CN114748696A

CN114748696A - Double-bond cross-linked biological valve material after co-cross-linking, and preparation method and application thereof

Info

Publication number: CN114748696A
Application number: CN202210273149.1A
Authority: CN
Inventors: 王云兵; 郑城; 雷洋
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-11-17
Filing date: 2022-03-18
Publication date: 2022-07-15
Anticipated expiration: 2042-03-18
Also published as: CN115970060B; CN115970060A; CN115779149B; CN115645618A; CN114748697A; CN114748693A; CN114748695A; CN115779149A; CN115645618B; CN114748696B; CN114748693B; CN116271236A; CN114748694A; CN114748697B; CN114748695B

Abstract

The application discloses a double-bond cross-linked biological valve material after co-cross-linking, a preparation method and application thereof, wherein (1) the biological material is soaked in a solution containing a first functional monomer for physical permeation; the first functional monomer has at least one carbon-carbon double bond and at least one group which reacts with an aldehyde group; (2) adding an aldehyde crosslinking agent into the system in the step (1) for co-crosslinking; (3) soaking the biological material treated in the step (2) in a solution containing a second functional monomer for physical permeation; the second functional monomer has at least one carbon-carbon double bond; (4) and (4) adding an initiator into the system in the step (3) to initiate double bond polymerization. According to the method, a second functional monomer is added in a double-bond polymerization step for copolymerization to form more large polymer cross-linked networks, so that the cross-linking degree of the biological material can be improved, and the calcification resistance can be improved.

Description

Double-bond cross-linked biological valve material after co-cross-linking, and preparation method 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 market are prepared by crosslinking glutaraldehyde, which can crosslink collagen in pericardium, but the calcification-resistant and anticoagulation-resistant properties of the biological valve crosslinked by glutaraldehyde need to be improved.

Disclosure of Invention

The application provides a double-bond cross-linked biological valve material after co-cross-linking, a preparation method and application thereof, which can improve the calcification-resistant performance of the biological material.

A preparation method of a double-bond cross-linked biological valve material after co-cross-linking is characterized by comprising the following steps:

(1) soaking the biological material in a solution containing a first functional monomer for physical permeation; the first functional monomer has at least one carbon-carbon double bond and at least one group which reacts with an aldehyde group;

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

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

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

Optionally, the aldehyde-based cross-linking agent is glutaraldehyde or formaldehyde.

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

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

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

Optionally, in the step (2M), the solvent of the solution is water, physiological saline, pH neutral buffer solution 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.

Optionally, the groups of the first functional monomer and the third functional monomer that react with the aldehyde group are each independently selected from amino or hydrazide.

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

Optionally, the first functional monomer further has at least one functional group; the third functional monomer also has at least one carbon-carbon double bond and/or at least one functional group; the functional groups of the first functional monomer and the third functional monomer are respectively and independently selected from at least one of hydroxyl, carboxyl, amide and sulfonic acid.

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

Optionally, the second functional monomer is one of polyethylene glycol diacrylate, 1, 4-butanediol diacrylate, ethyl acrylate, N-methyl-2-acrylamide, N-2, 2-propenyl-2-acrylamide, N-ethylacrylamide, N' -vinylbisacrylamide, (ethane-1, 2-diylbis (oxy)) bis (ethane-2, 1-diyl) diacrylate, and double-bonded hyaluronic acid.

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

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

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

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

The application also provides a double-bond cross-linked biological valve material after co-cross-linking, which is prepared by the preparation method.

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

Optionally, the biological valve is a heart valve.

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

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

(2) the method can block partial residual aldehyde group while co-crosslinking, improve the calcification-resistant performance of the biological material, and further improve the crosslinking degree of the biological material;

(3) after the co-crosslinking is completed, carbon-carbon double bonds are introduced while the residual aldehyde groups are eliminated, so that more carbon-carbon double bonds are provided for subsequent double bond polymerization, and the crosslinking degree of the biological material is further improved.

