Heart valve modification method, artificial heart valve and application

Abstract

The invention discloses a heart valve modification method, an artificial heart valve and application, according to the heart valve modification method, nucleoside dialdehyde / nucleotide dialdehyde is used as a bioactive molecule to carry out functional modification on the heart valve, and the stability and biocompatibility of the heart valve are improved; the requirement of promoting growth of endothelial cells on the material surface is met; meanwhile, the stability of the valve is effectively improved, and the service life of the biological valve is prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial heart valves, in particular to a heart valve modification method, an artificial heart valve and applications. 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] S-adenosylhomocysteine ​​hydrolase (SAHH) catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine ​​(AdoHcy) into adenosine and L-homocysteine, and is involved in regulating many biological processes. SAHH regulates cellular autophagy, removes damaged and aged organelles, and maintains tissue homeostasis, so it plays a vital role in cells. In cardiovascular diseases, chronic elevation of SAHH levels may be a key pathological factor in homocysteine-related diseases, so regulating its activity is expected to achieve the treatment of cardiovascular diseases. .

[0004] As a derivative of natural products, nucleoside / nucleotide dialdehydes show excellent biocompatibility. They are also active inhibitors of SAHH, so they are expected to be used for heart valve modification to guide cell behavior and promote regeneration. At the same time, the aldehyde group in its molecular structure can also effectively improve the stability of the valve, thereby solving the obvious cytotoxicity caused by the treatment technology based on glutaraldehyde cross-linking biological valves in clinical practice, which makes it difficult to meet the needs of cell adhesion and proliferation, and is not conducive to the endothelialization of stent materials. 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. The development and exploration of new valve modification strategies 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.

[0005] Therefore, it is necessary to propose new modification methods to meet the requirements of low cost, ease of use and low cytotoxicity, so as to achieve the purpose of improving the stability and biocompatibility of biological valves; to meet the needs of promoting the growth of endothelial cells on the surface of the material and to extend the service life of the biological valves. Summary of the invention

[0006] The purpose of the present invention is to propose a method for modifying a heart valve, using a heart valve modified with a new functional active molecule to meet the requirements of low cost, ease of use and low cytotoxicity, and to improve the stability and biocompatibility of the biological valve; to meet the requirements of promoting the growth of endothelial cells on the surface of the material and to extend the service life of the biological valve.

[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 modifying a heart valve, the steps comprising:

[0009] a) removing tissue cells from the heart valve;

[0010] b) placing the treated heart valve in a 10-200 mM bioactive molecule solution for a first modification;

[0011] c) placing the heart valve after the first modification in a sealing agent solution for a second modification to obtain a modified heart valve;

[0012] The bioactive molecule is a nucleoside dialdehyde or a nucleotide dialdehyde, and has the following chemical formula:

[0013]

[0014] Where: R' is selected from -OH or

[0015] R is selected from

[0016] Furthermore, the bioactive molecule is obtained by modifying nucleosides or nucleotides by incubating in a HIO4 solution in the dark;

[0017] The nucleoside is selected from uridine, cytidine, adenosine or guanosine;

[0018] The nucleotide is selected from uracil nucleotide, cytosine nucleotide, adenine nucleotide or guanine nucleotide.

[0019] Preferably, during the nucleoside modification process, the molar ratio of nucleoside to HIO4 is 1:(1.05-2); during the nucleotide modification process, the molar ratio of nucleoside to HIO4 is 1:(1.05-2).

[0020] Furthermore, the conditions of the first modification and / or the second modification process are: modification on a shaking platform at 30-38° C. for 1-7 days.

[0021] Furthermore, the blocking agent is ethylenediamine with a concentration of 1-100 mM.

[0022] Furthermore, the heart valve is a porcine heart valve.

[0023] Furthermore, the decellularization process in step a) 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.

[0024] Preferably, the pH value of the first buffer and / or the second buffer is 7.8.

[0025] The second aspect of the present invention is to provide an artificial biological heart valve obtained by the modification method described in the first aspect.

