ANTI-THROMBOTIC AND ENDOTHELIALIZATION-ENHANCED BIOPROSTHETIC VALVE MATERIAL AND PREPARATION METHOD THEREOF

Abstract

An anti-thrombotic and endothelialization-enhanced bioprosthetic valve material and preparation thereof are provided. The preparation method includes the following steps: immersing a glutaraldehyde cross-linked bioprosthetic valve material in functional reagent solution for 1-24 h, adjusting pH value of the solution to 3-8, removing it from solution and cleaning with deionized water. The new valve material of the present invention can effectively reduce the toxicity and side effects of the existing glutaraldehyde cross-linked bioprosthetic valve material and effectively reduce thrombosis, along with enhanced endothelialization. The material prepared can be used for pulmonary valve, aortic valve, mitral valve, tricuspid valve, venous valve and other cardiovascular implantable medical devices.

Claims

1. A preparation method of an anti-thrombotic and endothelialization-enhanced bioprosthetic valve material, comprising the following steps: immersing a glutaraldehyde cross-linked bioprosthetic valve material in a recombinant human type III collagen solution for 1-24 h to obtain a first soaked material, taking the first soaked material out and cleaning the first soaked material with deionized water to obtain a deionized water-cleaned material.

2. The preparation method of the anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 1, wherein the glutaraldehyde cross-linked bioprosthetic valve material is prepared by immersing an animal-derived pericardial biomaterial in an aqueous solution or phosphate buffer saline (PBS) with a volume concentration of 0.1-10% glutaraldehyde for 4-38 h.

3. The preparation method of the anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 2, wherein the animal-derived pericardial biomaterial consists of bovine pericardium, porcine pericardium or sheep pericardium.

4. The preparation method of the anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 1, wherein a concentration of the recombinant human type III collagen solution is 0.1-30 mg/mL.

5. The preparation method of the anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 1, wherein a primary structure of recombinant human type III collagen in the recombinant human type III collagen solution is hydroxyproline-free and has cellular adhesion properties.

6. The preparation method of the anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 5, wherein an amino acid sequence of the recombinant human type III collagen comprises a collagen tripeptide fragment (GER), but does not comprise collagen-mimetic peptides (GFOGER).

7. The preparation method of the anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 6, wherein a core sequence of the amino acid sequence of the recombinant human type III collagen includes but is not limited to GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

8. The preparation method of the anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 1, further comprising immersing the deionized water-cleaned material in a reducing agent solution for 0.1-24 h to obtain a second soaked material, then taking the second soaked material out and cleaning the second soaked material with deionized water.

9. The preparation method of the anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 8, wherein a reducing agent in the reducing agent solution is sodium borohydride, potassium borohydride or sodium cyanoborohydride.

10. An anti-thrombotic and endothelium-enhanced bioprosthetic valve material, wherein the anti-thrombotic and endothelium-enhanced bioprosthetic valve material is obtained by the preparation method according to claim 1.

11. The anti-thrombotic and endothelium-enhanced bioprosthetic valve material according to claim 10, wherein the glutaraldehyde cross-linked bioprosthetic valve material is prepared by immersing an animal-derived pericardial biomaterial in an aqueous solution or PBS with a volume concentration of 0.1-10% glutaraldehyde for 4-38 h.

12. The anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 11, wherein the animal-derived pericardial biomaterial is bovine pericardium, porcine pericardium or sheep pericardium.

13. The anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 10, wherein a concentration of the recombinant human type III collagen solution is 0.1-30 mg/mL.

14. The anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 10, wherein a primary structure of recombinant human type III collagen in the recombinant human type III collagen solution is hydroxyproline-free and has cellular adhesion properties.

15. The anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 14, wherein an amino acid sequence of the recombinant human type III collagen comprises a collagen tripeptide fragment (GER), but does not comprise collagen-mimetic peptides (GFOGER).

16. The anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 15, wherein a core sequence of the amino acid sequence of the recombinant human type III collagen includes but is not limited to GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

17. The anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 10, wherein further comprising immersing the deionized water-cleaned material in a reducing agent solution for 0.1-24 h to obtain a second soaked material, then taking the second soaked material out and cleaning the second soaked material with deionized water.

18. The anti-thrombotic and endothelialization-enhanced bioprosthetic valve material according to claim 17, wherein a reducing agent in the reducing agent solution is sodium borohydride, potassium borohydride or sodium cyanoborohydride.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1A is a scanning electron microscope (SEM) image showing a platelet adhesion experiment of animal-derived collagen;

[0024] FIG. 1B is a SEM image showing a platelet adhesion experiment of recombinant human type III collagen and animal-derived collagen;

[0025] FIG. 2A is a SEM image showing a platelet adhesion experiment of a control group of a glutaraldehyde cross-linked bioprosthetic valve;

[0026] FIG. 2B is an enlarged image of FIG. 2A;

[0027] FIG. 3A is a SEM image showing a platelet adhesion experiment of the experimental group in example 1; and

