FOLD RESISTANT DEHYDRATED CROSS-LINKED BIOLOGICAL MATERIAL, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF

Abstract

A biological material is provided and prepared by performing (a) oxidation, (b) first cross-linking, (c) second cross-linking, and (d) dehydration on a biological material for preparation; wherein (b) is performed after (a), and (a), (b), and (c) are all performed prior to (d); during oxidation, an oxidizing agent able to cause hydroxy groups to be converted to aldehyde groups is utilized, the hydroxy groups coming from mucopolysaccharides in the biological material; during first cross-linking, a first cross-linking agent able to cause cross-linking between aldehyde groups of mucopolysaccharides is utilized; and during second cross-linking, a second cross-linking agent able to cause cross-linking between collagen fibers in the biological material is utilized.

Claims

1. A biological material, prepared by composite crosslinking, using a preparation method comprising: performing (a) oxidation, (b) first crosslinking, (c) second crosslinking and (d) dehydration on the biological material, wherein (b) is performed after (a), and (a), (b) and (c) are all performed prior to (d); and wherein the oxidation uses an oxidant capable of converting hydroxyl groups of mucopolysaccharides of the biological material into aldehyde groups, the first crosslinking uses a first crosslinking agent capable of causing cross-linking between the aldehyde groups of the mucopolysaccharides, and the second crosslinking uses a second crosslinking agent capable of causing cross-linking between collagen fibers of the biological material.

2. The biological material according to claim 1, wherein the biological material is originated from pig, cattle, horse or sheep, and selected from pericardium, heart valve, blood vessel, ligament, muscle, intestine or skin.

3. The biological material according to claim 1, wherein the oxidation comprises exposing the biological material into a solution containing the oxidant.

4. The biological material according to claim 3, wherein the hydroxyl group is an ortho-hydroxyl group from the mucopolysaccharide.

5. The biological material according to claim 3, wherein the oxidant is sodium periodate.

6. The biological material according to claim 3, wherein the oxidant in the solution containing the oxidant has a mass percentage concentration ranging from 0.1% to 1%, and the method comprising exposing the biological material into the solution containing the oxidant for 1 to 12 hours by static contact or dynamic contact.

7. The biological material according to claim 1, wherein the method comprising performing (a) oxidation, (b) first crosslinking and (c) second crosslinking on the biological material in sequence: exposing the biological material after oxidation to a first crosslinking agent solution and a second crosslinking agent solution.

8. The biological material according to claim 7, wherein the first crosslinking agent has at least two active groups capable of reacting with the aldehyde groups, and the second crosslinking agent is different from the first crosslinking agent and capable of reacting with the active groups of the first crosslinking agent.

9. The biological material according to claim 8, wherein the first crosslinking agent is a small molecular substance having at least two amino groups.

10. The biological material according to claim 9, wherein the first crosslinking agent is lysine, ethylenediamine or hexamethylenediamine.

11. The biological material according to claim 7, wherein the first crosslinking agent in the first crosslinking agent solution has a mass percentage concentration ranging from 0.05% to 5%, and the method comprising exposing the biological material into the first crosslinking agent solution for 0.5 to 12 hours.

12. The biological material according to claim 7, wherein the second crosslinking agent is glutaraldehyde, the second crosslinking agent in the second crosslinking agent solution has a mass percentage concentration ranging from 0.05% to 25%, the crosslinking time ranges from 6 h to 3 weeks, and the crosslinking temperature ranges from 0 to 37° C.

13. The biological material according to claim 7, wherein the method further comprising end-capping of exposing the cross-linked biological material into a solution containing a capping substance to block residual active groups from the second crosslinking agent.

14. The biological material according to claim 1, wherein the method comprising performing (c) second crosslinking, (a) oxidation and (b) first crosslinking on the biological material in sequence: exposing the biological material into a second crosslinking agent solution, a solution containing the oxidant and a first crosslinking agent solution.