[0026] 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.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The heart valve modification method described in the present invention uses nucleoside dialdehydes / nucleotide dialdehydes as bioactive molecules for the first time to replace glutaraldehyde modification, meet the requirements of being cheap, easy to operate and avoiding cytotoxicity, and improve the stability and biocompatibility of the biological valve; obtain a decellularized valve stent material with good biomechanical properties, meet the requirement of promoting the growth of endothelial cells on the surface of the material, and extend the service life of the biological valve.

[0029] The present invention uses ethylenediamine to perform secondary modification on the valve modified with nucleoside dialdehyde / nucleotide dialdehyde, further improving the structural stability of the decellularized valve, while sealing the residual aldehyde groups, effectively reducing the calcification degree and thrombosis formation of the valve after implantation in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of H NMR spectrum analysis of nucleosides / nucleoside dialdehydes prepared using adenine as an example in Example 1 of the present invention.

[0031] Figure 2 Schematic diagram of the nucleoside dialdehyde / nucleotide dialdehyde and decellularized valve modification process described in Example 2 of the present invention.

[0032] Figure 3 The figures are comparative results of in vitro cytotoxicity evaluation of heart valves in different groups in the experimental examples of the present invention.

[0033] Figure 4 The figures are comparative evaluation results of the in vivo biocompatibility and regenerative ability of heart valves in different groups in the experimental examples of the present invention.

[0034] Figure 51 is a comparative evaluation result of heart valve calcification in vivo in different groups in the experimental examples of the present invention.

[0035] Figure 6 The results are a comparison of the cell adhesion, migration, proliferation and differentiation abilities of heart valves in different groups in the experimental examples of the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1 Preparation of nucleoside dialdehyde or nucleotide dialdehyde

[0038] 1. Preparation of nucleoside dialdehydes: Dissolve uridine (U), cytosine (T), adenine (T) and guanosine (G) (10 mmol) in 10 ml of water and 10 ml of HIO4 solution (1.5 M), incubate at 20°C in the dark for 1 h. Add 50 ml of ion exchange resin (HCO 3- ), after 30 min, the resin was filtered and rinsed with water twice. The water was evaporated to dryness in vacuo, and the residue was co-evaporated with methanol (2×10 mL) at 30° C. The remaining material was freeze-dried in vacuo to obtain the final product.

[0039] 2. Preparation of nucleotide dialdehydes: Uracil nucleotide (UMP), cytosine nucleotide (TMP), adenine nucleotide (AMP) and guanine nucleotide (GMP) (2 mmol) were dissolved in 2.5 ml of water and 4.2 ml of HIO4 solution (0.5 M), and incubated at 20°C in the dark for 1 h. The reaction mixture was allowed to stand at 0°C for 24 h, and the precipitated inorganic salts were filtered to obtain a 0.3 M nucleotide dialdehyde solution.

[0040] In step 3-4, the preparation reaction process of nucleoside dialdehyde or nucleotide dialdehyde is as follows:

[0041]

[0042] Where: R' is selected from -OH or

[0043] R is selected from

[0044] Taking adenine as an example, the nucleosides and nucleoside dialdehydes prepared by the above method were analyzed by nuclear magnetic resonance spectroscopy. Figure 1 As shown, Figure 1 A is the NMR image of adenine nucleoside; Figure 1 B is the NMR image of adenine nucleoside dialdehyde, which can indicate that the corresponding nucleoside dialdehyde / nucleotide dialdehyde can be prepared by the above method using adenine as a raw material.

[0045] Example 2 Decellularized Valve Modification

[0046] 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.

[0047] 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.

[0048] 3. Decellularized valve modification: Place the decellularized valve in different concentrations (10mM-200mM) of nucleoside dialdehyde or nucleotide dialdehyde solution and modify it in a 37℃ constant temperature shaker for different time periods of 1d-7d; rinse with physiological saline at room temperature for 24h, change the solution every 6h, and then store in 4℃ physiological saline.

[0049] 4. Residual aldehyde group sealing and secondary modification: Soak the different nucleoside / nucleotide modified heart valves obtained in step 5 in ethylenediamine solutions of different concentrations (1mM-100mM) and modify them in a constant temperature shaker at 37°C for different time periods of 1d-7d; rinse with physiological saline at room temperature for 24h, change the solution every 6h, and then store in 4°C physiological saline.

