperature environment, the temperature does not exceed the adaptive temperature of a human body, and the double bond polymerization process 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 valve material in an amino-double bond compound solution (namely a functional monomer and simultaneously has functional groups);

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

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

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

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

In some preferred embodiments, the method further comprises a step of introducing a carbon-carbon double bond twice between the co-crosslinking step and the second polymerization step, and one embodiment of the method comprising introducing a carbon-carbon double bond twice comprises the following steps:

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 at 4 ℃ and 100RPM (revolution speed) oscillation 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 10-500 mM, 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 scheme can further improve the degree of crosslinking of the pericardium while introducing the allyl group.

In another embodiment of the scheme comprising a double incorporation of a carbon-carbon double bond, the following steps are included: 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 realizing effective decellularization of 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-proportion amplification manner according to actual needs);

s4, dissolving polylysine in deionized water, and then mixing the solution with the following ratio of 1:1.5-1:5 (glycidyl methacrylate: amino group) glycidyl methacrylate was added in a molar ratio. 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 (the molar concentration is 100mM-500 mM) prepared in the S4 for 1-3 days to ensure that the solution reaches physical permeation close to saturation, so that the partially double-bonded polylysine is introduced as much as possible, and then adding glutaraldehyde to the aqueous solution until the mass concentration of the solution 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, finally soaking and cleaning with distilled water to remove 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 intervening a biological valve, such as through minimally invasive intervention; it may also be used for surgical biological valves, for example by surgical implantation.

The valve is made of 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 examples are given by way of illustration and not by way of limitation:

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 of an aqueous solution of DL-2-amino-4-pentenoic acid 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 calcium content is mu g/mg
		Glutaraldehyde control 1	168.595±9.973
Examples	43.220±10.873

The infrared spectra of the pericardium (GA) of sample 1 and control 1 are shown in fig. 4; the results of quantifying elastin in pericardium (GA) rats after subcutaneous implantation in samples 1 and control 1 are schematically 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 harvested pig hearts were washed with distilled water at 4 ℃ under 100RPM shaking for 2 hours, then soaked in 180mM modified polylysine aqueous solution at room temperature for 12 hours, then glutaraldehyde solution was added to a mass concentration of 2.5%, the reaction was carried out on a shaker at 37 ℃ for 24 hours, the pericardial material was taken out, washed, then soaked in 50mg/ml modified hyaluronic acid aqueous solution 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 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: collecting fresh rabbit blood, centrifuging 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, 100 μ L of platelet rich plasma was added and soaked for 1h at 37 ℃. 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 value 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 of 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 give control 3.

Example 3

Washing the freshly collected pig hearts with distilled water for 2 hours under the conditions of 4 ℃ and 100RPM rotational speed oscillation;

then soaking the pig heart envelope in 30mM 2-amino-7-ene-octanoic acid solution at 37 deg.C for 4 hr, adding glutaraldehyde to give a final concentration of 100mM, and soaking at 37 deg.C under 100RPM shaking for 24 hr;

taking out the porcine pericardium, and cleaning by using distilled water;

soaking the pig heart envelope in 30mM 2-amino-7-ene-caprylic acid solution for 4 hours;

and (3) cleaning, soaking in deionized water, adding ammonium persulfate and a sodium bisulfite initiator for initiation, wherein the molar concentrations of the ammonium persulfate and the sodium bisulfite are both 30mM, and reacting at 37 ℃ for 24 hours to obtain a sample marked as No. 3.

Example 4

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

then soaking the pig heart envelope in 20mM 4- (1-amino-2-methyl-propyl) -hepta-1, 6-dien-4-ol aqueous solution at 37 deg.C for 24 hours;

adding glutaraldehyde to make the final concentration of glutaraldehyde 100mM, and soaking at 37 deg.C under 100RPM rotation speed oscillation for 24 hr;

taking out the porcine pericardium, and cleaning by using distilled water;

soaking pig heart envelope in 30mM 4- (1-amino-2-methyl-propyl) -hepta-1, 6-diene-4-alcohol aqueous solution for 4 hr;

and (3) cleaning, soaking in deionized water, adding ammonium persulfate and a sodium bisulfite initiator for initiation, wherein the molar concentrations of the ammonium persulfate and the sodium bisulfite are both 30mM, and reacting at 37 ℃ for 24 hours to obtain a sample marked as sample No. 4.