15. The biological material according to claim 1, wherein the method comprising performing (a) oxidation, (c) second crosslinking and (b) first crosslinking on the biological material in sequence: exposing the biological material into a solution containing the oxidant, a second crosslinking agent solution and a first crosslinking agent solution.

16. The biological material according to claim 14, wherein the first crosslinking agent in the first crosslinking agent solution has a mass percentage concentration ranging from 0.5% to 10%, and the method comprising exposing the biological material into the first crosslinking agent solution for 0.5 to 12 hours.

17. The biological material according to claim 14, wherein the second crosslinking agent is glutaraldehyde, the second crosslinking agent in the second crosslinking agent solution has a mass percentage concentration ranging from 0.05% to 25%, the crosslinking time ranges from 6 h to 3 weeks, and the crosslinking temperature ranges from 0 to 37° C.

18. The biological material according to claim 14, wherein the method further comprising post-crosslinking of exposing the biological material treated with the first crosslinking agent into a third crosslinking agent solution to crosslink residual active groups from the first crosslinking agent.

19. A biological valve, comprising a stent and a valve provided on the stent, wherein the valve is the biological material according to claim 1.

20. The biological valve according to claim 19, wherein the biological material has a maximum breaking force n ranging from 25 to 30 N, and the biological material is configured to be crimped and maintained for at least 72 hours and become flattened without creases.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0090] FIG. 1 is a schematic diagram of oxidation;

[0091] FIG. 2 is a schematic diagram of crosslinking with a first crosslinking agent and a second crosslinking agent;

[0092] FIG. 3 is a schematic diagram of oxidation, crosslinking with the first crosslinking agent and crosslinking with the second crosslinking agent in sequence;

[0093] FIG. 4 is a flow chart of (a) oxidation, (b) first crosslinking, (c) second crosslinking and (d) dehydration performed in sequence;

[0094] FIG. 5 is a flow chart of (c) second crosslinking, (a) oxidation, (b) first crosslinking and (d) dehydration in sequence;

[0095] FIG. 6 is a flow chart of (a) oxidation, b) first crosslinking, (c) second crosslinking and (d) dehydration performed in sequence;

[0096] FIG. 7 is a flowchart of Example 1;

[0097] FIG. 8 is a graph comparing the maximum breaking force of the cross-linked pericardium prepared in Example 1 and the conventional glutaraldehyde cross-linked pericardium;

[0098] FIG. 9 is a graph showing the result of the crease resistance test of the cross-linked pericardium prepared in Example 1;

[0099] FIG. 10 is a graph comparing the maximum breaking force of the cross-linked pericardium prepared in Example 2 and the conventional glutaraldehyde cross-linked pericardium;

[0100] FIG. 11 is a graph showing the result of the crease resistance test of the cross-linked pericardium prepared in Example 2;

[0101] FIG. 12 is a graph comparing the maximum breaking force of the cross-linked pericardium prepared in Example 4 and the conventional glutaraldehyde cross-linked pericardium; and

[0102] FIG. 13 is a graph showing the result of the crease resistance test of the cross-linked pericardium prepared in Example 4.

DDESCRIPTION OF EMBODIMENTS

[0103] The technical solutions according to the embodiments of the present disclosure will be described clearly and fully in combination with the drawings according to the embodiments of the present disclosure. Obviously, the described embodiments are not all embodiments of the present disclosure, but only part of the embodiments of the present disclosure. Based on the disclosed embodiments, all other embodiments obtained by those skilled in the art without creative work fall into the scope of this invention.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art. The terms in the description of the present disclosure are used to describe specific embodiments, and not to limit the present disclosure.

