FUNCTIONALIZED BIOLOGICAL MATRIX MATERIAL, PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

Disclosed are a functionalized biological matrix material, a preparation method therefor and use thereof, which belong to the technical field of medical materials. In the present invention, by means of the hybridization of a biological matrix material with 3-sulfopropyl methacrylate, the cross-linking and functionalization of the biological matrix material are achieved at the same time. A specific method comprises modifying carbon-carbon double-bond structures such as allyl, methallyl in a biological matrix material, immersing the biological matrix material in an aqueous solution containing 3-sulfopropyl methacrylate, and finally performing cross-linking and functionalization on the biological matrix material by means of radical polymerization, and using the biological matrix material to prepare materials such as valves. The present invention achieves multi-site and long-range cross-linking of a biological matrix material by means of a polymer network, and at the same time introduces corresponding functional functional groups so as to achieve functionalization of the biological matrix material.

Claims

1. A method for preparing a functionalized biological matrix material, comprising steps of: cleaning a biological matrix material, immersing the biological matrix material in glycidyl methacrylate or methacrylic anhydride aqueous solution, and then in 3-sulfopropyl methacrylate aqueous solution, and adding an initiator to initiate polymerization.

2. The method for preparing a functionalized biological matrix material according to claim 1, wherein the biological matrix material is an animal tissue obtained from swine, ovine or bovine and subjected to decellularized treatment, and the animal tissue comprises a blood vessel, heart valve and pericardium, and the main constituent thereof is at least one of collagen, elastin and glycosaminoglycan.

3. The method for preparing a functionalized biological matrix material according to claim 1, wherein the methacrylic anhydride aqueous solution has a concentration of 1-20 wt %.

4. The method for preparing a functionalized biological matrix material according to claim 1, wherein the cleaned biological matrix material is immersed the methacrylic anhydride aqueous solution at 4-60° C. for 6-72 h.

5. The method for preparing a functionalized biological matrix material according to claim 1, wherein the 3-sulfopropyl methacrylate aqueous solution has a concentration of 10-500 mmol/L.

6. The method for preparing a functionalized biological matrix material according to claim 1, wherein the biological matrix material is immersed in the 3-sulfopropyl methacrylate aqueous solution at 4-60° C. for 2-72 h.

7. (canceled)

8. The method for preparing a functionalized biological matrix material according to claim 1, wherein the initiator is a photoinitiator or a thermal initiator, and wherein when the thermal initiator is used, the polymerization is performed at 20-45° C. for 18-48 h, and when the photoinitiator is used, the polymerization is performed at room temperature for 5-30 min under an ultraviolet light source.

9. The method for preparing a functionalized biological matrix material according to claim 1, wherein the method further comprises, after initiating the polymerization: crosslinking and fixing the obtained material from the polymerization by using a mixed solution of carbodiimide and N-hydroxysuccinimide; and immersing the obtained material from the crosslinking and fixing in a polyphenol solution.

10. The method for preparing a functionalized biological matrix material according to claim 9, wherein the material immersed in the polyphenol solution is rinsed, and stored with an antimicrobial solvent, or stored after dehydration and drying with an alcohol solution.

11-13. (canceled)

14. The method for preparing a functionalized biological matrix material according to claim 9, wherein the “immersing the obtained material from the crosslinking and fixing in a polyphenol solution” comprises: immersing the obtained material from the crosslinking and fixing in 0.01-10 mM polyphenol compound aqueous solution for 1-24 hours, and wherein the polyphenol compound is at least one of curcumin, procyanidin, quercetin, resveratrol, aloin, aloe emodin, tannic acid, epigallocatechin gallate, pentagalloylglucose and genipin.

15. A functionalized biological matrix material prepared by the method according to claim 1.

16. Use of the functionalized biological matrix material according to claim 15 in preparation of a medical material, wherein the medical material is a thoracotomy biological valve, an interventional biological valve, a tissue engineering heart valve, a biological patch, an artificial blood vessel or a tissue engineering blood vessel.

