A hydrogel-modified biological valve and its preparation method

Abstract

The present application discloses a hydrogel-modified biological valve and a preparation method thereof. The preparation method includes: Step 1, crosslinking a biological valve with an aqueous glutaraldehyde solution to obtain a crosslinked membrane; Step 2, successively soaking the crosslinked membrane in an aqueous ethanol solution and an aqueous glycerol solution for drying treatment to obtain a dried membrane; Step 3, placing the dried membrane in a mixed solution and carrying out a polymerization reaction at 50-70 °C for 24-48 h to obtain a hydrogel-modified biological valve; the mixed solution uses water as a solvent, and the solutes include methacrylated gelatin, polyvinyl alcohol, acrylamide, N,N'-methylenebisacrylamide, an initiator, and an accelerator. The hydrogel-modified biological valve and the preparation method thereof provided by the present application utilize a variety of polymers to form a gel structure on the surface of the biological valve, effectively improving the mechanical properties of the biological valve, enhancing the anti-fatigue property, and not easily generating inflammation and thrombus.

Description

Technical Field

[0001] The present application relates to the technical field of medical materials, and in particular to a hydrogel-modified biological valve and a preparation method thereof. Background Art

[0002] There is no effective drug treatment for lesions such as aortic valve calcification. Aortic valve replacement can improve the patient's survival rate and quality of life, but due to the patient's own factors after implantation and problems with the valve manufacturing process, there is no guarantee that the biological valve can reach the expected service life.

[0003] The biological valves currently used in clinical practice are usually made of porcine pericardium or bovine pericardium cross-linked with glutaraldehyde. The presence of glutaraldehyde residues and aldehyde groups can lead to poor endothelialization, inflammatory reactions, and rough surfaces. After implantation in the body, thrombosis, thickening of the valve leaflets, and changes in mechanical properties are likely to occur due to the adhesion of platelets, cellulose, and the infiltration of blood cells and plasma components. Therefore, the processing methods of biological valves need to be further improved. Summary of the invention

[0004] Based on this, the present application provides a hydrogel-modified biological valve and a preparation method thereof, which utilizes a variety of polymers to form a gel structure on the surface of the biological valve, thereby effectively improving the mechanical properties of the biological valve, enhancing fatigue resistance, and making it less likely to cause inflammation and thrombosis.

[0005] A method for treating a biological valve, comprising:

[0006] Step 1, cross-linking the biological valve using a glutaraldehyde aqueous solution to obtain a cross-linked membrane sheet;

[0007] Step 2, soaking the cross-linked membrane in an ethanol aqueous solution and a glycerol aqueous solution in sequence for drying to obtain a dried membrane;

[0008] Step 3, placing the dried membrane in the mixed solution, polymerizing at 50-70° C. for 24-48 hours to obtain a hydrogel-modified bioprosthesis;

[0009] The mixed solution uses water as solvent, and the solutes include methacrylated gelatin, polyvinyl alcohol, acrylamide, N,N'-methylenebisacrylamide, an initiator and an accelerator.

[0010] In the present application, hydrogel modification is performed on the surface of the diaphragm after drying treatment, which can effectively improve the toughness of the diaphragm, improve the mechanical properties and fatigue resistance of the diaphragm, and extend the service life of the diaphragm.

[0011] The solvents in the mixed solution of the present application improve the performance of the membrane by at least the following interactions:

[0012] (1) Glycerol remains on the surface of the dried diaphragm. There is a hydrogen bond interaction between glycerol and polyvinyl alcohol (PVA). Under the bridging effect of glycerol, polyvinyl alcohol forms a physical connection with the surface of the dried diaphragm. At the same time, the local crystallized area in polyvinyl alcohol can play a role of physical cross-linking, thereby improving the mechanical properties of the dried diaphragm. Here, the physical cross-linking between glycerol and PVA in the dry film, i.e. the dry diaphragm, can effectively improve the formation of the network structure and the fatigue resistance of the dry diaphragm. It can be effectively used for pre-installation, etc., greatly improving the fatigue resistance of the dry diaphragm.