[0105] In the first embodiment, a biological material is subjected to oxidation, crosslinking and dehydration in sequence. The crosslinking includes a first crosslinking and a second crosslinking performed in sequence. During the oxidation, an oxidant oxidizes ortho-hydroxyl groups of mucopolysaccharides of the biological material into aldehyde groups, thereby connecting the polysaccharides to the collagen fibers; then by means of crosslinking, the polysaccharides and the collagen fibers are respectively cross-linked to form a one-piece cross-linked network, and within the final network, cross-linking occurs between polysaccharides, polysaccharide and collagen fiber, and collagen fibers.

[0106] After the second crosslinking, end-capping, post-crosslinking and reduction can be performed, followed by dehydration. In the end-capping, the residual active groups of the second crosslinking agent are blocked, in the post-crosslinking, the residual active groups of the capping substance are cross-linked, and in the reduction, the schiff-base chemical bonds are converted into stable carbon-nitrogen single bonds.

[0107] The flow chart of this embodiment is shown in FIG. 4, where the biological material is sequentially subjected to oxidation, first crosslinking, second crosslinking, end-capping, post-crosslinking, reduction and dehydration.

Oxidation

[0108] Take a fresh pericardium, and cut and hang the same on a board, wherein the pericardium is a pig pericardium or a bovine pericardium. Then the pericardium is exposed to an oxidant solution by soaking or spraying, wherein the oxidant can be sodium periodate, and the mass percentage concentration thereof in the oxidant solution is 0.1% to 1%, and the oxidation time is 1 to 12 hours.

[0109] After oxidation, the ortho-hydroxyl groups of the mucopolysaccharides of the pericardium are oxidized into aldehyde groups, and the aldehyde groups react with the amino groups of the collagen fibers, thereby connecting the mucopolysaccharides to the collagen fibers.

First Crosslinking

[0110] After the mucopolysaccharides are connected to the collagen fibers, the mucopolysaccharides are cross-linked through aldehyde groups. The first crosslinking agent should have a group that can react with the aldehyde group, and preferably uses a bisamino compound. The mucopolysaccharides are cross-linked with each other through the reaction of the amino groups with the aldehyde groups.

[0111] The bisamino compound can be selected from lysine, ethylenediamine or hexamethylenediamine. The mass percentage concentration of the bisamino compound in the bisamino compound solution is 0.05% to 5%, the exposure time of the biological material in the bisamino compound solution is 0.5 to 12h, and the exposure method can be soaking or spraying.

Second Crosslinking

[0112] The collagen fibers of the biological material are cross-linked by a second crosslinking agent, and the second crosslinking agent can be glutaraldehyde. In this step, the biological material is exposed to the glutaraldehyde solution by soaking or spraying. The mass percentage concentration of glutaraldehyde in the glutaraldehyde solution is 0.05% to 25%, the crosslinking time is 6 h to 3 weeks, and the crosslinking temperature is 0 to 37° C.

End-Capping

[0113] After crosslinking using the glutaraldehyde, there are residual aldehyde groups in the biological material. In this step, end-capping is performed through a capping substance. The capping substance is preferably a polyamino substance, and the polyamino substance can be selected from polyethyleneimine, polylysine, ε-polylysine, lysine, hexamethylenediamine, etc.

[0114] In this step, the biological material is exposed to the polyamino substance solution. The mass percentage concentration of the polyamino substance in the polyamino substance solution is 0.5% to 10%, the exposure time is 0.5 h to 12 h, and the exposure method can be soaking or spraying.

Third Crosslinking

[0115] In the previous end-capping step, the polyamino substance is used as the capping substance. The residual amino groups are then cross-linked by a third crosslinking agent in this step. The third crosslinking agent is polyethylene glycol diglycidyl ether. The cross-linked network formed by polyethylene glycol improves the high elasticity of the pericardium. On the one hand, the high symmetry of the polyethylene glycol chain improves the high elasticity of the biological tissue, so that the ability of the biological material to deform elastically can be fully restored. On the other hand, water is a benign solvent for the polyethylene glycol, so that after being soaked in water, the biological material exhibits a high elasticity, further improving the crease resistance thereof.