17. A method for preparing a non-glutaraldehyde preloadable dry biological valve material, comprising steps of: a, obtaining an animal pericardium material; b, immersing the pericardium material in glycidyl methacrylate or acrylic anhydride solution to introduce carbon-carbon double-bond structure; c, immersing the material treated in step b in a 3-sulfopropyl methacrylate aqueous solution; d, adding an initiator to the material treated in step c for double-bond polymerization and crosslinking; e, crosslinking and fixing the material obtained in step d by using a mixed solution of carbodiimide and N-hydroxysuccinimide; f, immersing the material obtained in step e in a polyphenol solution; and g, rinsing the material treated in step f, storing the same with an antimicrobial solvent, or storing the same after dehydration and drying with an alcohol solution.

18. The method for preparing a non-glutaraldehyde preloadable dry biological valve material according to claim 17, wherein step a comprises decellularizing the animal pericardium material.

19-20. (canceled)

21. The method for preparing a non-glutaraldehyde preloadable dry biological valve material according to claim 17, wherein step b comprises: immersing the pericardium in 1-10 wt % glycidyl methacrylate solution for 3-7 days at 25-45° C. or in 1-5 wt % methacrylic anhydride aqueous solution for 12-48 hour.

22. The method for preparing a non-glutaraldehyde preloadable dry biological valve material according to claim 17, wherein step c comprises: immersing the material in 0.01-1M 3-sulfopropyl methacrylate aqueous solution for 12-48 h at 25-45° C.

23. The method for preparing a non-glutaraldehyde preloadable dry biological valve material according to claim 17, wherein step d comprises: thermally initiating double-bond polymerization at 20-45° C. for 12-48 h, and wherein the initiator is at least one of potassium persulfate, ammonium persulfate, sodium bisulfite and tetramethylethylenediamine.

24. The method for preparing a non-glutaraldehyde preloadable dry biological valve material according to claim 17, wherein step e comprises: immersing the material treated by the double-bond polymerization and crosslinking in a pH buffer of 10-60 mM carbodiimide and 1-20 mM N-hydroxysuccinimide at 25-45° C. for 24-48 h.

25. The method for preparing a non-glutaraldehyde preloadable dry biological valve material according to claim 17, wherein step f comprises: immersing the obtained material from the crosslinking and fixing in 0.01-10 mM polyphenol compound aqueous solution for 1-24 hours, and wherein the polyphenol compound is at least one of curcumin, procyanidin, quercetin, resveratrol, aloin, aloe emodin, tannic acid, epigallocatechin gallate, pentagalloylglucose and genipin.

26-28. (canceled)

29. A non-glutaraldehyde preloadable dry biological valve material prepared by the method according to claim 17.

30. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly explain that technical solutions of the examples of the present invention, the drawings accompanying the examples will be briefly described. It can be understood that the following drawings show are only some examples of the present invention, and should not be construed as limiting the scope. Other related drawings can be obtained from these drawings without any creative effort by those skilled in the art.

[0069] FIG. 1 shows the weight loss rate by the degrading elastase;

[0070] FIG. 2 shows the calcium content;

[0071] FIG. 3 shows the relative adhesion rates of endothelial cells; and

[0072] FIG. 4 shows the results of the water immersion and flattening test.

DESCRIPTION OF THE EMBODIMENTS

[0073] In order to make the objects, technical solutions and advantages of the present invention more clear and apparent, the present invention will be described in further detail with reference to the drawings and examples. It should be appreciated that the specific examples described herein are merely illustrative of the invention and are not intended to limit the invention. In other words, the described examples are only part of, but not all of, the examples of the invention. Generally, the components in the examples of the invention described and illustrated in the drawings herein can be arranged and designed in a variety of different configurations.

[0074] Therefore, the following detailed description of the examples of the invention referring to the drawings is not intended to limit the scope of the claimed invention, but is merely representative of selected examples of the invention. Based on the examples of the present invention, all other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present invention.

[0075] It should be noted that terms such as “the first” and “the second” are used only to distinguish one object or step from another object or step, without necessarily representing or implying any such relationship or order between these objects or steps in practice. Moreover, terms “comprising”, “including”, or the like, are intended to cover non-exclusive inclusions such that a process, method, object or apparatus comprising elements includes not only said elements, but also includes other elements that are not explicitly listed or are inherent to such process, method, object or apparatus. Without further limitation, an element defined by the statement “comprising a/an . . . ” does not preclude a further identical element presented in the process, method, object or apparatus comprising said element.

