Method for enzymatic cross-linking treatment of heart valves, artificial biological heart valve and use

Abstract

The application discloses a method for enzymatic cross-linking treatment of a heart valve, an artificial biological heart valve and application, and the cross-linking method of the heart valve adopts surface sulfydryl modification based on thiol oxidase cross-linking of a decellularized valve, meets the requirements of low cost and easy operation and avoidance of cell toxicity, and the decellularized valve support material has good biomechanical properties; meanwhile, the valve surface is subjected to amidation modification of epsilon-polylysine and alpha-polylysine, the broad-spectrum antibacterial performance and the ability of promoting cell adhesion, proliferation and differentiation of the epsilon-polylysine and the alpha-polylysine are utilized, endothelialization of the valve is accelerated, and the service life of the valve is prolonged.

Description

TECHNICAL FIELD

[0001] The present application 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 application. BACKGROUND

[0002] Heart valve disease is a cardiovascular disease with high morbidity and mortality. The most effective method for treating heart valve disease is valve replacement surgery. It is estimated that 850,000 valve replacements will be performed worldwide every year by 2050. Biological valves are one of the most commonly used valve substitutes in clinical practice. They have good blood compatibility and do not require long-term anticoagulant therapy. However, their service life is limited, and patients often need surgery again due to degradation and calcification of biological valves.

[0003] Current biological valve treatment techniques mainly use glutaraldehyde cross-linking. Although this cross-linking method effectively improves the stability of biological valves, it exhibits significant cytotoxicity, making it 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 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. According to the molecular configuration, it can be divided into epsilon-lysine and alpha-lysine. Among them, epsilon-lysine has broad-spectrum antibacterial properties, and alpha-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 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 for scientific research and the development of related industries. SUMMARY

[0006] The present application aims to provide a method for enzymatic cross-linking treatment of heart valves to avoid the toxic side effects of glutaraldehyde in existing technology and improve the structural stability, blood compatibility, and long-term functional stability of heart valves.

[0007] In view of the above, the present application provides the following solutions:

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

[0009] The heart valve is subjected to de-tissue cell treatment;

[0010] The surface of the treated heart valve is subjected to amidation modification and thiolation modification; the amidation modification uses epsilon-polylysine and alpha-polylysine;

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

[0012] Further, the reagent used in the sulfhydrylation modification is at least one of cysteine, sodium mercaptoacetate, mercaptododecyl glycol carboxylic acid, and HS-PEG-SH.

[0013] Preferably, the reagent used in the sulfhydrylation modification is cysteine with a concentration of 10-100 mM, and / or the concentrations of the epsilon-lysine and alpha-lysine are both 10-100 mM.

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

[0015] Further, the surface modification process is performed at 30-38℃ for 6-72 h on a shaker, and / or the sulfhydryl oxidation cross-linking treatment process is performed at 30-38℃ for 6-72 h on a shaker.

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

[0017] Further, the decellularization process is performed by using a first buffer solution containing 1-5% CHAPS and 1-5 mM TnBP to perform oscillation treatment at room temperature for 20-30 h, during which rinsing is performed; and then using a second buffer solution containing 1-5% CHAPS, 1-5 mM TnBP, 0.5-2% ASB-14, and 1-5% thiobetaine 10 to perform oscillation treatment at room temperature for 20-30 h.

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

[0019] The second aspect of the present application is to provide an artificial biological heart valve obtained by the method for performing enzymatic cross-linking treatment on a heart valve according to the first aspect.

[0020] The third aspect of the present application is to provide the use of the artificial biological heart valve according to the second aspect in the preparation of a biological stent material.

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

[0022] The heart valve crosslinking method of the application adopts surface thiol modification to crosslink the decellularized valve based on thiol oxidase, meets the needs of inexpensive and easy operation and avoids cell toxicity, and the decellularized valve support material has good biomechanical performance; at the same time, the surface of the valve is subjected to amidation modification of epsilon-polylysine and alpha-polylysine, the broad-spectrum antibacterial performance and the ability to promote cell adhesion, proliferation and differentiation are utilized, the valve endothelialization is accelerated, and the service life of the valve is prolonged. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure is a schematic diagram of the enzymatic crosslinking process of the heart valve described in the embodiments of the application.