[0116] The mass percentage concentration of the third crosslinking agent in the third crosslinking agent solution is 0.01% to 2%, and the crosslinking time is 10 to 48 h.

Reduction

[0117] The amino groups react with the aldehyde groups to form schiff-base chemical bonds, but the schiff-base chemical bonds are reversible. By means of reduction, the schiff-base chemical bonds can be converted into stable carbon-nitrogen single bonds, avoiding the chemical bond exchange reaction during the crimping process.

[0118] In this step, the biological material is exposed to a reductant solution, and the reductant can be sodium cyanoborohydride or sodium borohydride. The mass percentage concentration of the reductant in the reductant solution is 0.01% to 2%, the exposure time is 1 to 12 hours, and the exposure method can be soaking or spraying.

Dehydration

[0119] After soaking in glycerol, ethanol dehydration or freeze drying is performed.

[0120] In the second embodiment, a biological material is subjected to a second crosslinking, oxidation, first crosslinking and dehydration in sequence. During the second crosslinking, the collagen fibers of the biological material are cross-linked with each other. During the oxidation, an oxidant oxidizes ortho-hydroxyl groups of mucopolysaccharides of the biological material into aldehyde groups, thereby connecting the polysaccharides to the collagen fibers. Then, during the first crosslinking, polysaccharides and polysaccharides are cross-linked with each other. In the final network, cross-linking occurs between the collagen fiber and the collagen fiber, the polysaccharide and the collagen fiber, and the polysaccharide and the polysaccharide.

[0121] After the first crosslinking, post-crosslinking and reduction can be performed, followed by dehydration, without end-capping. In the post-crosslinking, the residual active groups of the first crosslinking agent are cross-linked, and in the reduction, the schiff-base chemical bonds are converted into stable carbon-nitrogen single bonds.

[0122] The flow chart of this embodiment is shown in FIG. 5, where the biological material is sequentially subjected to second crosslinking, oxidation, first crosslinking, post-crosslinking, reduction and dehydration.

Second Crosslinking

[0123] The collagen fibers of the biological material are cross-linked by a second crosslinking agent, and the second crosslinking agent can be glutaraldehyde. In this step, the biological material is exposed to the glutaraldehyde solution by soaking or spraying. The mass percentage concentration of glutaraldehyde in the glutaraldehyde solution is 0.05% to 25%, the crosslinking time is 6 h to 3 weeks, and the crosslinking temperature is 0 to 37° C.

Oxidation

[0124] Take a fresh pericardium, and cut and hang the same on a board, wherein the pericardium is a pig pericardium or a bovine pericardium. Then the pericardium is exposed to an oxidant solution by soaking or spraying, wherein the oxidant can be sodium periodate, and the mass percentage concentration thereof in the oxidant solution is 0.1% to 1%, and the oxidation time is 1 to 12 hours.

[0125] After oxidation, the ortho-hydroxyl groups of the mucopolysaccharides of the pericardium are oxidized into aldehyde groups, and the aldehyde groups react with the amino groups of the collagen fibers, thereby connecting the mucopolysaccharides to the collagen fibers.

First Crosslinking

[0126] After the mucopolysaccharides are connected to the collagen fibers, the mucopolysaccharides are cross-linked through aldehyde groups. The first crosslinking agent should have a group that can react with the aldehyde group, and preferably uses a polyamino substance, for example, being selected from lysine, ethylenediamine or hexamethylenediamine, polyethyleneimine, polylysine, or ε-polylysine. The mucopolysaccharides are cross-linked with each other through the reaction of the amino groups with the aldehyde groups.

[0127] The mass percentage concentration of the polyamino substances in the polyamino substance solution is 0.5% to 10%, the exposure time of the biological material in the polyamino substance solution is 0.5 to 12 h, and the exposure method can be soaking or spraying.