[0076] The features and performances of the present invention are described in further detail below with reference to examples.

EXAMPLE 1

[0077] A method for preparing a functionalized biological matrix material provided by a preferred example of the present invention comprises the following steps:

[0078] 1. Provide a fresh porcine pericardium and store it at 4° C. in wet;

[0079] 2. Immerse the animal tissue material in 0.1% EDTA solution for 1 hour, and then in 0.1% SDS solution for 24 hours to perform decellularization treatment to prepare a decellularized matrix;

[0080] 3. Clean the decellularized matrix by ultrasonic cleaning at room temperature with a cleaning solution prepared by distilled water and alcohol with a volume ratio of 1:1;

[0081] 4. Immerse the washed decellularized matrix in 1 wt % methacrylic anhydride aqueous solution at 25° C. for 24 hours;

[0082] 5. Immerse the above material in 10 mmol/L 3-sulfopropyl methacrylate aqueous solution at 25° C. for 24 hours after ultrasonic cleaning at room temperature with the above cleaning solution; and

[0083] 6. Add 5 mM potassium persulfate therein, reacting for 24 hours at 40° C., and clean the material by ultrasonic cleaning with distilled water, thereby obtaining the functionalized biological matrix material.

EXAMPLE 2

[0084] A method for preparing a functionalized biological matrix material provided by a preferred example of the present invention comprises the following steps:

[0085] 1. Provide a fresh porcine pericardium and store it at 4° C. in wet;

[0086] 2. Immerse the animal tissue material in 0.1% EDTA solution for 1 hour, and then in 0.1% SDS solution for 24 hours to perform decellularization treatment to prepare a decellularized matrix;

[0087] 3. Clean the decellularized matrix by ultrasonic cleaning at room temperature with a cleaning solution prepared by distilled water and alcohol with a volume ratio of 1:1;

[0088] 4. Immerse the washed decellularized matrix in 5 wt % methacrylic anhydride aqueous solution at 20° C. for 24 hours;

[0089] 5. Immerse the above material in 50 mmol/L 3-sulfopropyl methacrylate aqueous solution at 30° C. for 24 hours after ultrasonic cleaning at room temperature with the above cleaning solution; and

[0090] 6. Add 20 mM potassium persulfate therein, reacting for 24 hours at 37° C., and clean the material by ultrasonic cleaning with distilled water, thereby obtaining the functionalized biological matrix material.

EXAMPLE 3

[0091] A method for preparing a functionalized biological matrix material provided by a preferred example of the present invention comprises the following steps:

[0092] 1. Provide a fresh porcine pericardium and store it at 4° C. in wet;

[0093] 2. Immerse the animal tissue material in 0.1% EDTA solution for 1 hour, and then in 0.1% SDS solution for 24 hours to perform decellularization treatment to prepare a decellularized matrix;

[0094] 3. Clean the decellularized matrix by ultrasonic cleaning at room temperature with a cleaning solution prepared by distilled water and alcohol with a volume ratio of 1:1;

[0095] 4. Immerse the washed decellularized matrix in 3 wt % methacrylic anhydride aqueous solution at 30° C. for 24 hours;

[0096] 5. Immerse the above material in 50 mmol/L 3-sulfopropyl methacrylate aqueous solution at 37° C. for 24 hours after ultrasonic cleaning at room temperature with the above cleaning solution; and

[0097] 6. Add 20 mM potassium persulfate therein, reacting for 48 hours at 20° C., and clean the material by ultrasonic cleaning with distilled water, thereby obtaining the functionalized biological matrix material.