Example 5

then soaking in 20mM 2-amino pent-4-enoic acid aqueous solution at 37 deg.C for 4 hr;

taking out the porcine pericardium, and cleaning by using distilled water;

and after washing, soaking the valve in deionized water, adding an initiator of ammonium persulfate and sodium bisulfite, reacting for 24 hours at 37 ℃ for initiating by adding molar concentrations of both ammonium persulfate and sodium bisulfite, transferring the valve into glycerol, and dehydrating to obtain a dry valve sample marked as No. 5 sample.

Example 6

Washing freshly collected pig hearts with distilled water at 4 deg.C under 100RPM shaking conditions for 2 hours, and then soaking in 20mM 2-aminopent-4-enoic acid aqueous solution at 37 deg.C for 4 hours;

and adding ammonium persulfate and sodium bisulfite initiator to initiate reaction, wherein the molar concentration of the ammonium persulfate and the molar concentration of the sodium bisulfite are both 30mM, and reacting for 24 hours at 37 ℃ to obtain a sample marked as sample No. 6.

Enzyme degradation experiments (characterization of degree of crosslinking)

Sample No. 5, sample No. 6 and control group 3 were cut into circular sheets having a diameter of 1cm, and 6 parallel samples were set for each group. 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. Each sample was weighed on a one-hundred-thousandth balance as an initial weight (W0) and placed back in a 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 samples were 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 frozen product 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 by the 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:

TABLE 6

	Weight loss rate of enzyme degradation
		Control group 3	7.06％
Sample No. 5	5.81％
		Sample No. 6	4.53％

The enzyme degradation experiments of the sample nos. 5 and 6 and the control group 3 were performed to characterize the degree of cross-linking of the respective groups of samples, and the enzyme degradation weight loss ratios of the respective groups of samples calculated after the sample nos. 5 and 6 and the control group 3 were treated with collagenase i are shown in table 6. The enzyme degradation weight loss rates of the No. 5 sample and the No. 6 sample are lower than those of the control group 3, which shows that the enzyme degradation stabilities of the No. 5 sample and the No. 6 sample are higher than those of the control group 3, namely the crosslinking degrees of the No. 5 sample and the No. 6 sample are higher. The enzyme degradation experiment result shows that the method for preparing the biological valve material by double-bond post-crosslinking can improve the crosslinking degree of the biological valve material.

Example 7

transferring the pig heart envelope into 50mM 2-amino-pent-4-enoic acid aqueous solution, and soaking for 4 hr;

and adding ammonium persulfate and sodium bisulfite initiator to initiate reaction, wherein the molar concentration of the ammonium persulfate and the molar concentration of the sodium bisulfite are both 30mM, and reacting for 24 hours at 37 ℃ to obtain a sample marked as sample No. 7.

Example 8

Washing freshly collected pig hearts with distilled water at 4 deg.C under 100RPM shaking for 2 hours, and then soaking in 20mM 2-aminopent-4-enoic acid aqueous solution at 37 deg.C for 4 hours;

taking out the porcine pericardium, and cleaning by using distilled water;

4 hours in 50mM aqueous 2-aminopent-4-enoic acid;

taking out the porcine pericardium, and cleaning by using distilled water;

soaking the pericardium in distilled water, adding ammonium persulfate and sodium bisulfite initiator to initiate, reacting for 24 hours at 37 ℃ with the molar concentration of both ammonium persulfate and sodium bisulfite, then washing with distilled water, and dehydrating with glycerol to obtain a dry film sample marked as sample No. 8.

Example 9

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 9. Compared with the control sample 2, the water contact angle of the sample 9 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.

The samples obtained in comparative example 3 and examples 3 to 8 (sample No. 3, sample No. 4, sample No. 5, sample No. 6, sample No. 7, sample No. 8 and control sample No. 3) were subjected to rat subcutaneous implantation test, and samples of each group were taken out 30 days after implantation to perform alizarin red staining test to characterize the calcification degree of the samples of each group 30 days after rat subcutaneous implantation.