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

Drawings

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

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

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

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

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

FIG. 6 is a schematic diagram showing the detection of the amount of calcium attached to pericardium (GA) rats after subcutaneous implantation in samples 1 and control 1 of example 1;

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

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

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

FIG. 10 is a basic schematic diagram of embodiment 3

FIG. 11 is a photograph of alizarin red stained sections taken 30 days after the control samples of control group 3 were implanted;

FIG. 12 is a photograph of alizarin red stained sections taken 30 days after sample 3 implantation;

FIG. 13 is a photograph of alizarin red stained sections taken 30 days after sample 4 implantation;

FIG. 14 is a photograph of alizarin red stained sections taken 30 days after sample 5 implantation;

FIG. 15 is a photograph of alizarin red stained sections taken 30 days after sample 6 implantation;

fig. 16 is a photograph of alizarin red stained sections taken 30 days after sample 7 implantation.

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 herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The biological valve on the market is mainly glutaraldehyde cross-linked biological membrane, and glutaraldehyde can cross-link collagen in the pericardium, however, the calcification resistance and the anticoagulation resistance of the glutaraldehyde cross-linked biological valve need to be improved. The mechanical property, the calcification resistance and the anticoagulation property of the glutaraldehyde crosslinking membrane are improved by improving a crosslinking means on the basis of glutaraldehyde crosslinking.

According to the improved crosslinking scheme, a functional monomer with a carbon-carbon double bond and a group reacting with aldehyde group is introduced before glutaraldehyde crosslinking, the functional monomer physically permeates into the biological material and then is subjected to co-crosslinking with an aldehyde crosslinking agent, the amino group of the functional monomer reacts with the aldehyde group, and the carbon-carbon double bond and the functional group are introduced into the biological material simultaneously; and introducing a part of carbon-carbon double bonds through physical permeation of a second functional monomer, and finally initiating polymerization of the carbon-carbon double bonds on the biological material and the carbon-carbon double bonds of the second functional monomer to form a cross-linked network, thereby further improving the cross-linking degree of the biological material.

Specifically, the method comprises the following steps:

(1) soaking the biological material in a solution containing a first functional monomer for physical permeation; the first functional monomer has at least one carbon-carbon double bond and at least one group which reacts with residual aldehyde groups on the biomaterial;

The reaction principle of the present application:

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

1) aldehyde groups at two ends of a part of cross-linking agents react with amino groups of the biological materials; 2) 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: the second functional monomer is firstly physically permeated into the biological material after completing co-crosslinking and introducing the carbon-carbon double bond for one time, the carbon-carbon double bond is further introduced, the step of introducing the carbon-carbon double bond is physical permeation, after the second functional monomer is permeated, the carbon-carbon double bond of the second functional monomer is initiated to be copolymerized with the carbon-carbon double bond on the surface of the biological material, and secondary crosslinking is carried out to form a crosslinking network.

According to the method, the biological material is subjected to crosslinking treatment through aldehyde group co-crosslinking and double bond polymerization secondary crosslinking, and the biological material obtained through the secondary crosslinking treatment has good crosslinking degree; this application is crosslinked and is introduced carbon-carbon double bond and then further introduced carbon-carbon double bond through second functional monomer again, and the carbon-carbon double bond that introduces through physical permeation for the second time makes extra functional monomer participate in the copolymerization in the double bond polymerization process, forms bigger polymer crosslinked network, is favorable to promoting biological valve cross linking degree and anti calcification performance.

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

The biomaterial adopted in the application is a biomaterial which is conventional in the existing glutaraldehyde crosslinking process. The collagen content of the biomaterial 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 preferred embodiment, optionally, before step (3), a step (2M) is further included: soaking the biological material treated in the step (2) in a solution containing a third functional monomer to eliminate residual aldehyde groups on the residual part; the third functional monomer of this step has at least one group that reacts with an aldehyde group.

The first functional monomer has at least one group which reacts with aldehyde group, and in the co-crosslinking process, the first functional monomer reacts with partial residual aldehyde group on the biological material through the group to introduce carbon-carbon double bonds into the biological material. Alternatively, the group in the first functional monomer that reacts with the aldehyde group includes, but is not limited to, an amino group and a hydrazide.