Post-Crosslinking

[0128] In the previous first crosslinking step, the polyamino substance is used as the first crosslinking agent. The residual amino groups are then cross-linked by a third crosslinking agent in this step. The third crosslinking agent is polyethylene glycol diglycidyl ether. The cross-linked network formed by polyethylene glycol improves the high elasticity of the pericardium. On the one hand, the high symmetry of the polyethylene glycol chain improves the high elasticity of the biological tissue, so that the ability of the biological material to deform elastically can be fully restored. On the other hand, water is a benign solvent for the polyethylene glycol, so that after being soaked in water, the biological material exhibits a high elasticity, further improving the crease resistance thereof.

[0129] The mass percentage concentration of the third crosslinking agent in the third crosslinking agent solution is 0.01% to 2%, and the crosslinking time is 10 to 48 h.

Reduction

[0130] The amino groups react with the aldehyde groups to form schiff-base chemical bonds, but the schiff-base chemical bonds are reversible. By means of reduction, the schiff-base chemical bonds can be converted into stable carbon-nitrogen single bonds, avoiding the chemical bond exchange reaction during the crimping process.

[0131] In this step, the biological material is exposed to a reductant solution, and the reductant can be sodium cyanoborohydride or sodium borohydride. The mass percentage concentration of the reductant in the reductant solution is 0.01% to 2%, the exposure time is 1 to 12 hours, and the exposure method can be soaking or spraying.

Dehydration

[0132] After soaking in glycerol, ethanol dehydration or freeze drying is performed.

[0133] In the third embodiment, a biological material is subjected to oxidation, second crosslinking, first crosslinking and dehydration in sequence. During the oxidation, an oxidant oxidizes ortho-hydroxyl groups of mucopolysaccharides of the biological material into aldehyde groups, thereby connecting the polysaccharides to the collagen fibers. During the second crosslinking, the collagen fibers of the biological material are cross-linked with each other. Then, during the first crosslinking, polysaccharides and polysaccharides are cross-linked with each other. In the final network, cross-linking occurs between the collagen fiber and the collagen fiber, the polysaccharide and the collagen fiber, and the polysaccharide and the polysaccharide.

[0134] After the first crosslinking, post-crosslinking and reduction can be performed, followed by dehydration, without end-capping. In the post-crosslinking, the residual active groups of the first crosslinking agent are cross-linked, and in the reduction, the schiff-base chemical bonds are converted into stable carbon-nitrogen single bonds.

[0135] The flow chart of this embodiment is shown in FIG. 6, where the biological material is sequentially subjected to oxidation, second crosslinking, first crosslinking, post-crosslinking, reduction and dehydration.

Oxidation

[0136] Take a fresh pericardium, and cut and hang the same on a board, wherein the pericardium is a pig pericardium or a bovine pericardium. Then the pericardium is exposed to an oxidant solution by soaking or spraying, wherein the oxidant can be sodium periodate, and the mass percentage concentration thereof in the oxidant solution is 0.1% to 1%, and the oxidation time is 1 to 12 hours.

[0137] After oxidation, the ortho-hydroxyl groups of the mucopolysaccharides of the pericardium are oxidized into aldehyde groups, and the aldehyde groups react with the amino groups of the collagen fibers, thereby connecting the mucopolysaccharides to the collagen fibers.

Second Crosslinking

[0138] The collagen fibers of the biological material are cross-linked by a second crosslinking agent, and the second crosslinking agent can be glutaraldehyde. In this step, the biological material is exposed to the glutaraldehyde solution by soaking or spraying. The mass percentage concentration of glutaraldehyde in the glutaraldehyde solution is 0.05% to 25%, the crosslinking time is 6 h to 3 weeks, and the crosslinking temperature is 0 to 37° C.