[0050] The schematic diagram of the above modification process is as follows Figure 2 shown.

[0051] Experimental example

[0052] Verify the comparison results of decellularized heart valves (DHV) modified by different methods. A performance comparison experiment was conducted using glutaraldehyde cross-linked heart valves (GV group) and heart valves modified with adenine nucleoside dialdehyde / nucleotide dialdehyde (HDHV group) using the method described in Example 1-2 as examples.

[0053] 1. In vitro cytotoxicity assessment verified by CCK8 assay

[0054] The cytotoxicity of human umbilical vein endothelial cells (HUVEC) was verified by co-culture with decellularized heart valves (DHV group), glutaraldehyde cross-linked heart valves (GV group), and nucleoside dialdehyde / nucleotide dialdehyde modified adenine (HDHV group). Figure 3 After 48 hours of culture, the results showed that the GV group showed higher cytotoxicity, while the HDHV group and the DHV group had no obvious cytotoxicity, showing good biocompatibility and good application potential.

[0055] 2. In vivo biocompatibility and valve calcification characterization

[0056] A rat subcutaneous implantation model was established to evaluate the in vivo tissue compatibility of the scaffold. After 4 weeks, the specimens were removed and the ECM changes and calcification were observed using HE, Masson, EVG and Von Kossa staining.

[0057] 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. Figure 4 As shown in the results, it can be seen that the GV group had no obvious cell infiltration due to the overly dense modification and inherent cytotoxicity. On the contrary, the DHV group and the HDHV group showed a higher degree of cell infiltration in the tissue, meeting the needs of cell infiltration and migration.

[0058] In vivo calcification was evaluated using an abdominal aorta transplantation model. Tissues were sectioned 28 days after transplantation and stained with Von Kossa. Figure 5 As shown in the results, it can be seen that the GV group had a large amount of aldehyde residues due to glutaraldehyde modification, which led to obvious calcification and valve decay. However, the DHV group and HDHV group did not undergo calcification, showing good anti-calcification ability, which will greatly extend the service life of the valve.

[0059] 3. Cell adhesion, migration, proliferation and differentiation ability

[0060] like Figure 6As shown. In the figure, A is CD31 (endothelial cells) and α-SMA (interstitial cells) staining; B is the statistical data of endothelialization degree. 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 cytotoxicity and dense modification, there was no cell infiltration into the tissue in the GV group, which made it difficult to meet the needs of tissue regeneration. Although there was a large amount of cell infiltration in the DHV group, no specific maker of endothelial cells or interstitial cells was shown, 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 the HDHV group, we observed the remodeling of the complete endothelial cell layer and the appearance of interstitial cells, indicating that the HDHV group can promote cell migration, proliferation and differentiation, and accelerate the recellularization and regeneration of the valve.

[0061] 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 modifying a heart valve, characterized in that the steps include: a) removing tissue cells from the heart valve; b) placing the treated heart valve in a 10-200 mM bioactive molecule solution for a first modification; c) placing the heart valve after the first modification in a sealing agent solution for a second modification to obtain a modified heart valve; The bioactive molecule is a nucleoside dialdehyde or a nucleotide dialdehyde, and has the following chemical formula: Where: R' is selected from -OH or R is selected from 2. The method according to claim 1, characterized in that The bioactive molecule is obtained by modifying nucleosides or nucleotides by incubating in a HIO4 solution in the dark; The nucleoside is selected from uridine, cytidine, adenosine or guanosine; The nucleotide is selected from uracil nucleotide, cytosine nucleotide, adenine nucleotide or guanine nucleotide.

3. The method according to claim 2, characterized in that In the nucleoside modification process, the molar ratio of nucleoside to HIO4 is 1:(1.05-2); in the nucleotide modification process, the molar ratio of nucleoside to HIO4 is 1:(1.05-2).

4. The method according to claim 1, characterized in that: The conditions of the first modification and / or the second modification process are: modification on a shaking platform at 30-38° C. for 1-7 days.

5. The method according to claim 1, characterized in that The blocking agent is ethylenediamine, and the concentration is 1-100 mM.

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 in step a) 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 modification 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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