[0024] Figure 2 The figure is a comparison result of the biocompatibility and cell adhesion detection of different biological valves in the experimental examples of the application.

[0025] Figure 3 The figure is a comparison result of the in vivo cytotoxicity and regeneration ability evaluation of different biological valves in the experimental examples of the application.

[0026] Figure 4 The figure is a comparison result of the in vivo calcification evaluation of different biological valves in the experimental examples of the application.

[0027] Figure 5 The figure is a comparison result of the biological cell adhesion, migration, proliferation and differentiation of different biological valves in the experimental examples of the application. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the application will be described clearly and completely in combination with the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0029] EMBODIMENT

[0030] The method of enzymatic crosslinking treatment of heart valve is proposed, and the steps include:

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

[0032] 2. Preparation of decellularized heart valve (DHV): Porcine aortic valve was placed in TRIS-HCI buffer (40 mM, pH 7.8) containing 2% 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) and 2 mmol / l tributylphosphine (TnBP) for 24 h of decellularization at room temperature with continuous shaking. It was rinsed with sterile water for 6 times, 10 min each time. Then it was placed in TRIS-HCI buffer (40 mM, pH 7.8) containing 2% CHAPS, 2 mmol / l TnBP, 1% amidite sulfobetaine (ASB-14) and 2% sulfobetaine 10 (SB 3-10) for 24 h of decellularization at room temperature with continuous shaking.

[0033] 3. Cysteine, ε-lysine and α-lysine modification: The decellularized heart valve was placed in mixed aqueous solution of cysteine (10 mM-100 mM), ε-lysine (10 mM-100 mM) and α-lysine (10 mM-100 mM) with different concentrations (10 mM-200 mM) for static soaking for 30 min. Carbodiimide (EDC) / N-hydroxysuccinimide (NHS) was added to the above mixed solution to initiate the decellularization surface modification process of the three amino acids, with a final concentration of EDC / NHS of 0.01-1 M. Crosslinking was performed at 37°C for different time periods of 6 h-72 h; physiological saline was used for room temperature rinsing for 24 h, with liquid change every 6 h, and then it was placed in physiological saline at 4°C for storage.

[0034] 4. Enzymatic crosslinking of heart valve: The decellularized heart valve modified by cysteine, ε-lysine and α-lysine obtained in step three was soaked in thiol oxidase solution with different concentrations (0.01-10 U / ml). Crosslinking was performed at 37°C for different time periods of 6 h-72 h; physiological saline was used for room temperature rinsing for 24 h, with liquid change every 6 h, and then it was placed in physiological saline at 4°C for storage.

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

[0036] Experimental Example

[0037] Performance comparison tests were carried out on the decellularized heart valve (DHV), glutaraldehyde crosslinked decellularized heart valve (GV) and the heart valve crosslinked by thiol oxidase (SDHV) according to the above embodiment, respectively.

[0038] 1. Biocompatibility and cell adhesion detection

[0039] The cytotoxicity of the materials was detected by co-culturing different samples with HUVEC for 48 h by CCK8 method. The number of cells adhered to the material surface was evaluated by CCK8 method. To evaluate the cytotoxicity of the new materials, HUVEC were co-cultured with DHV, glutaraldehyde cross-linked DHV (GV) and DHV cross-linked based on thiol oxidase (SDHV) to verify the cytotoxicity. To verify the effect of alpha-polylysine on promoting cell adhesion, the materials were punched into 10 mm diameter patches and placed in a 96-well plate. After the cell suspension was added to the surface of the valve and incubated for 30 min, complete medium was added for 1 day, and the number of cells on the surface of the valve was detected by CCK8 method. The biocompatibility and cell adhesion detection results are shown in Figure 2 Figure 2 A is the biocompatibility detection result. After 48 h of culture, it can be seen from the result that GV shows high cytotoxicity, while SDHV and DHV are similar to the cell growth state of the blank control, showing low cytotoxicity and good biocompatibility, and having good application potential. This is also very important for the ideal tissue engineering valve. Figure 2 B is the cell adhesion detection result, which can be clearly seen that GV shows less cell adhesion due to toxicity. DHV shows a large number of cell adhesion sites due to the natural extracellular matrix, so it shows a large number of cell adhesion, while SDHV shows higher cell adhesion capacity, indicating that the modification of alpha-polylysine can further promote cell adhesion, and exhibit the potential to promote tissue regeneration.