In the embodiment comprising step (2M), the third functional monomer has at least one group that reacts with aldehyde group, and reacts with residual aldehyde group on the biomaterial during the soaking process. Alternatively, the group in the third functional monomer that reacts with the aldehyde group includes, but is not limited to, an amino group and a hydrazide.

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

The third functional monomer of the application has at least one amino group, and in a preferable scheme, the third functional monomer also has at least one carbon-carbon double bond, and when the biological material is treated by the third functional monomer solution again, the carbon-carbon double bond can be introduced again while the residual aldehyde group is sealed through the reaction of the amino group on the functional monomer and the residual aldehyde group on the biological membrane, so that the double bond base number of the subsequent double bond polymerization is increased.

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

The first functional monomer and the third functional monomer of the present application may further include a functional group in addition to the carbon-carbon double bond and the amino group, and optionally, the functional group in the first functional monomer and the third functional monomer is independently selected from at least one of a hydroxyl group, a carboxyl group, an amide group, and a sulfonic acid group.

The introduction of hydroxyl groups can improve the hydrophilicity of the biological material; the introduction of carboxyl groups can enable the biological material to be electrically neutral; 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 monomer satisfying both of at least one amino group, at least one carbon-carbon double bond and at least one functional group as described above, in one embodiment, a commercially available product can be directly used. Optionally, the first functional monomer and the third functional monomer are each independently selected from one of 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, and 4- (1-amino-ethyl) -hepta-1, 6-dien-4-ol.

The first functional monomer and the third functional monomer can be prepared by double bond modification, such as double-bonded polylysine, besides the commercial routes shown above.

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

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

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

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

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

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

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

And (3) after the first 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 optionally.

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 human body adaptive temperature, and optionally, the temperature is within 36-37 ℃; the reaction time of co-crosslinking is preferably complete as much as possible, and optionally, under the condition that the concentration of the crosslinking agent is 10-800 mM, the co-crosslinking time is 10-30 h.

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

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

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

Further optionally, in the step (2M), the solvent in the solution is water, physiological saline, pH neutral buffer solution or aqueous solution of ethanol, wherein in the aqueous solution of ethanol, ethanol and water may be mixed according to any proportion, and about 50% ethanol is usually used; the concentration of the third functional monomer in the solution is 20-50 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 third functional monomer solution; or directly transferring the biological material treated in the step (2) into a third functional monomer solution.

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

After step (2) or step (2M) is completed, further introducing a carbon-carbon double bond by a second functional monomer, wherein the introduction process is physical infiltration, and the second functional monomer does not react with the biological material in the step, and in one embodiment, the second functional monomer is optionally one of polyethylene glycol diacrylate, 1, 4-butanediol diacrylate, ethyl acrylate, N-methyl-2-acrylamide, N-2, 2-propenyl-2-acrylamide, N-ethylacrylamide, N' -vinyl bisacrylamide, and (ethane-1, 2-diylbis (oxy)) bis (ethane-2, 1-diylbis) diacrylate.

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

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

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

weighing 2g of sodium hyaluronate with the molecular weight of 10000, dissolving the sodium hyaluronate with the molecular weight of 20ml of PBS, and sequentially adding 6-12ml of glycidyl methacrylate and 4-8ml of triethylamine. Standing 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);

one 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 group). 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 carbon-carbon double bonds are reintroduced after the co-crosslinking or step (2M) is completed, and in an alternative embodiment, the second functional monomer is introduced directly after the co-crosslinking or step (2M) is completed. The scheme is commonly called as a one-pot method, namely, a second functional monomer is directly added into a reaction system of co-crosslinking or step (2M) after the co-crosslinking or step (2M) is finished, the second functional monomer is directly added into the reaction system to initiate double bond polymerization reaction after penetrating into the biological material, and the process of taking out and cleaning the biological material is not needed.