First Crosslinking

[0139] After the mucopolysaccharides are connected to the collagen fibers, the mucopolysaccharides are cross-linked through aldehyde groups. The first crosslinking agent should have a group that can react with the aldehyde group, and preferably uses a polyamino substance, for example, being selected from lysine, ethylenediamine or hexamethylenediamine, polyethyleneimine, polylysine, or ε-polylysine. The mucopolysaccharides are cross-linked with each other through the reaction of the amino groups with the aldehyde groups.

[0140] The mass percentage concentration of the polyamino substances in the polyamino substance solution is 0.5% to 10%, the exposure time of the biological material in the polyamino substance solution is 0.5 to 12 h, and the exposure method can be soaking or spraying.

Post-Crosslinking

[0141] In the previous first crosslinking step, the polyamino substance is used as the first crosslinking agent. The residual amino groups are then cross-linked by a third crosslinking agent in this step. The third crosslinking agent is polyethylene glycol diglycidyl ether. The cross-linked network formed by polyethylene glycol improves the high elasticity of the pericardium. On the one hand, the high symmetry of the polyethylene glycol chain improves the high elasticity of the biological tissue, so that the ability of the biological material to deform elastically can be fully restored. On the other hand, water is a benign solvent for the polyethylene glycol, so that after being soaked in water, the biological material exhibits a high elasticity, further improving the crease resistance thereof.

[0142] The mass percentage concentration of the third crosslinking agent in the third crosslinking agent solution is 0.01% to 2%, and the crosslinking time is 10 to 48 h.

Reduction

[0143] The amino groups react with the aldehyde groups to form schiff-base chemical bonds, but the schiff-base chemical bonds are reversible. By means of reduction, the schiff-base chemical bonds can be converted into stable carbon-nitrogen single bonds, avoiding the chemical bond exchange reaction during the crimping process.

[0144] In this step, the biological material is exposed to a reductant solution, and the reductant can be sodium cyanoborohydride or sodium borohydride. The mass percentage concentration of the reductant in the reductant solution is 0.01% to 2%, the exposure time is 1 to 12 hours, and the exposure method can be soaking or spraying.

Dehydration

[0145] After soaking in glycerol, ethanol dehydration or freeze drying is performed.

[0146] In the above three embodiments, the solvent for preparing the solution uses a conventional solvent for treating a biological material, which will not negatively affect the biological material, for example, water, PBS or physiological saline.

[0147] Specific examples will be described below:

[0148] In the following examples, the concentrations expressed as percentage represent mass percentage concentrations unless otherwise specified; the solutions mentioned in the following examples are all prepared by PBS unless otherwise specified.

Example 1

[0149] Referring to the flow chart shown in FIG. 7, a porcine pericardium was soaked in 1% sodium periodate solution at room temperature for 2 h, soaked in 2% lysine aqueous solution for 2 h, subsequently, cross-linked in 0.05% glutaraldehyde solution for 5 days, washed with 0.9% physiological saline, soaked in 2% ε-polylysine aqueous solution for 2 h, soaked in 2% polyethylene glycol diglycidyl ether aqueous solution added with 0.1% tetramethylethylenediamine catalyst for 24 h, washed with 0.9% physiological saline three times, soaked in 1% sodium cyanoborohydride aqueous solution for 2 h, soaked in 40% glycerol aqueous solution after being washed 3 times, and then freeze-dried.

[0150] The pericardium prepared in Example 1 was subjected to breaking force test and water absorption and flattening test.

[0151] Breaking force test: after the dry pericardium was hydrated, it was cut into 1 * 5 cm strips, and the maximum breaking force was measured by a universal tensile machine. A conventional glutaraldehyde cross-linked pericardium was used as a control group. The results are shown in FIG. 8. The breaking force of the pericardium prepared in Example 1 is 25 to 30 N, which is significantly greater than the conventional glutaraldehyde cross-linked pericardium.