[0013] (2) See Figure 1 As shown, polyvinyl alcohol (PVA), acrylamide (AM), methacrylylated gelatin (GelMA) and N,N'-methylenebisacrylamide (MBAA) are soaked in one pot at the same time, which effectively allows acrylamide capable of double bond polymerization and cross-linking agent N,N'-methylenebisacrylamide (MBAA) to fully immerse into the internal structure of the membrane, and the double bond polymerization reaction occurs more fully under the conditions of initiator and promoter and high temperature. At the same time, in the mixed solution, polyvinyl alcohol, acrylamide and cross-linking agent N,N'-methylenebisacrylamide are fully mixed, acrylamide is polymerized, and the cross-linked polyacrylamide and polyvinyl alcohol obtained under the action of the cross-linking agent are interspersed with each other to form a double-network interpenetrating hydrogel system. There is a hydrogen bond intermolecular interaction between polyacrylamide and polyvinyl alcohol, which can effectively improve the mechanical properties of the membrane after drying.

[0014] (3) See Figure 2 As shown, at the same time, methacryloyl gelatin is the reaction product of gelatin and methacrylic anhydride. On the one hand, methacryloyl gelatin can react with the aldehyde groups remaining on the membrane. On the other hand, methacryloyl gelatin contains double bonds and can participate in the double bond polymerization reaction in (2), forming a chemical bond connection between the membrane and the hydrogel, so that the hydrogel can be more firmly attached to the membrane surface.

[0015] After the surface of the membrane is modified with hydrogel, the aldehyde residue on the surface of the membrane can be reduced, making it easier to endothelialize and less prone to inflammation. At the same time, the hydrogel also makes the surface of the biological valve smooth, and it is not easy to be adhered by platelets and cellulose and cause thrombosis after implantation. The valve also has good toughness and shows excellent performance in fatigue tests.

[0016] The polymerization reaction of the present application is carried out at 50-70°C. The membrane can be immersed in the mixed solution for a period of time at room temperature to allow the substances in the mixed solution to be evenly dispersed on the outer surface of the membrane. Physical crosslinking is first formed between polyvinyl alcohol and glycerol based on hydrogen bonding, and then the temperature is increased to initiate the subsequent double bond polymerization reaction.

[0017] Several optional methods are also provided below, but they are not intended to be additional limitations on the above-mentioned overall solution, but are merely further supplements or preferences. Under the premise that there are no technical or logical contradictions, each optional method can be combined with the above-mentioned overall solution separately, and multiple optional methods can also be combined.

[0018] Optionally, in step 1, cross-linking the biological valve using a glutaraldehyde aqueous solution includes: placing the biological valve in a 0.2-0.5 wt % glutaraldehyde aqueous solution for cross-linking for 24-72 hours.

[0019] When the biological valve is treated with glutaraldehyde aqueous solution, it is carried out at room temperature. Preferably, the concentration of the glutaraldehyde aqueous solution is 0.2-0.3wt%, the cross-linking time is 48h, and after the cross-linking is completed, it is washed several times with 0.9wt% saline.

[0020] Optionally, in step 2, a pretreatment step is further included before the cross-linked membrane is dried, and the pretreatment step includes:

[0021] The cross-linked membrane was immersed in an aqueous solution containing 1-3 wt% glutaraldehyde and 15-25 wt% isopropanol for 24-48 hours.

[0022] The pretreatment step is mainly used to reduce the bioburden. The cross-linked membrane is immersed in an aqueous solution containing glutaraldehyde and isopropanol. The pretreatment is carried out at room temperature. After the pretreatment, it is washed several times with 0.9wt% saline.

[0023] Optionally, in step 2, the concentration of the ethanol aqueous solution is 50-85wt%, and the immersion time in the ethanol aqueous solution is 20-60min; the concentration of the glycerol aqueous solution is 80-99wt%, and the immersion time in the glycerol aqueous solution is 1-24h.

[0024] Step 2 is a drying treatment, which is mainly to remove moisture from the membrane. After soaking in ethanol aqueous solution and glycerol aqueous solution in turn, the membrane after drying treatment is obtained. Step 2 is also carried out at room temperature.