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

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

Optionally, the solvent in the solution containing the second functional monomer is water, normal saline, 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%; the mass percentage concentration of the second functional monomer is 1-10%.

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

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

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

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

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

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

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

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

Optionally, the double bond polymerization process can be carried out at 20-50 ℃, preferably, the temperature does not need to be controlled particularly, the double bond polymerization process can be carried out at room temperature, preferably not exceeding the human body adaptive temperature, and preferably carried out at 36-37 ℃. The time for polymerizing the double bond is preferably 2 to 48 hours, more preferably 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 a more preferred flow shown in fig. 1 as an example:

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

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

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

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

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

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

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

A more specific embodiment, comprising the steps of:

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

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

S3, soaking the biological material cleaned 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 full physical permeation of DL-2-amino-4-pentenoic acid;

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 the 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 distilled water, and removing unreacted DL-2-amino-4-pentenoic acid and glutaraldehyde.

S6, soaking the biological material processed 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 biomaterial processed in the step S6 into ammonium persulfate and sodium bisulfite initiator to initiate, wherein the molar concentration of the ammonium persulfate and the sodium bisulfite is 10-100 mM.

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 DL-2-amino-4-pentenoic acid/glutaraldehyde/pericardium co-crosslinking 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 more specific embodiment, the method comprises the steps of:

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

s3, weighing 2g of sodium hyaluronate with the molecular weight of 10000, dissolving the sodium hyaluronate with the molecular weight of 20ml of PBS, and sequentially adding 6-12ml of glycidyl methacrylate and 4-8ml of triethylamine. Standing 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 adding glycidyl methacrylate in a molar ratio of 1:1.5-1:5 (glycidyl methacrylate: amino group). 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 S2 in an aqueous solution of partially double-bonded polylysine (with a molar concentration of 100mM-500mM) prepared in 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 to a 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/N, N, N ', N' -tetramethyl ethylenediamine, wherein the concentration of the double-bonded hyaluronic acid is 20mg/ml-60 mg/ml. Reacting at 37 deg.c for 12-24 hr;

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

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

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

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

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

3) compared with most researches of using polysaccharide to perform hydrophilic modification on the surface interface, the hyaluronic acid and the pericardium material are combined in a chemical covalent manner, so that the method has higher stability.

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; and may also be used to surgically implant biological valves, such as by surgery.

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

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

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

The following is a description of specific examples:

example 1

In this example, freshly collected pig hearts were washed with distilled water at 4 ℃ under 100RPM shaking for 2 hours, then soaked in 30mM DL-2-amino-4-pentenoic acid aqueous solution at 37 ℃ for 12 hours, then glutaraldehyde was added to give a concentration of 100mM, and washed with distilled water after soaking at 37 ℃ under 100RPM shaking for 24 hours. After cleaning, soaking the sample in a 5% aqueous solution of polyethylene glycol diacrylate, soaking the sample for 12 hours at 37 ℃ to ensure sufficient physical permeation of the polyethylene glycol diacrylate, adding ammonium persulfate and sodium bisulfite initiator for initiation, wherein the molar concentrations of the ammonium persulfate and the sodium bisulfite are both 40mM, and reacting the mixture for 24 hours at 37 ℃, and marking the reaction product as a 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 the analysis of the relative activities of lactate dehydrogenase in example 1 and glutaraldehyde control 1 are shown in Table 1, and the amount of calcium attachment is shown in Table 2.

TABLE 1

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

TABLE 2

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

The infrared spectrograms of the pericardium (GA) of the sample 1 and the control 1 sample are shown in fig. 4; results of elastin quantification after pericardium (GA) rat subcutaneous implantation of sample 1 and control 1 sample are shown in fig. 5; a schematic diagram of the detection of calcium loading 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: 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 cutoff of 1000, and freeze-drying to obtain the partially double-bonded polylysine;

In this example, freshly collected pig pericardium was washed with distilled water at 4 ℃ under 100RPM shaking for 2 hours, then soaked in 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 pericardium 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 1 × 1cm square pieces, flattened between two glass sheets, vacuum freeze-dried and subjected to water contact angle test.

