An anticoagulant proendothelial tissue-engineered valve material and a preparation method thereof

Abstract

The application discloses an anticoagulant proendothelization tissue engineering valve material and a preparation method thereof. First, a biological valve material is soaked in a solution containing double-bonded silk fibroin, so that the valve material is fully contacted with components in the solution and adsorbed; the double-bonded silk fibroin is obtained by modifying silk fibroin and contains carbon-carbon double bonds; then, a double-bond reagent is added to the solution obtained in the step, so that the double-bond reagent is fully dispersed, and the valve material is continuously soaked; the double-bond reagent is one of polyethylene glycol diacrylate, 1,4-butanediol diacrylate and ethane-1,2-diyl diacrylate; an initiator is further added to the solution, a double-bond polymerization reaction is carried out, and the tissue engineering valve material is prepared. In the application, the double-bonded silk fibroin and the double-bond reagent are in-situ polymerized on a valve material matrix, a polymer network is embedded between collagen fibers of the valve material matrix, and the anticoagulant proendothelization tissue engineering valve material is prepared.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cardiovascular repair materials, and particularly relates to an anticoagulant endothelium-promoting tissue engineering valve material and a preparation method thereof. BACKGROUND

[0002] Tissue engineering valves are usually prepared from animal aortic valves and animal pericardial membranes, and are used to replace damaged autologous heart valves in the treatment of valvular heart disease. Compared with mechanical heart valves, tissue engineering valves have a series of advantages: the tissue engineering valve has a more flexible texture and a structure closer to the original heart valve than the traditional mechanical valve, so that the hemodynamic performance of the tissue engineering valve is excellent; the thrombogenicity of the tissue engineering valve is lower than that of the mechanical valve, so that the patient does not need to take anticoagulant drugs for life after implantation, reducing the burden of the patient and the risk of bleeding caused by long-term use of anticoagulant drugs; in addition, the tissue engineering valve not only bears the valve function of the original valve after implantation, but also serves as a site for valve tissue regeneration, acting as a tissue engineering scaffold to promote valve regeneration, so as to achieve permanent repair of the valve and the valve function. These advantages make the tissue engineering valve a research hotspot in the field of valve materials.

[0003] At present, tissue engineering valves based on animal tissues (aortic valves and pericardial membranes) still have a series of challenges. These animal tissue-based tissue engineering valves are collagen-based biomaterials, which naturally adsorb blood components and cause coagulation reactions, leading to thrombus formation; thrombus formation will seriously hinder the movement of the valve leaflet, resulting in loss of valve function and accelerated degradation of the valve. As a long-term implant, endothelialization is a powerful means to ensure the long-term blood performance of the valve, which can reduce the interaction between the tissue engineering valve matrix and blood by forming an endothelial layer with excellent blood compatibility, thereby reducing the risk of thrombus formation.

[0004] At present, tissue engineering valves based on animal tissues are still the main source of tissue engineering valves due to their wide source and hemodynamic performance close to that of the original valve. In view of the thrombus problem existing in the current tissue engineering valve and the importance of endothelialization, it is of great significance to modify the tissue engineering valve for anticoagulation and endothelialization. SUMMARY

[0005] In order to solve the thrombus problem existing in the current tissue engineering valve, the present application provides a preparation method of an anticoagulant endothelium-promoting tissue engineering valve material.

[0006] The preparation method of the anticoagulant endothelium-promoting tissue engineering valve material provided by the present application comprises the following steps:

[0007] S1, soaking the biological valve material in a solution containing double-bonded silk fibroin, so that the valve material is in contact with the components in the solution and adsorbs them.

[0008] The biological valve material is selected from animal tissues, including pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessels, skin, amniotic membrane or ligament.

[0009] The double-bonded silk fibroin is a carbon-carbon double-bond-containing silk fibroin obtained by modifying the original silk fibroin. The preparation method of the double-bonded silk fibroin is as follows:

[0010] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are added to the original silk fibroin solution, and after stirring at room temperature for 2 hours, 2-aminoethyl methacrylate hydrochloride is added, the pH is adjusted to ≥8, and the reaction is continued for 24 hours. Finally, dialysis is performed to obtain double-bonded silk fibroin powder.