[0025] Optionally, in the mixed solution, the concentration of methacryloyl gelatin is 10-20wt%, the degree of amino substitution in the methacryloyl gelatin is not more than 30%, and the molecular weight is 100-200kDa.

[0026] Optionally, in the mixed solution, the concentration of polyvinyl alcohol is 1-5wt%.

[0027] Optionally, in the mixed solution, the concentration of acrylamide is 30-50 wt %, and the concentration of N,N'-methylenebisacrylamide is 0.01-0.1 wt %.

[0028] Optionally, in step 3, the dried membrane is placed in the mixed solution and soaked at room temperature for 1 to 24 hours, and then the temperature is raised to 50 to 70° C. for polymerization reaction.

[0029] In step 3, the membrane is first immersed in the mixed solution at room temperature for a period of time, so that the substances in the mixed solution are evenly dispersed on the outer surface of the membrane, and the polyvinyl alcohol and glycerol first form physical crosslinks based on hydrogen bonds, and then the temperature is increased to initiate the polymerization reaction.

[0030] Optionally, the initiator in the mixed solution is ammonium persulfate, and the concentration of the initiator is 0.05-0.15wt%; the accelerator in the mixed solution is tetramethylethylenediamine, and the concentration of the accelerator is 0.05-0.15wt%.

[0031] The present application also provides a hydrogel-modified biological valve, which is obtained by treatment using the above-mentioned treatment method.

[0032] The hydrogel-modified biological valve and preparation method provided in the present application utilize a variety of polymers to form a gel structure on the surface of the biological valve, so that the mechanical properties of the biological valve are effectively improved, the fatigue resistance is enhanced, and it is not easy to cause inflammation and thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the formation of double network gel by polyvinyl alcohol, acrylamide and N,N'-methylenebisacrylamide;

[0034] Figure 2 is a schematic diagram of the structure of methacryloyl gelatin;

[0035] Figure 3 A schematic diagram of applying a biological valve to a stent;

[0036] Figure 4 Schematic diagram of the partial release of the valve stent from the sheath. DETAILED DESCRIPTION

[0037] Example 1

[0038] A method for treating a biological valve comprises the following steps:

[0039] (1) Cross-linking: Fresh porcine pericardium was cut into a certain size and placed in a 0.25wt% glutaraldehyde aqueous solution for cross-linking at room temperature for 48h. After cross-linking, it was washed three times with 0.9wt% saline;

[0040] (2) Reducing bioburden: The cross-linked membrane was placed in an aqueous solution containing 1 wt% glutaraldehyde and 20 wt% isopropanol and soaked at room temperature for 24 h. After soaking, it was washed three times with 0.9 wt% saline.

[0041] (3) Drying: After reducing the bioburden, the membrane was first placed in a 75 wt% ethanol aqueous solution and soaked at room temperature for 30 min, and then transferred to a 95 wt% glycerol aqueous solution and soaked at room temperature for 2 h;

[0042] (4) Polyvinyl alcohol (PVA) (Sigma-Aldrich.81381, weight average molecular weight of about 31000), acrylamide (AM), methacryloyl gelatin (GelMA, amino substitution degree 30%) and N,N'-methylenebisacrylamide (MBAA) were dissolved in deionized water to form a mixed solution, in which the concentration of polyvinyl alcohol was 2wt%, the concentration of acrylamide was 40wt%, the concentration of methacryloyl gelatin was 15wt%, and the concentration of N,N'-methylenebisacrylamide was 0.04wt%;

[0043] (5) Weigh a certain amount of ammonium persulfate (APS) and tetramethylethylenediamine (TMED) and dissolve them in deionized water, with ammonium persulfate serving as an initiator and tetramethylethylenediamine serving as an accelerator. Add the initiator and accelerator solutions to the mixed solution of step (4) and stir evenly. In the obtained mixed solution, the concentration of ammonium persulfate is 0.1 wt %, and the concentration of tetramethylethylenediamine is 0.1 wt %.

[0044] (6) The mixed solution of step (5) is transferred to a silicone mold that is of a size that matches the pig pericardium, and the membrane sheet that has been dried in step (3) is placed in the mixed solution. The mixture is polymerized at 60° C. for 24 hours, and then washed after solidification to obtain a hydrogel-modified biological heart valve.