EXAMPLE 4

[0098] A method for preparing a functionalized biological matrix material provided by a preferred example of the present invention comprises the following steps:

[0099] 1. Provide a fresh porcine pericardium and store it at 4° C. in wet;

[0100] 2. Immerse the animal tissue material in 0.1% EDTA solution for 1 hour, and then in 0.1% SDS solution for 24 hours to perform decellularization treatment to prepare a decellularized matrix;

[0101] 3. Clean the decellularized matrix by ultrasonic cleaning at room temperature with a cleaning solution prepared by distilled water and alcohol with a volume ratio of 1:1;

[0102] 4. Immerse the washed decellularized matrix in 3 wt % methacrylic anhydride aqueous solution at 40° C. for 18 hours;

[0103] 5. Immerse the above material in 200 mmol/L 3-sulfopropyl methacrylate aqueous solution at 40° C. for 18 hours after ultrasonic cleaning at room temperature with the above cleaning solution; and

[0104] 6. Add Irgacure 2959 photoinitiator therein, irradiating for 10 min in an UV crosslink box, and clean the material by ultrasonic cleaning with distilled water, thereby obtaining the functionalized biological matrix material.

EXAMPLE 5

[0105] A method for preparing a functionalized biological matrix material provided by a preferred example of the present invention comprises the following steps:

[0106] 1. Provide a fresh porcine pericardium and store it at 4° C. in wet;

[0107] 2. Immerse the animal tissue material in 0.1% EDTA solution for 1 hour, and then in 0.1% SDS solution for 24 hours to perform decellularization treatment to prepare a decellularized matrix;

[0108] 3. Clean the decellularized matrix by ultrasonic cleaning at room temperature with a cleaning solution prepared by distilled water and alcohol with a volume ratio of 1:1;

[0109] 4. Immerse the washed decellularized matrix in 1 wt % methacrylic anhydride aqueous solution at 40° C. for 18 hours;

[0110] 5. Immerse the above material in 500 mmol/L 3-sulfopropyl methacrylate aqueous solution at 35° C. for 18 hours after ultrasonic cleaning at room temperature with the above cleaning solution; and

[0111] 6. Add Irgacure 2959 photoinitiator therein, irradiating for 10 min in an UV crosslink box, and clean the material by ultrasonic cleaning with distilled water, thereby obtaining the functionalized biological matrix material.

EXAMPLE 6

[0112] A method for preparing a functionalized biological matrix material provided by a preferred example of the present invention comprises the following steps:

[0113] 1. Provide a fresh porcine pericardium and store it at 4° C. in wet;

[0114] 2. Clean the extracellular matrix by ultrasonic cleaning at room temperature with a cleaning solution prepared by distilled water and alcohol with a volume ratio of 1:1;

[0115] 3. Immerse the washed extracellular matrix in 2 wt % methacrylic anhydride aqueous solution at 30° C. for 24 hours;

[0116] 4. Immerse the above material in 500 mmol/L 3-sulfopropyl methacrylate aqueous solution at 25° C. for 24 hours after ultrasonic cleaning at room temperature with the above cleaning solution;

[0117] 6. Add 5 mM ammonium persulfate therein, reacting at 30° C. for 24 hours; and

[0118] 7. Clean the material by ultrasonic cleaning with distilled water.

Experimental Example 1

[0119] Glutaraldehyde Group Samples:

[0120] The samples are prepared by treating decellularized biological matrix materials by traditional glutaraldehyde crosslinking, as glutaraldehyde valves for short: the porcine pericardium is immersed in 0.1 wt % ethylenediaminetetraacetic acid disodium solution for 1 h, then in 0.1 wt % sodium dodecyl sulfate solution for 24 h, rinsed in sterile PBS solution for 1 h, and then successively immersed in 0.1 vt %, 0.5 vt %, 1 vt % glutaraldehyde PBS solution for crosslinking for 24 h.

[0121] Experimental Group Samples:

[0122] The materials prepared in Examples 1-3, respectively, as hybrid valves for short.

[0123] Blank Control Group Samples:

[0124] The decellularized biological matrix materials, as decellularized valves for short.

[0125] 1. The weight losses of the glutaraldehyde group samples, the samples prepared in Example 1 and the blank control group samples after being degraded by elastase were measured, and the results are shown in FIG. 1.

[0126] As can be seen from FIG. 1, the method in Example 1 can effectively protect elastin in the decellularized matrix, potentially improving the mechanical properties and prolonging the service life thereof. This is because the density of crosslinks is improved by an interpenetrating network of the biomaterial prepared according to the method of the present invention with the polymer, thereby protecting the elastin.

