Method for enzymatic crosslinking treatment of heart valve, artificial biological heart valve and application

Abstract

The invention discloses a method for enzymatic crosslinking treatment of a heart valve, an artificial biological heart valve and application. According to the heart valve crosslinking method, a decellularized valve is crosslinked based on sulfydryl oxidase through surface sulfhydrylation modification, the requirements for low cost, easy implementation and cytotoxicity avoiding are met, and a decellularized valve stent material is good in biomechanical property; meanwhile, the surface of the valve is subjected to amidation modification of epsilon-polylysine and alpha-polylysine, and the broad-spectrum antibacterial performance and the cell adhesion, proliferation and differentiation promoting capacity of the valve are utilized, so that endothelialization of the valve is accelerated, and the service life of the valve is prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial heart valves, in particular to a method for enzymatic cross-linking treatment of heart valves, an artificial biological heart valve and applications thereof. Background Art

[0002] Heart valve disease is a cardiovascular disease with high morbidity and mortality. Currently, the most effective treatment for heart valve disease is valve replacement surgery. It is estimated that 850,000 valve replacements will be required worldwide each year by 2050. Bioprosthetic valves are one of the most commonly used valve substitutes in clinical practice. They have good blood compatibility and do not require long-term anticoagulation therapy, but their service life is limited. Patients often need reoperation due to bioprosthetic valve degradation and calcification.

[0003] At present, the main processing technology for biological valves is glutaraldehyde cross-linking. Although this cross-linking method effectively improves the stability of biological valves, it shows obvious cytotoxicity, which is difficult to meet the needs of cell adhesion and proliferation, and is not conducive to the endothelialization of stent materials. However, valve endothelialization is generally considered to be a prerequisite for achieving long-term anticoagulation, anti-calcification, and maintaining long-term functional stability of the valve.

[0004] Lysine is one of the essential amino acids for the human body. It can be divided into two types, ε-lysine and α-lysine, according to its molecular configuration. ε-lysine has broad-spectrum antibacterial properties, and α-lysine can effectively promote cell adhesion, proliferation and differentiation. It has great application prospects in the field of promoting valve re-endothelialization.

[0005] Therefore, developing and exploring new valve cross-linking strategies to avoid the toxic side effects of glutaraldehyde while improving the structural stability, blood compatibility and long-term functional stability of heart valves is of great significance to scientific research and the development of related industrial fields. Summary of the invention

[0006] The purpose of the present invention is to propose a method for enzymatic cross-linking treatment of heart valves, avoid the toxic side effects of glutaraldehyde used in the prior art, and improve the structural stability, blood compatibility and long-term functional stability of the heart valves.

[0007] In view of this, the scheme of the present invention is:

[0008] The first aspect of the present invention is to provide a method for enzymatic cross-linking of a heart valve, the steps comprising:

[0009] Decellularization of heart valves;

[0010] The treated heart valve surface is subjected to amidation modification and thiol modification; the amidation modification uses ε-polylysine and α-polylysine;

[0011] The modified heart valve is immersed in a 0.01-10U / ml sulfhydryl oxidase solution to perform sulfhydryl oxidation cross-linking treatment to obtain a cross-linked heart valve.

[0012] Furthermore, the reagent used for the thiolation modification is at least one selected from cysteine, sodium thioglycolate, thiododecyl glycol carboxylic acid, and HS-PEG-SH.

[0013] Preferably, the reagent used for the thiol modification is cysteine, and the concentration is 10-100 mM; and / or, the concentrations of ε-lysine and α-lysine are both 10-100 mM.

[0014] Furthermore, the heart valve surface modification process uses a first catalyst and a second catalyst; the first catalyst is carbodiimide, preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the second catalyst is selected from at least one of N-hydroxysuccinimide, 1-hydroxybenzotriazole, and 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate; preferably N-hydroxysuccinimide.

[0015] Furthermore, the surface modification process conditions are: shaking reaction at 30-38°C for 6-72h; and / or, the thiol oxidation cross-linking treatment process conditions are: shaking reaction at 30-38°C for 6-72h.

[0016] Furthermore, the heart valve is a porcine heart valve.

[0017] Furthermore, the decellularization process is to use a first buffer containing 1-5% CHAPS and 1-5mM TnBP to shake at room temperature for 20-30 hours, during which rinsing is performed; then use a second buffer containing 1-5% CHAPS, 1-5mM TnBP, 0.5-2% ASB-14, and 1-5% thiobetaine 10 to shake at room temperature for 20-30 hours.

[0018] Preferably, the pH value of the first buffer and / or the second buffer is 7.8.

[0019] The second aspect of the present invention is to provide an artificial biological heart valve obtained by the method of enzymatic cross-linking treatment of a heart valve as described in the first aspect.