[0045] Example 2

[0046] A method for treating a biological valve comprises the following steps:

[0047] (1) Cross-linking: Fresh porcine pericardium was cut into a certain size and placed in a 0.25wt% glutaraldehyde aqueous solution for cross-linking at room temperature for 48h. After cross-linking, it was washed three times with 0.9wt% saline;

[0048] (2) Reducing bioburden: The cross-linked membrane was placed in an aqueous solution containing 1 wt% glutaraldehyde and 20 wt% isopropanol and soaked at room temperature for 24 h. After soaking, it was washed three times with 0.9 wt% saline.

[0049] (3) Drying: After reducing the bioburden, the membrane was first placed in a 75 wt% ethanol aqueous solution and soaked at room temperature for 30 min, and then transferred to a 95 wt% glycerol aqueous solution and soaked at room temperature for 2 h;

[0050] (4) Polyvinyl alcohol (PVA) (Sigma-Aldrich.81381, weight average molecular weight of about 31000), acrylamide (AM), methacryloyl gelatin (GelMA, amino substitution degree 30%) and N,N'-methylenebisacrylamide (MBAA) were dissolved in deionized water to form a mixed solution, in which the concentration of polyvinyl alcohol was 1wt%, the concentration of acrylamide was 30wt%, the concentration of methacryloyl gelatin was 10wt%, and the concentration of N,N'-methylenebisacrylamide was 0.04wt%;

[0051] (5) Weigh a certain amount of ammonium persulfate (APS) and tetramethylethylenediamine (TMED) and dissolve them in deionized water, with ammonium persulfate serving as an initiator and tetramethylethylenediamine serving as an accelerator. Add the initiator and accelerator solutions to the mixed solution of step (4) and stir evenly. In the obtained mixed solution, the concentration of ammonium persulfate is 0.1 wt %, and the concentration of tetramethylethylenediamine is 0.1 wt %.

[0052] (6) The mixed solution of step (5) is transferred to a silicone mold that is of a size that matches the pig pericardium, and the membrane sheet that has been dried in step (3) is placed in the mixed solution. The mixture is polymerized at 50° C. for 24 hours, and then washed after solidification to obtain a hydrogel-modified biological heart valve.

[0053] Example 3

[0054] A method for treating a biological valve comprises the following steps:

[0055] (1) Cross-linking: Fresh porcine pericardium was cut into a certain size and placed in a 0.25wt% glutaraldehyde aqueous solution for cross-linking at room temperature for 48h. After cross-linking, it was washed three times with 0.9wt% saline;

[0056] (2) Reducing bioburden: The cross-linked membrane was placed in an aqueous solution containing 1 wt% glutaraldehyde and 20 wt% isopropanol and soaked at room temperature for 24 h. After soaking, it was washed three times with 0.9 wt% saline.

[0057] (3) Drying: After reducing the bioburden, the membrane was first placed in a 75 wt% ethanol aqueous solution and soaked at room temperature for 30 min, and then transferred to a 95 wt% glycerol aqueous solution and soaked at room temperature for 2 h;

[0058] (4) Polyvinyl alcohol (PVA) (Sigma-Aldrich.81381, weight average molecular weight of about 31000), acrylamide (AM), methacryloyl gelatin (GelMA, amino substitution degree 30%) and N,N'-methylenebisacrylamide (MBAA) were dissolved in deionized water to form a mixed solution, in which the concentration of polyvinyl alcohol was 3wt%, the concentration of acrylamide was 35wt%, the concentration of methacryloyl gelatin was 15wt%, and the concentration of N,N'-methylenebisacrylamide was 0.04wt%;

[0059] (5) Weigh a certain amount of ammonium persulfate (APS) and tetramethylethylenediamine (TMED) and dissolve them in deionized water, with ammonium persulfate serving as an initiator and tetramethylethylenediamine serving as an accelerator. Add the initiator and accelerator solutions to the mixed solution of step (4) and stir evenly. In the obtained mixed solution, the concentration of ammonium persulfate is 0.1 wt %, and the concentration of tetramethylethylenediamine is 0.1 wt %.