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

(3) Hemolysis rate test

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

(4) Calcification testing

An incision was made in the back of 45-50g male SD rats, and subcutaneous tissue was separated with a blunt instrument to create a cavity, the control group and the sample of example 1 were placed in the cavity, then the skin was sutured, the sample was 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 subjected to inductively coupled plasma atomic emission spectroscopy 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 rate (%)
			Glutaraldehyde control 2	0.41	1.54
Example 2	0.24	0.38

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

TABLE 5

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

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

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

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

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

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

Control group 3

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

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 immersed in a 20mM aqueous solution of 2-methylallylamine at 37 ℃ for 2 hours;

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

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

soaking pig heart bags in 20mM 2-methylallyl amine water solution for 2 hours;

after cleaning, the substrate is soaked in a 5 wt% aqueous solution of N, N '-vinyl bisacrylamide and soaked for 12 hours at 37 ℃ to ensure that the N, N' -vinyl bisacrylamide is fully and physically permeated.

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

Example 4

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;

cleaning, soaking in 5 wt% 1, 4-butanediol diacrylate aqueous solution, and soaking at 37 deg.C for 12 hr to ensure sufficient physical permeation of 1, 4-butanediol diacrylate;

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

Example 5

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

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

After cleaning, the (ethane-1, 2-diylbis (oxy)) bis (ethane-2, 1-diyl) diacrylate was soaked in a 2 wt% aqueous solution of (ethane-1, 2-diylbis (oxy)) bis (ethane-2, 1-diyl) diacrylate and the physical permeation of (ethane-1, 2-diylbis (oxy)) bis (ethane-2, 1-diyl) diacrylate was sufficiently ensured at 37 ℃ for 12 hours.

Adding ammonium persulfate and sodium bisulfite initiator to initiate reaction at the molar concentration of 30mM and 37 ℃ for 24 hours;

washing with distilled water, soaking in glycerol, and dehydrating to obtain dry film. To facilitate the differentiation of the samples prepared in the examples, the sample obtained in this example was designated as sample 5.

Example 6

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

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

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

N, N '-vinyl bisacrylamide was added to a final concentration of 5%, and the mixture was immersed at 37 ℃ for 12 hours to ensure sufficient physical permeation of N, N' -vinyl bisacrylamide.

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

Example 7

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

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

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

soaking pericardium in 50mM 2-amino pent-4-enoic acid water solution for 4 hr;

Initiating by adding ammonium persulfate and sodium bisulfite initiator, wherein the molar concentration of the ammonium persulfate and the sodium bisulfite is 30mM, and reacting for 24 hours at 37 ℃. To facilitate the differentiation of the samples prepared in the examples, the sample obtained in this example was designated as sample 7.

Example 8

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

The samples obtained in comparative example 3 and examples 3 to 6 were subjected to measurement of the collagenase degradation weight loss ratio.

Enzyme degradation experiments:

sample 3, sample 4, sample 5, sample 6, sample 7 and control 3 were cut into circular sheets of 1cm diameter, with 6 replicates per set. These circular sheet samples were placed in 48-well plates, frozen overnight at minus 80 ℃ and then transferred to a vacuum lyophilizer for 48 hours. The weight of each sample was weighed as the initial weight (W0) on a one-hundred-thousandth balance and placed back in the 48-well plate. 0.5mL of collagenase I in PBS was added to each well of the 48-well plate using a pipette gun and the biological valve 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, discarding 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 collagenase solution was weighed on a one-hundred-thousand balance and recorded as the final weight (Wt). The weight loss rate of enzyme degradation is calculated by the following formula:

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

Table 6 collagenase degradation weight loss ratio.