[0028] FIG. 3B is an enlarged image of FIG. 3A.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1

[0029] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0030] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 0.6% for 24 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0031] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 30 mg/mL recombinant human type III collagen solution for 24 h, then removed from the solution and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

Example 2

[0032] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0033] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 0.6% for 12 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0034] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 15 mg/mL recombinant human type III collagen solution for 24 h, then removed from the solution and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

Example 3

[0035] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0036] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 0.6% for 12 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0037] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 30 mg/mL recombinant human type III collagen solution for 1 h, then removed from the solution and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0038] (3) The material obtained in step (2) is soaked in a 50 mmol/L sodium cyanoborohydride (NaCNBH.sub.3)-containing PBS for 12 h, and cleaned with distilled water.

Example 4

[0039] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0040] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 0.6% for 24 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0041] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 1 mg/mL recombinant human type III collagen solution for 24 h, then removed from the solution and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0042] (3) The material obtained in step (2) is soaked in a 50 mmol/L sodium borohydride (NaBH.sub.4)-containing PBS for 24 h, and cleaned with distilled water.

Example 5

[0043] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0044] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 0.6% for 24 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0045] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 5 mg/mL recombinant human type III collagen solution for 5 h, then removed from the solution and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0046] (3) The material obtained in step (2) is soaked in a 100 mmol/L potassium borohydride (KBH.sub.4)-containing PBS for 1 h, and cleaned with distilled water.

Example 6

[0047] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0048] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 2% for 12 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0049] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 2 mg/mL recombinant human type III collagen solution for 12 h, then removed from the solution and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0050] (3) The material obtained in step (2) is soaked in a 20 mmol/L NaCNBH.sub.3-containing PBS for 5 h, and cleaned with distilled water.

Example 7

[0051] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0052] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 2% for 12 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0053] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 10 mg/mL recombinant human type III collagen solution for 5 h, then removed from the solution and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0054] (3) The material obtained in step (2) is soaked in a 50 mmol/L KBH.sub.4-containing PBS for 1 h, and cleaned with distilled water.

Example 8

[0055] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0056] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 0.5% for 24 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0057] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 20 mg/mL recombinant human type III collagen solution for 10 h, then removed from the solution and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0058] (3) The material obtained in step (2) is soaked in a 20 mmol/L KBH.sub.4-containing PBS for 24 h, and cleaned with distilled water.

Example 9

[0059] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0060] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 1% for 24 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0061] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 25 mg/mL recombinant human type III collagen solution for 15 h, then taken out and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0062] (3) The material obtained in step (2) is soaked in a 60 mmol/L NaBH.sub.4-containing PBS for 12 h, and cleaned with distilled water.

Example 10

[0063] A preparation method of an anti-thrombotic and endothelium-enhanced bioprosthetic valve material includes the following steps.

[0064] (1) Fresh porcine pericardium is collected and cleaned, and then soaked in a glutaraldehyde aqueous solution with a volume concentration of 2% for 24 h, to obtain a glutaraldehyde cross-linked animal-derived pericardial biomaterial.

[0065] (2) The glutaraldehyde cross-linked animal-derived pericardial biomaterial is cleaned and soaked in a 10 mg/mL recombinant human type III collagen solution for 24 h, then removed from the solution and cleaned with distilled water, where, the amino acid sequence of the recombinant human type III collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0066] (3) The material obtained in step (2) is soaked in a 80 mmol/L NaBH.sub.4-containing PBS for 16 h, and cleaned with distilled water.

Comparative Example

[0067] Preparation of a glutaraldehyde cross-linked bioprosthetic valve: fresh porcine pericardium is collected and cleaned, soaked in a glutaraldehyde aqueous solution with a volume concentration of 0.6% for 24 h, then removed from the solution and cleaned with distilled water.

Experimental Example: Platelet Adhesion Experiment

[0068] The material prepared in example 1 is cut into an appropriate size and incubated with platelet-rich plasma for 1 h. The adhesion of platelets on the material is observed by scanning electron microscope. By scanning electron microscope observation, the SEM image of the platelet adhesion experiment on animal-derived collagen shows that there is a lot of platelet adhesion (FIG. 1A), while the SEM image of the platelet adhesion experiment on recombinant human collagen shows that there is almost no platelet adhesion (FIG. 1B). In FIGS. 1A-1B, the small image in the upper right corner of each image is an enlarged view showing a part of the each image.

[0069] Compared with the control group treated with 0.6% glutaraldehyde (FIGS. 2A-2B), the number of platelets adhered on the surface of the recombinant human collagen-modified bioprosthetic valve (FIGS. 3A-3B) is significantly reduced, indicating that the recombinant human collagen-modified bioprosthetic valve has better anticoagulant performance than the bioprosthetic valve treated with glutaraldehyde alone, which can potentially solve coagulation-related problems such as short service life of bioprosthetic valves due to blood coagulation.