Composite crosslinked bioprosthetic valve

Abstract

The present application belongs to the technical field of biomaterials, and in particular relates to a composite cross-linked biological valve. The preparation method of the composite cross-linked biological dry valve comprises the following steps: (1) soaking biomaterials in a cationic self-polymer solution at room temperature for 24-36 hours, and then pre-treating the solution by ultrasonic treatment at 25-35 DEG C for 30-40 hours; (2) soaking the pre-treated biomaterials in an active microgel solution for 10-20 hours, and then adding an aldehyde-based cross-linking agent into the system, and performing co-cross-linking at 30-40 DEG C for 20-30 hours; and (3) taking out the co-cross-linked biomaterials and performing freeze-drying to obtain a composite cross-linked biological valve. The cross-linked biological valve has good anti-calcification, and also has excellent anti-coagulation performance and biocompatibility.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a composite cross-linked biological valve. BACKGROUND

[0002] A human heart has four heart valves, which normally open and close in cooperation with the contraction of myocardium to make blood circulate in the whole body and maintain normal life activities. Valvular heart disease is the most common heart disease, and the number of patients diagnosed every year worldwide can reach about 100 million. The preferred treatment method for this disease is valve replacement.

[0003] Biological heart valves are usually prepared from porcine or bovine pericardium and used to replace the functionally defective human own heart valves; biological heart valves have many advantages compared with mechanical heart valves: patients do not need to take anticoagulants for a long time after implantation of biological heart valves, and biological heart valves can be used in minimally invasive surgical procedures. These advantages make biological heart valves gradually become the market mainstream in clinical application.

[0004] Almost all biological valve products on the current market are prepared by cross-linking with glutaraldehyde, which can cross-link collagen in pericardium. However, biological valves cross-linked with glutaraldehyde have an inescapable thrombosis problem, which seriously threatens the quality of life and life of patients.

[0005] Therefore, how to provide a new cross-linking method to provide a biological valve that meets the use needs is a technical problem to be solved by those skilled in the art. SUMMARY

[0006] The present application aims to overcome the problems in the prior art and provide a composite cross-linked biological valve. The composite cross-linked biological valve of the present application has good anti-calcification properties, and also has excellent anticoagulant properties and biocompatibility.

[0007] The object and technical problem of the present application are achieved by using the following technical solutions.

[0008] One aspect of the present application provides a preparation method of a composite cross-linked biological dry valve, comprising:

[0009] (1) the biomaterial is immersed in a cationic self-polymer solution at room temperature for 24-36 hours, and then the solution is warmed to 25-35°C and ultrasonicated for 30-40 hours for pretreatment; wherein the cationic self-polymer solution is prepared by the following method: a propenyl quaternary ammonium salt cationic monomer is added to deionized water to form an aqueous solution, nitrogen is introduced for protection, and the temperature of the system is adjusted to 5-10°C by ice bath or liquid nitrogen, 0.1wt% of a water-soluble oxidation-reduction system initiator is added, and solution self-polymerization is carried out for 1-1.5 hours, then the reaction system is warmed to 30-50°C and reacted for another 1-1.5 hours, and the cationic self-polymer solution is obtained after the reaction is completed;

[0010] (2) the pretreated biomaterial is immersed in an active microgel solution for 10-20 hours, and then an aldehyde-based crosslinking agent is added to the system and co-crosslinked at 30-40°C for 20-30 hours; wherein the active microgel solution is prepared by the following method: gelatin is added to a 5-10wt% dilute acid solution, and then N,N-methylenebisacrylamide, potassium permanganate and acrylamide are sequentially dissolved in water, and the solution is reacted at 50-70°C for 3-5 hours, and after the reaction is completed, the solution is filtered, and the obtained solution is the active microgel solution;

[0011] (3) the co-crosslinked biomaterial is taken out and freeze-dried to obtain a composite crosslinked biomaterial valve.

[0012] Further, in step (1), the propenyl quaternary ammonium salt cationic monomer is mixed with deionized water in a mass-volume ratio of 1g:5-9mL.

[0013] Further, in step (1), the propenyl quaternary ammonium salt cationic monomer is selected from any one or a mixture of two or more of dimethyldiallylammonium chloride, acryloyloxyethyltrimethylammonium chloride, dimethylaminoethyl methacrylate, methacryloyloxyethyltrimethylammonium chloride, and dimethylaminoethyl acrylate.

[0014] Further, in step (1), the oxidizing agent in the water-soluble oxidation-reduction system initiator includes hydrogen peroxide, persulfate, hydroperoxide, and the reducing agent includes ferrous sulfate and sodium sulfite.

[0015] Further, in step (2), the aldehyde-based crosslinking agent is glutaraldehyde or formaldehyde.

[0016] Further, in step (2), the active microgel solution and the aldehyde-based crosslinking agent are added in a volume ratio of 1:0.2-0.6.

[0017] Further, in step (2), the average molecular weight of the gelatin is 8×10 4 ~ 10×10 4 Da.