[0011] S2, adding a double-bonding agent to the solution of step S1, dispersing the double-bonding agent, and continuing to soak the valve material; the double-bonding agent is selected from one or more of polyethylene glycol diacrylate, 1,4-butanediol diacrylate, ethane-1,2-diyl diacrylate, ethyl acrylate, N-methyl-2-acrylamide, N-2,2-propenyl-2-acrylamide, N-ethyl acrylamide, N,N'-vinyl bisacrylamide, (ethane-1,2-diyl bis(oxy))bis(ethane-2,1-diyl) diacrylate, N,N'-dimethyl acrylamide, N,N-dimethyl methacrylamide, acrylamide, 2-propenoic acid-2-methoxyethyl ester, and double-bonded polylysine.

[0012] S3, adding an initiator to the solution of step S2 to perform a double-bond polymerization reaction, thereby preparing a tissue engineering valve material.

[0013] Preferably, in step S1, the mass percentage concentration of double-bonded silk fibroin in the solution containing double-bonded silk fibroin is 0.5% to 1.5%, and the soaking time is 0.5 to 24 hours.

[0014] Further preferably, in step S1, the solution containing double-bonded silk fibroin can also contain original silk fibroin.

[0015] Preferably, in step S2, the mass percentage concentration of the double-bonding agent is 1% to 6%, and the soaking time is 0.5 to 24 hours.

[0016] In step S3, the initiator is selected from one of the following mixtures: ammonium persulfate and sodium bisulfite, ammonium persulfate and sodium sulfite, sodium persulfate and sodium sulfite, potassium persulfate and sodium sulfite, sodium persulfate and sodium bisulfite, potassium persulfate and sodium bisulfite, potassium persulfate and tetramethyl ethylenediamine, ammonium persulfate and tetramethyl ethylenediamine, and sodium persulfate and tetramethyl ethylenediamine.

[0017] The preparation principle of the tissue engineering valve material of the present application is as follows:

[0018] In the preparation method of the present application, the valve material is soaked in the double-bonded silk fibroin solution, so that the valve material is fully contacted with the double-bonded silk fibroin in the solution and absorbs a certain amount of double-bonded silk fibroin. Further, the double-bonded reagent is added to the solution to be fully dispersed between the valve material and the double-bonded silk fibroin. Finally, the initiator is added to initiate the polymerization between the double-bonded silk fibroin and the double-bonded reagent to form a network of the silk fibroin and the double-bonded reagent polymer on the valve material in situ, thereby preparing the tissue engineering valve with the anticoagulation and endothelialization functions. In the whole preparation process, the biological valve material does not participate in the chemical reaction, i.e., the biological valve material does not react with the double-bonded silk fibroin, nor does it react with the double-bonded reagent, nor does it participate in the polymerization reaction of the double-bonded silk fibroin and the double-bonded reagent. The polymer network formed does not have covalent connection with the biological valve material. Since the biological valve material does not participate in the polymerization reaction, there is no crosslinking of the biological valve material in the preparation method of the present application, and the prepared product is not a crosslinked biological valve, but an anticoagulation and endothelialization tissue engineering valve.

[0019] Compared with the prior art, the present application has the following advantages:

[0020] (1) The introduction of the silk fibroin into the tissue engineering valve material can promote the growth and proliferation of endothelial cells, thereby accelerating the endothelialization of the tissue engineering valve material. Moreover, the double-bonded reagents used all have good hydrophilicity. Through the in-situ polymerization of the double-bonded reagent and the double-bonded silk fibroin on the valve material, the introduction of the double-bonded reagent polymer can improve the hydrophilicity of the tissue engineering valve material, thereby inhibiting the adhesion of blood components on the tissue engineering valve material through the formation of a hydration layer and improving the anticoagulation performance of the tissue engineering valve material.

[0021] (2) Unlike the preparation of traditional biological heart valves, the chemical crosslinking of the valve material is not involved in the present application, i.e., no chemical reaction occurs between the valve material and the components. The present application aims to form a polymer network through the in-situ polymerization of the components on the valve material and the compounding of collagen on the valve material matrix, thereby obtaining the tissue engineering valve in which the components are compounded after polymerization.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0023] Figure 1 The process flow diagram for preparing the anticoagulant and endothelialization-promoting tissue-engineered valve material of this invention is shown in the figure.