[0127] 2. The calcium contents of the glutaraldehyde group samples and the samples prepared in Example 2 after subcutaneous implantation in rats for 30 days were measured, respectively, and the results are shown in FIG. 2.

[0128] As can be seen from FIG. 2, the calcium contents of the materials prepared in Example 2 are far less than those of the glutaraldehyde group samples. Therefore, the method of the present invention can effectively reduce the calcification reaction. This is because glutaraldehyde is not introduce into the decellularized biological matrix material prepared according to the method of the present invention, and at the same time, most residues in the decellularized matrix are buried, so that the ability of the decellularized matrix material to bind calcium ion is reduced, thereby reducing the calcification reaction.

[0129] 3. The relative adhesion rates of endothelial cells to the materials of the glutaraldehyde group samples, the samples prepared in Example 3 and the blank control group samples co-cultured with the endothelial cells for 1 day were measured, and the results are shown in FIG. 3.

[0130] As can be seen from FIG. 3, the method of the present invention can effectively improve the adhesion of endothelial cells on the decellularized biological matrix material. This is because the decellularized biological matrix material prepared according to the method of the present invention involves a biocompatible crosslinking agent, instead of a biotoxic crosslinking agent such as glutaraldehyde, and at the same time, functional monomers that promote the growth and migration of the endothelial cells are introduced to further improve the biocompatibility.

EXAMPLE 7

[0131] A method for preparing a non-glutaraldehyde preloadable dry biological valve material provided by a preferred example of the present invention comprises the following steps:

[0132] a. Immerse a porcine pericardium in 0.1 wt % ethylenediaminetetraacetic acid disodium solution for 1 h, then in 0.1 wt % sodium dodecyl sulfate solution for 24 h, and then rinsed in sterile PBS solution for 1 h.

[0133] b. Immerse the decellularized pericardium material in 5 wt % glycidyl methacrylate solution and incubate at 37° C. for 3 days to introduce carbon-carbon double-bond structure.

[0134] c. Immerse the material obtained in the above step in 0.1 M 3-sulfopropyl methacrylate aqueous solution at 37° C. for 24 h.

[0135] d. Add the material obtained in the above step into an aqueous solution of 20 mM ammonium persulfate and 20 mM sodium bisulfite for crosslinking at 37° C. for 24 hours.

[0136] e. Immerse the material obtained in the above step in a mixed solution of 60 mM carbodiimide and 12 mM N-hydroxysuccinimide for crosslinking and fixation at 37° C. for 24 h.

[0137] f. Immerse the material obtained in the above step in 0.1 mM curcumin aqueous solution at room temperature for 24 hours, and rinse the material.

[0138] g. Immerse the rinsed material in a mixed solution of 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and store the material at room temperature.

EXAMPLE 8

[0139] A method for preparing a non-glutaraldehyde preloadable dry biological valve material provided by a preferred example of the present invention comprises the following steps:

[0140] a. Immerse a porcine pericardium in 0.1 wt % ethylenediaminetetraacetic acid disodium solution for 1 h, then in 0.1 wt % sodium dodecyl sulfate solution for 24 h, and then rinsed in sterile PBS solution for 1 h.

[0141] b. Immerse the decellularized pericardium material in 2 wt % glycidyl methacrylate solution and incubate at 37° C. for 3 days to introduce carbon-carbon double-bond structure.

[0142] c. Immerse the material obtained in the above step in 0.1 M 3-sulfopropyl methacrylate aqueous solution at 37° C. for 24 h.

[0143] d. Add the material obtained in the above step into an aqueous solution of 20 mM ammonium persulfate and 20 mM sodium bisulfite for crosslinking at 37° C. for 24 hours.

[0144] e. Immerse the material obtained in the above step in a mixed solution of 60 mM carbodiimide and 12 mM N-hydroxysuccinimide for crosslinking and fixation at 37° C. for 24 h.

[0145] f. Immerse the material obtained in the above step in 0.1 mM curcumin aqueous solution at room temperature for 24 hours, and rinse the material.

[0146] g. Immerse the rinsed material in a mixed solution of 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and store the material at room temperature.