[0018] Further, in step (2), the gelatin, N,N-methylenebisacrylamide, potassium permanganate, acrylamide and dilute acid solution are mixed in a mass-volume ratio of 1g:0.03~0.05:0.01~0.03:0.03~0.05:20~40mL.

[0019] Furthermore, the acid mentioned in step (2) is selected from one of hydrochloric acid, phosphoric acid, acetic acid, citric acid, malic acid, fumaric acid, and tartaric acid.

[0020] Further, the freeze-drying conditions described in step (3) are: temperature -20 to -15°C, time 16 to 18 hours.

[0021] By employing the above technical solution, the present invention has at least the following advantages: The present invention uses a cationic self-polymer solution as a pretreatment solution for biomaterials. The cationic self-polymer uses propylene-based quaternary ammonium salt cations as monomers, and undergoes solution self-polymerization via a water-soluble redox initiator, introducing a large number of cations. By immersing the biomaterial in this solution and subjecting it to ultrasonic treatment at a certain temperature, a large number of positive charges can be carried on the surface of the biomaterial. Simultaneously, prolonged ultrasonic treatment allows the positive charges to be uniformly distributed on the surface of the biomaterial. As the main treatment agent for biomaterials, the active microgel solution differs from conventional gels in that it is a solution with a low gel concentration. The solution contains doped gels. Immersing biomaterials in this solution can maintain the surface charge properties of the biomaterials. After adding an aldehyde crosslinking agent, it can more easily undergo co-crosslinking with the aldehyde crosslinking agent, increasing the degree of crosslinking and improving the mechanical properties of the biomaterials. Simultaneously, the reaction environment of the microgel solution can also block some residual aldehyde groups on the biomaterials, improving their anti-calcification and anticoagulant properties, and further improving the crosslinking efficiency. The method of this invention is simple. The treatment with two solutions can enhance the physicochemical properties and biocompatibility of the valve, improve the service life of the valve, and at the same time enable the valve to maintain its flexibility in a dry state, thereby reducing the production and transportation costs of the valve and simplifying the valve usage process.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Implementation

[0023] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] Example 1: (1) The pig pericardium was soaked in a cationic self-polymer solution at room temperature for 30 h, and then the solution was heated to 30 °C and sonicated for 35 h for pretreatment. The cationic self-polymer solution was prepared as follows: the propylene-based quaternary ammonium salt cationic monomer dimethyl diallyl ammonium chloride was added to deionized water at a mass-volume ratio of 1 g: 7 mL to prepare an aqueous solution. Nitrogen gas was introduced for protection and the system temperature was adjusted to 8 °C using an ice bath. 0.1 wt% of a water-soluble redox initiator (hydrogen peroxide-ferrous sulfate) was added to carry out a solution self-polymerization reaction for 1.2 h. After that, the reaction system was heated to 40 °C and reacted for another 1.2 h. After the reaction was completed, the cationic self-polymer solution was obtained. (2) The pretreated pig pericardium was soaked in an active microgel solution for 15 h. Then, glutaraldehyde was added to the system at a volume ratio of 1:0.4 between the active microgel solution and the aldehyde crosslinking agent. Co-crosslinking was carried out at 35 °C for 25 h. The active microgel solution is prepared according to the following method: gelatin (average molecular weight 8×10⁻⁶) is used to prepare the microgel solution. 4 ~10×10 4 Da) was added to a dilute hydrochloric acid solution with a concentration of 8wt%, and then N,N-methylenebisacrylamide, potassium permanganate, and acrylamide were added sequentially and dissolved in water. The mixture was reacted at 60℃ for 4 hours. After the reaction was completed, the solution was filtered, and the resulting solution was the active microgel solution. The above gelatin, N,N-methylenebisacrylamide, potassium permanganate, acrylamide, and dilute hydrochloric acid solution were mixed in a mass-volume ratio of 1g:0.04:0.02:0.04:30mL. (3) The above co-crosslinked porcine pericardium was taken out and freeze-dried at -20℃ for 17 hours to obtain a composite crosslinked bio-valve.