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

[0041] By sewing different materials into valve rolls and performing abdominal aortic transplantation, after 28 days of transplantation, the tissues were sectioned and HE stained, and the results are shown in Figure 3 From the results, it can be seen that GV has no obvious cell infiltration due to the problem of too dense cross-linking and inherent cytotoxicity. On the contrary, DHV and SDHV exhibit high cell infiltration in the tissue, meeting the needs of cell infiltration and migration.

[0042] 3. In vivo calcification evaluation

[0043] In vivo calcification evaluation was performed by abdominal aortic transplantation model, after 28 days of transplantation, the tissues were sectioned and Von Kossa stained, and the results are shown in Figure 4 From the results, it can be seen that GV forms obvious calcification and valve failure due to the large number of aldehyde groups remaining caused by glutaraldehyde cross-linking. While DHV and SDHV do not calcify, showing good anti-calcification ability, which can greatly prolong the service life of the valve.

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

[0045] Figure 5 Results of cell adhesion, migration, proliferation and differentiation. Figure 5 A is the results of CD31 (endothelial cells) and a-SMA (mesenchymal cells) staining; Figure 5 B is the data statistics of endothelialization. After 28 days of implantation, the tissue was sectioned and stained for CD31 and a-SMA. It can be seen from the results that GV has no cell infiltration into the tissue due to cytotoxicity and dense crosslinking, making it difficult to meet the needs of tissue regeneration. Although DHV has a large number of cell infiltration, it does not show specific markers of endothelial cells or mesenchymal cells, indicating that DHV can promote cell migration to some extent, but does not have the ability to promote cell differentiation into endothelial cells or mesenchymal 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 mesenchymal 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 application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method of enzymatic cross-linking treatment of a heart valve, characterized in that the steps The application relates to a method for preparing a biological heart valve prosthesis. The method comprises the following steps: carrying out a decellularization treatment on a heart valve; carrying out surface modification on the treated heart valve by amide modification and thiol modification; the amide modification uses epsilon-polylysine and alpha-polylysine; and carrying out thiol oxidation cross-linking treatment on the modified heart valve by immersing the heart valve in a thiol oxidase solution with a concentration of 0.01-10 U / mL, so as to obtain a cross-linked heart valve. The reagent used in the thiol modification is at least one of cysteine, sodium mercaptoacetate, mercaptododecaglycol carboxylic acid and HS-PEG-SH. The reagent used in the thiol modification is cysteine with a concentration of 10-100 mM; and / or the concentrations of the epsilon-lysine and alpha-lysine are both 10-100 mM.

2. The method of claim 1, wherein, The surface modification process of the heart valve uses a first catalyst and a second catalyst; the first catalyst is a carbodiimide, and the second catalyst is at least one of N-hydroxysuccinimide, 1-hydroxybenzotriazole and 1H-benzotriazole-1-yl oxytripyrrolidinophosphonium hexafluorophosphate.

3. The method of claim 2, wherein, The surface modification process is carried out at 30-38 DEG C for 6-72 h on a shaking table; and / or the thiol oxidation cross-linking treatment process is carried out at 30-38 DEG C for 6-72 h on a shaking table.

4. The method of claim 1, wherein, The heart valve is a pig heart valve.

5. The method of claim 1, wherein, The decellularization treatment process is as follows: a first buffer solution containing 1-5% CHAPS and 1-5 mM TnBP is used for oscillation treatment at room temperature for 20-30 h, and rinsing is carried out during the oscillation treatment; and then a second buffer solution containing 1-5% CHAPS, 1-5 mM TnBP, 0.5-2% ASB-14 and 1-5% sulfobetaine 10 is used for oscillation treatment at room temperature for 20-30 h.

6. The method of claim 1, wherein, The pH value of the first buffer solution and / or the second buffer solution is 7.

8.

7. The method of claim 1, wherein, The method is obtained by any one of claims 1-8.

8. The method of claim 7, wherein, 10. The application of the artificial biological heart valve prosthesis in claim 9 in the preparation of a biological stent material.

9. Artificial biological heart valve, characterized in that, ​ ​

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