[0020] The third aspect of the present invention is to propose the use of the artificial biological heart valve described in the second aspect in the preparation of biological scaffold materials.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The heart valve cross-linking method of the present invention adopts surface thiol modification based on thiol oxidase cross-linking of decellularized valves, which meets the requirements of being cheap, easy to operate and avoiding cytotoxicity, and is a decellularized valve stent material with good biomechanical properties; at the same time, the valve surface is amidated with ε-polylysine and α-polylysine, and their broad-spectrum antibacterial properties and the ability to promote cell adhesion, proliferation and differentiation are utilized to accelerate valve endothelialization and extend the service life of the valve. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the process of enzymatic cross-linking treatment of heart valves described in an embodiment of the present invention.

[0024] Figure 2 The comparison results of biocompatibility and cell adhesion tests of different biological valves described in the experimental examples of the present invention are shown.

[0025] Figure 3 The figures are comparative results of in vivo cytotoxicity and regenerative ability evaluation of different bioprosthetic valves described in the experimental examples of the present invention.

[0026] Figure 4 The figures are comparative results of in vivo calcification assessment of different biological valves described in the experimental examples of the present invention.

[0027] Figure 5 The results are a comparison of the adhesion, migration, proliferation and differentiation of biological cells of different biological valves described in the experimental examples of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example

[0030] A method for enzymatic cross-linking of a heart valve is provided, the steps comprising:

[0031] 1. Obtaining porcine aortic valve: Cut fresh porcine aortic valve in a clean environment, rinse with heparin saline for 3 times, place in saline solution containing double antibiotics (100U / ml penicillin, 100mg / ml streptomycin), and store at 4℃ for later use.

[0032] 2. Preparation of decellularized valve (DHV): The porcine aortic valve was placed in TRIS-HCI buffer (40 mM, pH 7.8) containing 2% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and 2 mmol / l tributylphosphine (TnBP), and the decellularization treatment was continued at room temperature for 24 h with continuous shaking. It was rinsed with sterile water for 6 times, 10 minutes each time. It was then placed in TRIS-HCI buffer (40 mM, pH 7.8) containing 2% CHAPS, 2 mmol / l TnBP, 1% amidinosulfobetaine (ASB-14) and 2% sulfobetaine 10 (SB 3-10), and the decellularization treatment was continued at room temperature for 24 h with continuous shaking.

[0033] 3. Modification of cysteine, ε-lysine and α-lysine: Place the decellularized valve in a mixed aqueous solution of cysteine ​​(10mM-100mM), ε-lysine (10mM-100mM) and α-lysine (10mM-100mM) at different concentrations (10mM-200mM) and soak for 30 minutes. Add carbodiimide (EDC) / N-hydroxysuccinimide (NHS) to the above mixed solution to start the surface modification process of the decellularized valve with three amino acids, and the final concentration of EDC / NHS is 0.01-1M. Crosslink at 37℃ constant temperature shaker for different times of 6h-72h; rinse with saline at room temperature for 24h, change the solution every 6h, and then store in 4℃ saline.

[0034] 4. Enzymatic cross-linking of heart valves: Soak the decellularized valves modified with cysteine, ε-lysine and α-lysine obtained in step 3 in sulfhydryl oxidase solutions of different concentrations (0.01-10U / ml). Cross-link for different time periods of 6h-72h in a 37℃ constant temperature shaker; rinse with saline at room temperature for 24h, change the solution every 6h, and then store in saline at 4℃.

[0035] The schematic diagram of the above modification and cross-linking process is shown in Figure 1 shown.

[0036] Experimental example

[0037] The performance comparison test was conducted on the decellularized valve (DHV), the glutaraldehyde cross-linked decellularized valve (GV) and the heart valve cross-linked by sulfhydryl oxidase (SDHV) in the above-mentioned embodiment.

[0038] 1. Biocompatibility and cell adhesion testing

[0039] The cytotoxicity of the materials was compared by co-culturing different samples with HUVEC for 48 hours using the CCK8 method. The number of cells adhering to the surface of the material was evaluated by the CCK8 method. In order to evaluate the cytotoxicity of the new material, HUVEC was co-cultured with DHV, glutaraldehyde-crosslinked DHV (GV) and thiol oxidase-crosslinked DHV (SDHV) to verify the cytotoxicity. In order to verify the effect of α-polylysine in promoting cell adhesion, we punched the material into original sheets with a diameter of 10 mm and spread them in a 96-well plate. After the cell suspension was dropped on the surface of the valve and incubated statically for 30 minutes, complete culture medium was added and cultured for 1 day. The number of cells on the surface of the valve was detected by the CCK8 method. The results of biocompatibility and cell adhesion tests are shown in Figure 2 shown. Figure 2 A is the result of biocompatibility test. After 48 hours of culture, it can be seen from the results that GV showed high cytotoxicity, while SDHV and DHV had similar cell growth status to the blank control, showing low cytotoxicity and good biocompatibility, and had good application potential. This is also extremely important for an ideal tissue engineering valve. Figure 2 B is the result of cell adhesion test, which clearly shows that GV exhibits less cell adhesion due to its toxicity. DHV exhibits a large amount of cell adhesion due to the large number of cell adhesion sites in the natural extracellular matrix, while SDHV exhibits a higher cell adhesion ability, indicating that the modification of α-polylysine can further promote cell adhesion and show the potential to promote tissue regeneration.