[0024] Figure 2 This is a schematic diagram illustrating the principle of preparing coagulation-promoting endothelialization tissue-engineered valve materials according to the present invention.

[0025] Figure 3 This is a scanning electron microscope image of blood adhesion in the control group.

[0026] Figure 4 Scanning electron microscope image of blood adhesion in tissue-engineered valve 1 prepared in Example 1.

[0027] Figure 5 This is a fluorescence staining image of endothelial cells on the surface of the control group.

[0028] Figure 6 The image shows fluorescent staining of endothelial cells on the surface of the tissue-engineered valve 2 prepared in Example 2. Detailed Implementation

[0029] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0030] Figure 1 This is a flowchart illustrating the preparation process of the anticoagulant and endothelialization-promoting tissue-engineered valve material of the present invention. Figure 2 This is a schematic diagram illustrating the principle of preparing coagulation-promoting endothelialization tissue-engineered valve materials according to the present invention. Specific examples of the preparation method are as follows.

[0031] Example 1

[0032] A method for preparing an anticoagulant and endothelialization-promoting tissue-engineered valve material:

[0033] Freshly harvested porcine aortic valves were rinsed with physiological saline to remove surface residue;

[0034] Preparation of double-bonded silk fibroin: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, both at a concentration of 100 nM, were added to a 2% (w / w) silk fibroin solution. After stirring at room temperature for 2 hours, 2-aminoethyl methacrylate hydrochloride (concentration set at 100 nM) was added, the pH was adjusted to above 8, and the reaction was carried out for 24 hours. Finally, the double-bonded silk fibroin powder was obtained by dialysis.

[0035] The double-bonded silk fibroin powder is dissolved in PBS buffer, and the concentration of the double-bonded silk fibroin is set to be 0.5% (w / w); the porcine aortic valve is soaked in the solution at room temperature for 12 hours.

[0036] Then, polyethylene glycol diacrylate is added to the solution, and the concentration is set to be 2% (w / w), and the soaking is continued for 6 hours.

[0037] Then, ammonium persulfate and sodium sulfite are added to the solution, and the concentration is set to be 20 mM, and the reaction is carried out at 37°C for 8 hours; the porcine aortic valve is taken out and washed with PBS to obtain the tissue engineering valve 1.

[0038] Example 2

[0039] A preparation method of an anticoagulant and endothelium-promoting tissue engineering valve material:

[0040] The freshly removed porcine aortic valve is washed with physiological saline to remove surface residues.

[0041] Preparation of double-bonded silk fibroin: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are added to a silk fibroin solution with a concentration of 2% (w / w), and the concentration of each is 100 nM; after stirring at room temperature for 2 hours, 2-aminoethyl methacrylate hydrochloride is added (the concentration is set to be 100 nM), the pH is adjusted to be above 8, and the reaction is carried out for 24 hours; finally, the double-bonded silk fibroin powder is obtained after dialysis.

[0042] The double-bonded silk fibroin powder is dissolved in PBS buffer, and the concentration of the double-bonded silk fibroin is set to be 0.8% (w / w); at the same time, the concentration of the silk fibroin is set to be 1.5% (w / w), and the porcine aortic valve is soaked in the solution at room temperature for 12 hours.

[0043] Then, N-methyl-2-acrylamide is added to the solution, and the concentration is set to be 0.7% (w / w), and the soaking is continued for 6 hours.

[0044] Then, potassium persulfate (10 mM) and sodium bisulfite (8 mM) are added to the solution, and the reaction is carried out at 37°C for 8 hours; the porcine aortic valve is taken out and washed with PBS to obtain the tissue engineering valve 2.

[0045] Example 3

[0046] A preparation method of an anticoagulant and endothelium-promoting tissue engineering valve material:

[0047] The freshly removed porcine aortic valve is washed with physiological saline to remove surface residues.