[0060] (6) The mixed solution of step (5) is transferred to a silicone mold that is of a size that matches the pig pericardium, and the membrane sheet that has been dried in step (3) is placed in the mixed solution. The mixture is polymerized at 60° C. for 24 hours, and then washed after solidification to obtain a hydrogel-modified biological heart valve.

[0061] Example 4

[0062] A method for treating a biological valve comprises the following steps:

[0063] (1) Cross-linking: Fresh porcine pericardium was cut into a certain size and placed in a 0.25wt% glutaraldehyde aqueous solution for cross-linking at room temperature for 48h. After cross-linking, it was washed three times with 0.9wt% saline;

[0064] (2) Reducing bioburden: The cross-linked membrane was placed in an aqueous solution containing 1 wt% glutaraldehyde and 20 wt% isopropanol and soaked at room temperature for 24 h. After soaking, it was washed three times with 0.9 wt% saline.

[0065] (3) Drying: After reducing the bioburden, the membrane was first placed in a 75 wt% ethanol aqueous solution and soaked at room temperature for 30 min, and then transferred to a 95 wt% glycerol aqueous solution and soaked at room temperature for 2 h;

[0066] (4) Polyvinyl alcohol (PVA) (Sigma-Aldrich.81381, weight average molecular weight of about 31000), acrylamide (AM), methacryloyl gelatin (GelMA, amino substitution degree 30%) and N,N'-methylenebisacrylamide (MBAA) were dissolved in deionized water to form a mixed solution, in which the concentration of polyvinyl alcohol was 5wt%, the concentration of acrylamide was 50wt%, the concentration of methacryloyl gelatin was 20wt%, and the concentration of N,N'-methylenebisacrylamide was 0.06wt%;

[0067] (5) Weigh a certain amount of ammonium persulfate (APS) and tetramethylethylenediamine (TMED) and dissolve them in deionized water, with ammonium persulfate serving as an initiator and tetramethylethylenediamine serving as an accelerator. Add the initiator and accelerator solutions to the mixed solution of step (4) and stir evenly. In the obtained mixed solution, the concentration of ammonium persulfate is 0.1 wt %, and the concentration of tetramethylethylenediamine is 0.1 wt %.

[0068] (6) The mixed solution of step (5) is transferred to a silicone mold that is of a size that matches the pig pericardium, and the membrane sheet that has been dried in step (3) is placed in the mixed solution. The mixture is polymerized at 70° C. for 48 hours, and then washed after solidification to obtain a hydrogel-modified biological heart valve.

[0069] Comparative Example 1

[0070] A method for treating a biological valve comprises the following steps:

[0071] (1) Cross-linking: Fresh porcine pericardium was cut into a certain size and placed in a 0.25wt% glutaraldehyde aqueous solution for cross-linking at room temperature for 48h. After cross-linking, it was washed three times with 0.9wt% saline;

[0072] (2) Reducing bioburden: The cross-linked membrane was placed in an aqueous solution containing 1 wt% glutaraldehyde and 20 wt% isopropanol and soaked at room temperature for 24 h. After soaking, it was washed three times with 0.9 wt% saline.

[0073] (3) Drying: After reducing the bioburden, the membrane was first placed in a 75 wt% ethanol aqueous solution and soaked at room temperature for 30 min, and then transferred to a 95 wt% glycerol aqueous solution and soaked at room temperature for 2 h.

[0074] Performance Characterization

[0075] The bioprosthetic valves after treatment in each embodiment and comparative example were characterized as follows:

[0076] Accelerated aging testing is used to reflect the performance of the product after real-time aging, thereby determining the shelf life.

[0077] According to the accelerated aging theory, the accelerated aging temperature is set to 60℃, and the calculated accelerated time is 66 days, which is equivalent to 24 months in actual time; the accelerated time is 99 days, which is equivalent to 36 months in actual time. Therefore, the samples are subjected to accelerated aging tests at 60℃ for 0 days, 66 days, and 99 days, and the samples are tested for breaking force, breaking elongation, and fatigue performance after each stage.