Sample numbering	Collagenase degradation weight loss rate
		Control group 3	8.6％
Sample 3	2.3％
		Sample 4	3.7％
Sample No. 5	1.9％
		Sample No. 6	3.8％
Sample 7	3.1％

The samples 3, 4, 5, 6, 7 and 3 were subjected to enzyme degradation experiments to characterize the degree of cross-linking of the samples in each group, and the weight loss rate of enzyme degradation of the samples in each group was calculated after treating the samples 3, 4, 5, 6, 7 and 3 with collagenase i, and the results are shown in table 6. The enzyme degradation weight loss rates of the samples 3, 4, 5, 6 and 7 are all lower than that of the control group 3, which indicates that the enzyme degradation stabilities of the samples 3, 4, 5 and 6 are all higher than that of the control group 3, i.e. the cross-linking degrees of the samples 3, 4, 5 and 6 are higher. The enzyme degradation experiment result shows that the preparation method of the double-bond cross-linked biological valve material after co-cross-linking can improve the cross-linking degree of the biological valve material.

Alizarin red staining experiment:

samples of material (sample 3, sample 4, sample 5, sample 6, sample 7, and control 3) taken 30 days after subcutaneous implantation in mice were washed with PBS. After washing, the tissue was fixed in 4% (w/v) paraformaldehyde PBS tissue fixative 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 and control 3, which were implanted into the rat subcutaneously for 30 days, were stained by alizarin red staining test to characterize the degree of calcification of each group of samples. Alizarin red staining results of the sample sections 30 days after implantation into the rat skin are shown in fig. 11-16, where the darker the color of the alizarin red stained sample indicates the higher degree of calcification. Compared with the alizarin red staining result of the section of the control sample 3 (fig. 11), the alizarin red staining result of the samples 3, 4, 5, 6, and 7 is obviously lighter in color, which indicates that the calcification degree of the samples 3, 4, 5, 6, and 7 is lower than that of the control sample 3, i.e., the samples 3, 4, 5, 6, and 7 have a certain anti-calcification effect compared with the control group 3. Alizarin red staining results of the sample 3, the sample 4, the sample 5, the sample 6, the sample 7 and the control group 3 which are implanted into the subcutaneous tissues of rats for 30 days show that the preparation method of the double-bond cross-linked biological valve material after co-cross-linking can improve the anti-calcification performance of the biological valve.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is specific and detailed, but not to be understood 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 application shall be subject to the appended claims.

Claims

1. A preparation method of a double-bond cross-linked biological valve material after co-cross-linking is characterized by comprising the following steps:

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

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 first 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 co-crosslinking time is 10-30 h.

4. 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 a third functional monomer to eliminate residual aldehyde groups; the third functional monomer has at least one group that reacts with an aldehyde group.

5. The method according to claim 4, 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 third functional monomer in the solution is 10-100 mM; the soaking time is 2-48 h.

6. The method according to claim 1 or 4, wherein the group of the first functional monomer or the third functional monomer that reacts with the aldehyde group is independently selected from an amino group or a hydrazide.

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

8. The method according to claim 1 or 4, wherein the first functional monomer further has at least one functional group; the third functional monomer also has at least one carbon-carbon double bond and/or at least one functional group; the functional groups of the first functional monomer and the third functional monomer are respectively and independently selected from at least one of hydroxyl, carboxyl, amide and sulfonic acid.

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

10. The method according to claim 1, wherein the second functional monomer is one of polyethylene glycol diacrylate, 1, 4-butanediol diacrylate, ethyl acrylate, N-methyl-2-acrylamide, N-2, 2-propenyl-2-acrylamide, N-ethylacrylamide, N' -vinylbisacrylamide, (ethane-1, 2-diylbis (oxy)) bis (ethane-2, 1-diyl) diacrylate, and double-bonded hyaluronic acid.

11. The method according to claim 1, wherein in the step (3): adding a second functional monomer 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 a second functional monomer.

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

13. The preparation method according to claim 1, characterized in that 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

14. A double-bond cross-linked biological valve material after co-crosslinking, which is prepared by the preparation method of any one of claims 1 to 13.

15. A biological valve comprising a stent and a valve, wherein the valve is the cross-linked double-bond cross-linked biological valve material of claim 14.

16. The biological valve of claim 15, wherein the biological valve is a heart valve.