[0048] Preparation of double-bonded silk fibroin: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide were added to a silk fibroin solution with a concentration of 2% (w / w), both at a concentration of 100 nM. After stirring at room temperature for 2 hours, 2-aminoethyl methacrylate hydrochloride was added (concentration set to 100 nM), and the pH was adjusted to 8 or above. The reaction was allowed to proceed for 24 hours, and the double-bonded silk fibroin powder was obtained after dialysis.

[0049] The double-bonded silk fibroin powder was dissolved in PBS buffer, and the concentration of double-bonded silk fibroin was set to 1% (w / w). The porcine aortic valve was immersed in the solution and treated at room temperature for 12 hours.

[0050] Then, polyethylene glycol diacrylate was added to the solution at a concentration of 2.5% (w / w), and the immersion was continued for 3 hours.

[0051] Potassium persulfate and sodium bisulfite were then added to the solution at a concentration of 20 mM, and the reaction was allowed to proceed at 37°C for 8 hours. The porcine aortic valve was removed and washed with PBS to obtain the tissue-engineered valve 3.

[0052] Example 4

[0053] A method for preparing an anticoagulant pro-endothelialization tissue-engineered valve material:

[0054] The freshly harvested porcine aortic valve was rinsed with physiological saline to remove surface residues.

[0055] Preparation of double-bonded silk fibroin: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide were added to a silk fibroin solution with a concentration of 2% (w / w), both at a concentration of 100 nM. After stirring at room temperature for 2 hours, 2-aminoethyl methacrylate hydrochloride was added (concentration set to 100 nM), and the pH was adjusted to 8 or above. The reaction was allowed to proceed for 24 hours, and the double-bonded silk fibroin powder was obtained after dialysis.

[0056] The double-bonded silk fibroin powder was dissolved in PBS buffer, and the concentration of double-bonded silk fibroin was set to 1.5% (w / w). The porcine aortic valve was immersed in the solution and treated at room temperature for 12 hours.

[0057] Then, N,N'-dimethylacrylamide was added to the solution at a concentration of 1.5% (w / w), and the immersion was continued for 1 hour.

[0058] Potassium persulfate and sodium bisulfite were then added to the solution at a concentration of 15 mM, and the reaction was allowed to proceed at 37°C for 6 hours. The porcine aortic valve was removed and washed with PBS to obtain the tissue-engineered valve 4.

[0059] Performance characterization:

[0060] (1) To characterize the changes of the anti-coagulation and pro-endothelization properties of the valve materials before and after the modification, the in vitro blood adhesion experiment was performed, and the scanning electron microscope was used to observe the images of the blood cell adhesion on the tissue engineering valve materials to characterize the improvement of the anti-coagulation properties before and after the modification. The fresh pig aortic valve was directly used as the control group after being washed by the saline. Figure 3 The scanning electron microscope picture of the in vitro blood adhesion experiment of the control group. Figure 4 The scanning electron microscope picture of the in vitro blood adhesion experiment of the tissue engineering valve 1 prepared in Example 1. It can be seen that the blood cells adhered on the tissue engineering valve 1 prepared by the method of the application are obviously less than those of the control group, which indicates that the tissue engineering valve 1 has the anti-coagulation property, that is, the tissue engineering valve material prepared by the application has the anti-coagulation property.

[0061] (2) The endothelial cell growth experiment was performed on the valve materials, and after being cultured for 3 days, the fluorescence staining of the endothelial cells adhered and grown on the valve was performed, and the fluorescence microscope was used to observe the adhesion of the endothelial cells on the tissue engineering valve to characterize the endothelization property of the tissue engineering valve. Figure 5 The fluorescence staining picture of the endothelial cells on the surface of the control group. Figure 6 The fluorescence staining picture of the endothelial cells on the surface of the tissue engineering valve 2 prepared in Example 2. It can be seen that the endothelial cells adhered on the tissue engineering valve 2 prepared in Example 2 after the co-culture are obviously more than those of the control group, that is, the tissue engineering valve 2 is beneficial to the growth and proliferation of the endothelial cells, which indicates that the tissue engineering valve material prepared by the application has the pro-endothelization property.