[0078] The test methods for breaking force and breaking elongation are as follows:

[0079] (1) Use a laser cutting machine to cut the membrane into 10×50 (mm) rectangular pericardial strip samples;

[0080] (2) Fix both ends of the sample on the tensile tester and stretch it with the set parameters (spacing 25 mm, stretching rate 100 mm / min), and measure the corresponding breaking force and breaking elongation.

[0081] Fatigue performance test According to the requirements of ISO-5840, a special fixture is customized to perform fatigue performance test on valve products. The structure of valve products can be found in Figure 3 As shown, the valve product 100 includes a stent 120 and a valve leaflet 130. The biological valve is processed and used as the valve leaflet 130. The valve product 100 is compressible. It is loaded into the sheath tube 200 in a compressed state, and is released from the sheath tube after being delivered to the target site through the sheath tube 200. Figure 4 As shown, the distal end of valve product 100 is released from sheath 200 .

[0082] The breaking force and breaking elongation of each embodiment and comparative example at time 0 are shown in Table 1.

[0083] Table 1

[0084]

[0085] The breaking force, breaking elongation and fatigue times of each embodiment and comparative example at 66 days are shown in Table 2.

[0086] Table 2

[0087]

[0088] The breaking force, breaking elongation and fatigue times of each embodiment and comparative example at 99 days are shown in Table 3.

[0089] Table 3

[0090]

[0091] According to the characterization results in Tables 1 to 3, the present application utilizes hydrogel modification to significantly improve the mechanical properties and fatigue resistance of the valve, which is beneficial to extending the shelf life of the product.

[0092] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0093] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for preparing a hydrogel-modified biological valve, characterized in that: include: Step 1, cross-linking the biological valve using a glutaraldehyde aqueous solution to obtain a cross-linked membrane sheet; Step 2, soaking the cross-linked membrane in an ethanol aqueous solution and a glycerol aqueous solution in sequence for drying to obtain a dried membrane; Step 3, placing the dried membrane in the mixed solution, polymerizing at 50-70° C. for 24-48 hours to obtain a hydrogel-modified bioprosthesis; The mixed solution uses water as solvent, and the solutes include methacrylated gelatin, polyvinyl alcohol, acrylamide, N,N'-methylenebisacrylamide, an initiator and an accelerator; In the mixed solution, the concentration of methacrylated gelatin is 10-20wt%; In the mixed solution, the concentration of polyvinyl alcohol is 1-5wt%; In the mixed solution, the concentration of acrylamide is 30-50 wt %, and the concentration of N,N'-methylenebisacrylamide is 0.01-0.1 wt %.

2. The method for preparing a hydrogel-modified biological valve according to claim 1, characterized in that: In step 1, cross-linking the biological valve using a glutaraldehyde aqueous solution includes: placing the biological valve in a 0.2-0.5 wt % glutaraldehyde aqueous solution for cross-linking for 24-72 hours.

3. The method for preparing a hydrogel-modified biological valve according to claim 1, characterized in that: In step 2, a pretreatment step is also included before the cross-linked membrane is dried, and the pretreatment step includes: The cross-linked membrane was immersed in an aqueous solution containing 1-3 wt% glutaraldehyde and 15-25 wt% isopropanol for 24-48 hours.

4. The method for preparing a hydrogel-modified biological valve according to claim 1, characterized in that: In step 2, the concentration of the ethanol aqueous solution is 50-85wt%, the immersion time in the ethanol aqueous solution is 20-60min, the concentration of the glycerol aqueous solution is 80-99wt%, and the immersion time in the glycerol aqueous solution is 1-24h.

5. The method for preparing a hydrogel-modified biological valve according to any one of claims 1 to 4, characterized in that: In step 3, the dried membrane is placed in the mixed solution and soaked at room temperature for 1 to 24 hours, and then the temperature is raised to 50 to 70° C. for polymerization reaction.

6. The method for preparing a hydrogel-modified biological valve according to any one of claims 1 to 4, characterized in that: The initiator in the mixed solution is ammonium persulfate, and the concentration of the initiator is 0.05-0.15wt%; the accelerator in the mixed solution is tetramethylethylenediamine, and the concentration of the accelerator is 0.05-0.15wt%.

7. A hydrogel-modified bioprosthesis, characterized in that: The method is prepared according to any one of claims 1 to 6.

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