[0040] 2. Evaluation of in vivo cytotoxicity and regeneration capacity

[0041] Different materials were sewn into valve rolls and transplanted into the abdominal aorta. After 28 days of transplantation, the tissues were sliced ​​and stained with HE. The results were as follows: Figure 3 As shown in the results, it can be seen that GV has no obvious cell infiltration due to its overly dense cross-linking and inherent cytotoxicity. In contrast, DHV and SDHV show a high degree of cell infiltration inside the tissue, meeting the needs of cell infiltration and migration.

[0042] 3. In vivo Calcification Assessment

[0043] In vivo calcification was assessed using an abdominal aorta transplant model. Tissues were sectioned and stained with Von Kossa 28 days after transplantation. Figure 4 As shown in the results, it can be seen that a large amount of aldehyde residues in GV caused by glutaraldehyde cross-linking led to obvious calcification and valve decay. However, DHV and SDHV did not undergo calcification, showing good anti-calcification ability, which will greatly extend the service life of the valve.

[0044] 4. Cell adhesion, migration, proliferation and differentiation performance

[0045] Figure 5 Comparison results of cell adhesion, migration, proliferation and differentiation performance. Figure 5 Middle A shows the staining results of CD31 (endothelial cells) and α-SMA (stromal cells); Figure 5 B in the middle is the statistical data of the degree of endothelialization. 28 days after transplantation, the tissue was sectioned and stained for CD31 and α-SMA. From the results, it can be seen that due to the cytotoxicity and dense cross-linking of GV, there is no cell infiltration into the tissue, and it is difficult to meet the needs of tissue regeneration. Although DHV has a large number of cell infiltrations, it does not show specific makers of endothelial cells or interstitial cells, indicating that although DHV can promote cell migration to a certain extent, it does not have the ability to promote cell differentiation into endothelial cells or interstitial cells, so as to achieve tissue regeneration. On the contrary, in SDHV, we observed the remodeling of the complete endothelial cell layer and the appearance of interstitial cells, indicating that SDHV can promote cell migration, proliferation and differentiation, and accelerate the recellularization and regeneration of the valve.

[0046] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for enzymatic cross-linking of a heart valve, characterized in that the steps include: Decellularization of heart valves; The treated heart valve surface is subjected to amidation modification and thiol modification; the amidation modification uses ε-polylysine and α-polylysine; The modified heart valve is immersed in a 0.01-10U / ml sulfhydryl oxidase solution to perform sulfhydryl oxidation cross-linking treatment to obtain a cross-linked heart valve.

2. The method according to claim 1, characterized in that The reagent used for the thiolation modification is selected from at least one of cysteine, sodium thioglycolate, thiododecyl polyethylene glycol carboxylic acid, and HS-PEG-SH.

3. The method according to claim 2, characterized in that The reagent used for the thiol modification is cysteine, and the concentration is 10-100 mM; and / or, the concentrations of ε-lysine and α-lysine are both 10-100 mM.

4. The method according to claim 1, characterized in that: The heart valve surface modification process uses a first catalyst and a second catalyst; the first catalyst is carbodiimide, and the second catalyst is selected from at least one of N-hydroxysuccinimide, 1-hydroxybenzotriazole, and 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate.

5. The method according to claim 1, characterized in that The surface modification process conditions are: shaking reaction at 30-38° C. for 6-72 hours; and / or, the thiol oxidation cross-linking treatment process conditions are: shaking reaction at 30-38° C. for 6-72 hours.

6. The method according to claim 1, characterized in that The heart valve is a porcine heart valve.

7. The method according to claim 1, characterized in that The decellularization process is to use a first buffer containing 1-5% CHAPS and 1-5mM TnBP to shake at room temperature for 20-30 hours, during which rinsing is performed; then use a second buffer containing 1-5% CHAPS, 1-5mM TnBP, 0.5-2% ASB-14, and 1-5% sulfobetaine 10 to shake at room temperature for 20-30 hours.

8. The method according to claim 7, characterized in that The pH value of the first buffer and / or the second buffer is 7.

8.

9. A bioprosthetic heart valve, characterized in that: Obtained by the method according to any one of claims 1 to 8.

10. Use of the artificial biological heart valve according to claim 9 in preparing biological scaffold materials.

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