[0152] Water absorption and flattening test: 3 * 3 cm pericardium sheets were crimped and compressed into a 1 ml syringe (as shown in FIG. 9A), placed in an oven at 40° C. for 3 days, then released into physiological saline solution. Observe the flattening of the sheets. The results are shown in FIG. 9B and FIG. 9C, where FIG. 9B shows a conventional glutaraldehyde cross-linked dry pericardium which has a lot of creases, and FIG. 9C shows the HE dry pericardium (prepared in Example 1) which has no obvious visible crease after being released and soaked in water.

Example 2

[0153] Referring to the flow chart shown in FIG. 5, a porcine pericardium was cross-linked in 0.05% glutaraldehyde solution for 5 days, warshed with 0.9% physiological saline, soaked in 1% sodium periodate solution at room temperature for 2 h, subsequently, soaked in 2% ε-polylysine solution for 2 h, soaked in 2% polyethylene glycol diglycidyl ether added with 0.1% catalyst for 24 h, washed with 0.9% physiological saline three times, soaked in 1% sodium cyanoborohydride solution for 2 h, soaked in 40% isopropanol solution after being washed 3 times, and then blow-dried or freeze-dried.

[0154] The breaking force test was the same as in Example 1. The results are shown in FIG. 10, which shows that the breaking force of the pericardium prepared in Example 2 was significantly greater than that of the conventional glutaraldehyde cross-linked pericardium.

[0155] Water absorption and flattening test: 3 * 3 cm pericardium sheets were crimped and compressed into a 1 ml syringe (as shown in FIG. 11A), placed in an oven at 40° C. for 3 days, then released into physiological saline solution. Observe the flattening of the sheets. The results are shown in FIG. 10B and FIG. 10C, where FIG. 10B shows a conventional glutaraldehyde cross-linked dry pericardium which has a lot of creases, and FIG. 10C shows the HE dry pericardium (prepared in Example 2) which has no obvious visible crease after being released and soaked in water.

Example 3

[0156] A porcine pericardium was soaked in 1% sodium periodate solution for 2 h at room temperature, soaked in 2% lysine solution for 2 h, subsequently, cross-linked in 0.05% glutaraldehyde solution for 5 days, washed with 0.9% physiological saline, soaked in 2% bisamino polyethylene glycol solution for 24 h, washed with 0.9% physiological saline three times, soaked in 1% sodium cyanoborohydride solution for 2 h, soaked in 40% glycerol aqueous solution after being washed 3 times, and then freeze-dried.

Example 4

[0157] A porcine pericardium was soaked in 1% sodium periodate solution at room temperature for 2 h, cross-linked in 0.05% glutaraldehyde solution for 5 days and washed with 0.9% physiological saline, soaked in 2% polyethyleneimine solution for 2 h, subsequently, soaked in 2% bisamino polyethylene glycol aqueous solution for 24 h, washed with 0.9% physiological saline three times, soaked in 1% sodium cyanoborohydride solution for 2 h, soaked in 60% glycerin aqueous solution after being washed 3 times, and then blow-dried.

[0158] The breaking force test was the same as in Example 1. The results are shown in FIG. 12, which shows that the breaking force of the pericardium prepared in Example 4 was significantly greater than that of the conventional glutaraldehyde cross-linked pericardium.

[0159] Water absorption and flattening test: 3 * 3 cm pericardium sheets were crimped and compressed into a 1 ml syringe (as shown in FIG. 13A), placed in an oven at 40° C. for 3 days, then released into physiological saline solution. Observe the flattening of the sheets. The results are shown in FIG. 13B and FIG. 13C, where FIG. 13B shows a conventional glutaraldehyde cross-linked dry pericardium which has a lot of creases, and FIG. 13C shows the HE dry pericardium (prepared in Example 4) which has no obvious visible crease after being released and soaked in water.

[0160] The above-described embodiments only illustrate several embodiments of the present disclosure, and the description thereof is specific and detail, but should not be construed as limiting the scope of the patent disclosure. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present disclosure, all of which fall into the protection scope of the present disclosure. Therefore, the protection scope of the present application should be based on the appended claims.