EXAMPLE 9

[0147] A method for preparing a non-glutaraldehyde preloadable dry biological valve material provided by a preferred example of the present invention comprises the following steps:

[0148] a. Immerse a porcine pericardium in 0.2 wt % ethylenediaminetetraacetic acid disodium solution for 1 h, then in 0.2 wt % sodium dodecyl sulfate solution for 20 h, and then rinsed in sterile PBS solution for 3 h.

[0149] b. Immerse the decellularized pericardium material in 8 wt % glycidyl methacrylate solution and incubate at 37° C. for 4 days to introduce carbon-carbon double-bond structure.

[0150] c. Immerse the material obtained in the above step in 0.2 M 3-sulfopropyl methacrylate aqueous solution at 37° C. for 24 h.

[0151] d. Add the material obtained in the above step into an aqueous solution of 20 mM ammonium persulfate and 20 mM sodium bisulfite for crosslinking at 37° C. for 24 hours.

[0152] e. Immerse the material obtained in the above step in a mixed solution of 50 mM carbodiimide and 15 mM N-hydroxysuccinimide for crosslinking and fixation at 37° C. for 24 h.

[0153] f. Immerse the material obtained in the above step in 1 mM procyanidin aqueous solution at room temperature for 24 hours, and rinse the material.

[0154] g. Immerse the rinsed material in a mixed solution of 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and store the material at room temperature.

EXAMPLE 10

[0155] A method for preparing a non-glutaraldehyde preloadable dry biological valve material provided by a preferred example of the present invention comprises the following steps:

[0156] a. Immerse a porcine pericardium in 0.3 wt % ethylenediaminetetraacetic acid disodium solution for 2 h, then in 0.3 wt % sodium dodecyl sulfate solution for 24 h, and then rinsed in sterile PBS solution for 5 h.

[0157] b. Immerse the decellularized pericardium material in 3 wt % methacrylic anhydride solution and incubate at 37° C. for 40 h to introduce carbon-carbon double-bond structure.

[0158] c. Immerse the material obtained in the above step in 0.5 M 3-sulfopropyl methacrylate aqueous solution at 37° C. for 20 h.

[0159] d. Add the material obtained in the above step into an aqueous solution of 20 mM ammonium persulfate and 20 mM sodium bisulfite for crosslinking at 37° C. for 24 hours.

[0160] e. Immerse the material obtained in the above step in a mixed solution of 60 mM carbodiimide and 12 mM N-hydroxysuccinimide for crosslinking and fixation at 37° C. for 30 h.

[0161] f. Immerse the material obtained in the above step in 0.5 mM resveratrol aqueous solution at room temperature for 20 hours, and rinse the material.

[0162] g. Immerse the rinsed material in a mixed solution of 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and store the material at room temperature.

EXAMPLE 11

[0163] A method for preparing a non-glutaraldehyde preloadable dry biological valve material provided by a preferred example of the present invention comprises the following steps:

[0164] a. Immerse the pericardium material in 10 wt % glycidyl methacrylate solution and incubate at 40° C. for 3 days to introduce carbon-carbon double-bond structure.

[0165] b. Immerse the material obtained in the above step in 0.5 M 3-sulfopropyl methacrylate aqueous solution at 37° C. for 24 h.

[0166] c. Add the material obtained in the above step into an aqueous solution of 20 mM ammonium persulfate and 20 mM sodium bisulfite for crosslinking at 37° C. for 24 hours.

[0167] d. Immerse the material obtained in the above step in a mixed solution of 60 mM carbodiimide and 12 mM N-hydroxysuccinimide for crosslinking and fixation at 37° C. for 24 h.

[0168] e. Immerse the material obtained in the above step in 1 mM curcumin aqueous solution at room temperature for 24 hours, and rinse the material.

[0169] f. Immerse the rinsed material in a mixed solution of 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and store the material at room temperature.

Comparative 1

[0170] A biological valve material is prepared through the following steps:

[0171] a. Immerse a porcine pericardium in 0.1 wt % ethylenediaminetetraacetic acid disodium solution for 1 h, then in 0.1 wt % sodium dodecyl sulfate solution for 24 h, and then rinsed in sterile PBS solution for 1 h.