[0062] In summary, by introducing the silk fibroin with excellent biocompatibility and the polymer of the hydrophilic double bond reagent into the valve material, the unnecessary adhesion of the blood on the tissue engineering valve material can be reduced, and the anti-coagulation property of the tissue engineering valve can be improved by the complex of the polymer network and the collagen on the valve material matrix; the introduction of the silk fibroin can promote the growth and proliferation of the endothelial cells on the tissue engineering valve, and the endothelization property of the tissue engineering valve material can be improved.

[0063] The above description is only the preferred embodiment of the application, and does not limit the application in any form. Although the application has been disclosed as the above preferred embodiment, it is not intended to limit the application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content without departing from the technical solution of the application, and any simple modification, equivalent change and modification made on the basis of the technical essence of the application to the above embodiment are still within the scope of the technical solution of the application.

Claims

1. A method for preparing an anticoagulant and endothelialization-promoting tissue-engineered valve material, characterized in that, The steps are as follows: S1. Immerse the bio-valve material in a solution containing double-bonded silk fibroin to ensure that the valve material is in full contact with and adsorbs the components in the solution; the double-bonded silk fibroin is a modified silk fibroin containing carbon-carbon double bonds. S2. Add a double bond reagent to the solution from step S1 to fully disperse the double bond reagent, and continue to soak the valve material; the double bond reagent is selected from one or more of the following: polyethylene glycol diacrylate, 1,4-butanediol diacrylate, ethane-1,2-diyl diacrylate, ethyl acrylate, N-methyl-2-acrylamide, N-2,2-propenyl-2-acrylamide, N-ethylacrylamide, N,N'-vinylbisacrylamide, (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)diacrylate, N,N'-dimethylacrylamide, N,N-dimethylmethylacrylamide, acrylamide, 2-methoxyethyl acrylate, and double-bonded polylysine; S3. Add an initiator to the solution from step S2 to carry out a double bond polymerization reaction and prepare tissue-engineered valve material.

2. The method for preparing the anticoagulant and endothelialization-promoting tissue-engineered valve material as described in claim 1, characterized in that, The preparation method of the double-bonded silk fibroin is as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide were added to the original silk fibroin solution. After stirring at room temperature for 2 hours, 2-aminoethyl methacrylate hydrochloride was added to adjust the pH to ≥8. The reaction was continued for 24 hours, and finally dialyzed to obtain double-bonded silk fibroin powder.

3. The method for preparing the anticoagulant and endothelialization-promoting tissue-engineered valve material as described in claim 2, characterized in that, Step S1: The solution containing double-bonded silk fibroin has a mass percentage concentration of 0.5%-1.5% and a soaking time of 0.5-24 hours.

4. The method for preparing the anticoagulant and endothelialization-promoting tissue-engineered valve material as described in claim 3, characterized in that, Step S1: The solution containing double-bonded silk fibroin also contains the original silk fibroin.

5. The method for preparing the anticoagulant and endothelialization-promoting tissue-engineered valve material as described in claim 1, characterized in that, The bioprosthetic valve material is selected from animal tissues, including the pericardium, valves, intestinal membrane, meninges, pulmonary membrane, blood vessels, skin, amnion, or ligaments.

6. The method for preparing the anticoagulant and endothelialization-promoting tissue-engineered valve material as described in claim 1, characterized in that, In step S2, the mass percentage concentration of the double bond reagent is 1% to 6%, and the soaking time is 0.5 to 24 hours.

7. The method for preparing the anticoagulant and endothelialization-promoting tissue-engineered valve material as described in claim 1, characterized in that, In step S3, the initiator is selected from one of the following: a mixture of ammonium persulfate and sodium bisulfite, a mixture of ammonium persulfate and sodium bisulfite, a mixture of sodium persulfate and sodium bisulfite, a mixture of potassium persulfate and sodium bisulfite, a mixture of potassium persulfate and sodium bisulfite, a mixture of potassium persulfate and tetramethylethylenediamine, a mixture of ammonium persulfate and tetramethylethylenediamine, and a mixture of sodium persulfate and tetramethylethylenediamine.

8. A tissue-engineered valve material that promotes anticoagulation and endothelialization, characterized in that, It is prepared by the preparation method described in any one of claims 1-7.

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