Alizarin red staining experiment:

samples of the material (3, 4, 5, 6, 7, 8 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 at room temperature for 24 hours. And after the fixation is finished, the operation knife is taken out and is repaired and leveled, and then the operation knife is transferred into the dehydration box. The material samples were subjected to gradient dehydration 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 slide spreader to 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 with neutral gum and the staining images were collected on a pathological section scanner.

Samples 3, 4, 5, 6, 7, 8 and control 3, which were implanted in 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 alizarin red staining result images of the sample sections 30 days after the control samples 3 and 3, 4, 5, 6, 7 and 8 were implanted into the rat skin are shown in fig. 11-17, wherein the deeper the color of the alizarin red stained sample indicates the higher the calcification degree. Compared with the alizarin red staining result of the section of the control sample 3 (fig. 11), the alizarin red staining results of the samples 3, 4, 5, 6, 7 and 8 are obviously lighter in color, which indicates that the calcification degree of the samples 3, 4, 5, 6, 7 and 8 is lower than that of the control sample 3, i.e. the samples 3, 4, 5, 6, 7 and 8 have a certain anti-calcification effect compared with the control group 3. The results of alizarin red staining of the No. 3 sample, the No. 4 sample, the No. 5 sample, the No. 6 sample, the No. 7 sample, the No. 8 sample and the control group 3 which are implanted into the subcutaneous tissues of rats for 30 days show that the double-bond post-crosslinking method for preparing the biological valve material can improve the calcification-resistant performance of the biological valve.

Blood contact test:

the control samples No. 3 and No. 5 and No. 6, which had uniform surface and thickness, were cut into sheets having a diameter of 1cm, washed with physiological saline, drained and 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 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.

The scanning electron micrographs of the blood contact experiments of control samples No. 3, no. 5 and No. 6 are shown in FIGS. 18 to 20. More blood cell adhesion and aggregation were observed on the scanning electron micrograph of control 3 after incubation with rabbit blood, while the number 5 and 6 had fewer adhered blood cells, and only a few blood cells were dispersedly adhered to the surface. The results show that the sample No. 5 and the sample No. 6 can inhibit the adhesion of blood cells to a certain degree so as to reduce the risk of blood coagulation and have the effect of anticoagulation.

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 preparation method of a double-bond post-crosslinking biological valve material is characterized by comprising the following steps:

2. The method according to 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 production method according to claim 1, wherein in step (3): adding an initiator into the system treated in the previous step; or cleaning the biological material after the previous step and then soaking the biological material in a solution containing an initiator.

5. The preparation method according to claim 1, wherein 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 to 100mM; or

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

6. The method according to claim 1, further comprising a step (2M) before the step (3): soaking the biological material treated in the step (2) in a solution containing functional monomers to eliminate residual aldehyde groups; the 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.

7. The method according to claim 6, wherein in the step (2M), the solvent of the solution is water, physiological saline, 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 48h.

8. The method according to claim 6, wherein the functional monomer in the step (2M) further has at least one functional group and at least one carbon-carbon double bond; the functional group in the step (2M) is at least one selected from the group consisting of a hydroxyl group, a carboxyl group, an amide group and a sulfonic acid group.

9. The method according to claim 6, wherein the functional monomer in step (2M) is selected from the group consisting of 2-amino-7-ene-octanoic acid, 6-ene-heptanoic acid, 4- (1-amino-2-methyl-propyl) -hepta-1, 6-dien-4-ol, and 4- (1-amino-ethyl) -hepta-1, 6-dien-4-ol.

10. The production method according to claim 1, wherein the functional group in step (1) is at least one selected from the group consisting of a hydroxyl group, a carboxyl group, an amide group and a sulfonic acid group.

11. The method according to claim 1, wherein the functional monomer in step (1) is one selected from the group consisting of 2-amino-7-ene-octanoic acid, 6-ene-heptanoic acid, 4- (1-amino-2-methyl-propyl) -hepta-1, 6-dien-4-ol, and 4- (1-amino-ethyl) -hepta-1, 6-dien-4-ol.

12. A double-bond post-crosslinking biological valve material, which is characterized by being prepared by the preparation method of any one of claims 1 to 11.

13. A biological valve comprising a stent and a valve, wherein the valve is the double bond post-crosslinked biological valve material of claim 12.

14. The biological valve of claim 13, wherein the biological valve is a heart valve.