[0172] b. Immerse the material obtained in the above step in 5 wt % glycidyl methacrylate solution and incubate at 37° C. for 3 days.

[0173] c. Immerse the material obtained in the above step in 0.1 M 3-sulfopropyl methacrylate aqueous solution at 37° C. for 24 h.

[0174] d. Add the material obtained in the above step into an aqueous solution of 20 mM ammonium persulfate and 20 mM sodium bisulfite for crosslinking at 37° C. for 24 hours.

[0175] e. Immerse the material obtained in the above step in a mixed solution of 60 mM carbodiimide and 12 mM N-hydroxysuccinimide for crosslinking and fixation at 37° C. for 24 h.

[0176] f. Rinse the material obtained in the above step and immerse the rinsed material in a mixed solution of 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and store the material at room temperature.

Comparative 2

[0177] A biological valve material is prepared through the following steps:

[0178] a. Immerse a porcine pericardium in 0.1 wt % ethylenediaminetetraacetic acid disodium solution for 1 h, then in 0.1 wt % sodium dodecyl sulfate solution for 24 h, and then rinsed in sterile PBS solution for 1 h.

[0179] b. Immerse the material obtained in the above step in 2 wt % methacrylic anhydride solution and incubate at 37° C. for 24 hours.

[0180] c. Immerse the material obtained in the above step in 0.1 M 3-sulfopropyl methacrylate aqueous solution at 37° C. for 24 h.

[0181] d. Add the material obtained in the above step into an aqueous solution of 20 mM ammonium persulfate and 20 mM sodium bisulfite for crosslinking at 37° C. for 24 hours.

[0182] e. Immerse the material obtained in the above step in a mixed solution of 60 mM carbodiimide and 12 mM N-hydroxysuccinimide for crosslinking and fixation at 37° C. for 24 h.

[0183] f. Rinse the material obtained in the above step and immerse the rinsed material in a mixed solution of 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and store the material at room temperature.

Comparative 3

[0184] A biological valve material is prepared through the following steps:

[0185] a. Immerse a porcine pericardium in 0.1 wt % ethylenediaminetetraacetic acid disodium solution for 1 h, then in 0.1 wt % sodium dodecyl sulfate solution for 24 h, and then rinsed in sterile PBS solution for 1 h.

[0186] b. Immerse the material obtained in the above step in a mixed solution of 60 mM carbodiimide and 12 mM N-hydroxysuccinimide for crosslinking and fixation at 37° C. for 24 h.

[0187] c. Rinse the material obtained in the above step and immerse the rinsed material in a mixed solution of 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and store the material at room temperature.

Comparative 4

[0188] A biological valve material is prepared through the following steps:

[0189] a. Immerse a porcine pericardium in 0.1 wt % ethylenediaminetetraacetic acid disodium solution for 1 h, then in 0.1 wt % sodium dodecyl sulfate solution for 24 h, and then rinsed in sterile PBS solution for 1 h.

[0190] b. Immerse the material obtained in the above step in a mixed solution of 60 mM carbodiimide and 12 mM N-hydroxysuccinimide for crosslinking and fixation at 37° C. for 24 h.

[0191] c. Immerse the material obtained in the above step in 0.1 mM curcumin aqueous solution at room temperature for 24 hours.

[0192] d. Rinse the material obtained in the above step and immerse the rinsed material in a mixed solution of 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and store the material at room temperature.

Experimental Example 2

[0193] A control group was provided and the samples thereof as well as the materials prepared in Examples 7 and 8 and Comparatives 1 to 4 were subjected to anti-calcification test, elastin stability test, and water immersion and flattening test, respectively.

[0194] In the control group: the porcine pericardium is immersed in 0.1 wt % ethylenediaminetetraacetic acid disodium solution for 1 h, then in 0.1 wt % sodium dodecyl sulfate solution for 24 h, rinsed in sterile PBS solution for 1 h, and then successively immersed in 0.1 vt %, 0.5 vt %, 1 vt % glutaraldehyde PBS solution for crosslinking for 24 h, then in 20 vt % glycerol, 40 vt % ethanol and 40 vt % isopropanol for 4 h, followed by air dry, and stored at room temperature.

[0195] (1) Anti-Calcification Test

[0196] The samples from the example group, the comparative group and the control group were cut to the size of 1 cm*1 cm and cleaned. 0.1 mL of 3% pentobarbital sodium was intraperitoneally injected into a young SD rat of about 20 days for anesthesia. Shave the fur off the muscles on both sides of the spine and disinfect with iodine and alcohol. One sample from the experimental group was implanted subcutaneously into the right back, and one sample from the control group was implanted subcutaneously into the left back, with the skin incisions sutured. After 60 days, the animals were euthanized by cervical dislocation and the implants were removed therefrom. Carefully removed the host tissue from the surface of the implants and rinse the implants with saline. Weigh the dried implant after freeze drying, and then digest with 6N concentrated hydrochloric acid in a water bath at 95° C. until no visible solid particles were present, followed by quantitative analysis of calcium using an inductively coupled plasma-optical emission spectrometer.

[0197] The materials obtained in the Example groups and the Control groups were subjected to anti-calcification test, and the results are shown in Table 1 below. As can be seen from Table 1, the calcium contents of the materials prepared in Example 7 and Example 8 are greatly reduced compared to the control group, and the calcium contents of the materials prepared in Example 7 and Example 8 are less than those of Comparatives 1 to 4. Therefore, the material of the present invention has excellent anti-calcification property.

TABLE-US-00001 TABLE 1 Calcium Content Calcium content (μg/mg) Control group 19.41 ± 9.88  Example 7 0.40 ± 0.05 Example 8 0.44 ± 0.04 Comparative 1 0.57 ± 0.31 Comparative 2 0.56 ± 0.40 Comparative 3 2.99 ± 2.32 Comparative 4 2.24 ± 1.40

[0198] (2) Elastin Stability Test

[0199] After the biological valve was implanted into the body, it will contact the protease in the blood. Under the action of the protease, the collagen and elastin in the valve will be degraded, thus affecting the stability of the biological valve. Enzymatic degradation experiment in vitro is an effective method to test the resistance of the biological valve to protease degradation. The component stability of the biological valve can be tested by simulating the protease environment in vivo. The biological valve, after freeze drying, was incubated in a Tris buffer (0.1 M Tris, 1 mM CaCl.sub.2, pH=7.8) containing elastase (30 U/mL) at 37° C. for 24 h, and then rinsed, and weighed after freeze drying. The dry weight loss rate was calculated.

[0200] The elastin stability results are shown in Table 2 below. As can be seen from Table 2 below, the weight loss rates by the degrading elastase of Examples 7 and 8 are greatly reduced relative to the control group, while the weight loss rates by the degrading elastase of Comparatives 2, 3 and 4 were only slightly reduced relative to the control group. In conclusion, the elastin stability of the material prepared by the invention is greatly improved.

TABLE-US-00002 TABLE 2 Elastin Stability Weight loss rate by the degrading elastase (%) Control group 12.48 ± 0.44 Example 7  6.36 ± 0.04 Example 8  8.45 ± 0.49 Comparative 1  6.48 ± 0.32 Comparative 2 10.16 ± 0.33 Comparative 3 11.16 ± 0.32 Comparative 4 11.44 ± 0.36

[0201] (3) Water Immersion and Flattening Test

[0202] The materials prepared in Examples 7 and 8, Comparatives 1-4 and the control group were subjected to a folding and immersion test as follows: A plastic tube with an inner diameter of 5 mm was used for simulating a folding test; for each group, the materials were cut into a square sample with an area of about 3 cm*3 cm with scissors; the square samples were slowly inserted into the plastic tube with an inner diameter of 5 mm by tweezers, and then placed in a constant temperature and humidity box at 40° C. and 60% -80% for 72 hours; the materials were then removed from the plastic tube and immersed in PBS buffer, and the immersion and flattening results were observed and photographed.

[0203] As shown in FIG. 4, both the examples and the control group have a good water immersion and flattening performance, while the control group (glutaraldehyde) could not become flattened quickly and the creases were clear.

[0204] The above examples are merely preferred examples of the present invention and are not intended to limit the present invention, and any modifications, equivalent alternatives, developments, etc. made within the spirit and principle of the present invention, should fall within in the scope of protection of the present invention.