[0025] Example 2: (1) The pig pericardium was soaked in a cationic self-polymer solution at room temperature for 36 h, and then the solution was heated to 25 °C and sonicated for 40 h for pretreatment. The cationic self-polymer solution was prepared as follows: Acrylonitrile quaternary ammonium salt cationic monomer acryloyloxyethyltrimethylammonium chloride was added to deionized water at a mass-volume ratio of 1 g: 9 mL to prepare an aqueous solution. Nitrogen gas was introduced for protection and the system temperature was adjusted to 10 °C using an ice bath. 0.1 wt% of a water-soluble redox initiator (hydrogen peroxide-sodium sulfate) was added to carry out a solution self-polymerization reaction for 1 h. After that, the reaction system was heated to 30 °C and reacted for another 1.5 h. After the reaction was completed, the cationic self-polymer solution was obtained. (2) The pretreated pig pericardium was soaked in an active microgel solution for 20 h. Then, glutaraldehyde was added to the system at a volume ratio of 1:0.6 between the active microgel solution and the aldehyde crosslinking agent. Co-crosslinking was carried out at 30 °C for 30 h. The active microgel solution is prepared according to the following method: gelatin (average molecular weight 8×10⁻⁶) is used to prepare the microgel solution. 4 ~10×10 4 Da) was added to a 10wt% dilute acetic acid solution, and then N,N-methylenebisacrylamide, potassium permanganate, and acrylamide were added sequentially and dissolved in water. The mixture was reacted at 50℃ for 5 hours. After the reaction was completed, the solution was filtered, and the resulting solution was the active microgel solution. The above gelatin, N,N-methylenebisacrylamide, potassium permanganate, acrylamide, and dilute acetic acid solution were mixed in a mass-volume ratio of 1g:0.05:0.01:0.03:40mL. (3) The above co-crosslinked porcine pericardium was taken out and freeze-dried at -15℃ for 18 hours to obtain a composite crosslinked bio-valve.

[0026] Example 3: (1) The pig pericardium was soaked in a cationic self-polymer solution at room temperature for 24 hours, and then the solution was heated to 35°C and sonicated for 30 hours for pretreatment. The cationic self-polymer solution was prepared as follows: the propylene-based quaternary ammonium salt cationic monomer dimethylaminoethyl methacrylate was added to deionized water at a mass-volume ratio of 1 g: 5 mL to prepare an aqueous solution. Nitrogen gas was introduced for protection and the system temperature was adjusted to 5°C using an ice bath. 0.1 wt% of a water-soluble redox initiator (hydrogen peroxide-ferrous sulfate) was added to carry out a solution self-polymerization reaction for 1.5 hours. Then the reaction system was heated to 50°C and reacted for another hour. After the reaction was completed, the cationic self-polymer solution was obtained. (2) The pretreated pig pericardium was soaked in an active microgel solution for 10 hours. Then glutaraldehyde was added to the system at a volume ratio of 1:0.2 between the active microgel solution and the aldehyde crosslinking agent. Co-crosslinking was carried out at 40°C for 20 hours. The active microgel solution is prepared according to the following method: gelatin (average molecular weight 8×10⁻⁶) is used to prepare the microgel solution. 4 ~10×10 4Da) was added to a 5 wt% dilute citric acid solution, and then N,N-methylenebisacrylamide, potassium permanganate, and acrylamide were added sequentially and dissolved in water. The mixture was reacted at 70°C for 3 h. After the reaction was completed, the solution was filtered, and the resulting solution was the active microgel solution. The above gelatin, N,N-methylenebisacrylamide, potassium permanganate, acrylamide, and dilute citric acid solution were mixed in a mass-volume ratio of 1 g: 0.03: 0.03: 0.05: 20 mL. (3) The above co-crosslinked porcine pericardium was taken out and freeze-dried at -20°C for 16 h to obtain a composite crosslinked bio-valve.

[0027] Example 4: (1) The pig pericardium was soaked in a cationic self-polymer solution at room temperature for 30 h, and then the solution was heated to 28 °C and sonicated for 34 h for pretreatment. The cationic self-polymer solution was prepared as follows: the propylene-based quaternary ammonium salt cationic monomer methacryloyloxyethyltrimethylammonium chloride was added to deionized water at a mass-volume ratio of 1 g: 6 mL to prepare an aqueous solution. Nitrogen gas was introduced for protection and the system temperature was adjusted to 7 °C using an ice bath. 0.1 wt% of a water-soluble redox initiator (hydrogen peroxide-sodium sulfate) was added to carry out a solution self-polymerization reaction for 1.3 h. After that, the reaction system was heated to 50 °C and reacted for another 1.3 h. After the reaction was completed, the cationic self-polymer solution was obtained. (2) The pretreated pig pericardium was soaked in an active microgel solution for 15 h. Then, glutaraldehyde was added to the system at a volume ratio of 1:0.6 between the active microgel solution and the aldehyde crosslinking agent, and co-crosslinking was carried out at 40 °C for 30 h. The active microgel solution is prepared according to the following method: gelatin (average molecular weight 8×10⁻⁶) is used to prepare the microgel solution. 4 ~10×10 4 Da) was added to a 7wt% dilute malic acid solution, and then N,N-methylenebisacrylamide, potassium permanganate, and acrylamide were added sequentially and dissolved in water. The mixture was reacted at 50°C for 5 hours. After the reaction was completed, the solution was filtered, and the resulting solution was the active microgel solution. The above gelatin, N,N-methylenebisacrylamide, potassium permanganate, acrylamide, and dilute malic acid solution were mixed in a mass-volume ratio of 1g:0.05:0.01:0.03:20mL. (3) The above co-crosslinked porcine pericardium was taken out and freeze-dried at -15°C for 16 hours to obtain a composite crosslinked bio-valve.

[0028] Comparative Example 1: (1) Porcine pericardium was soaked in an active microgel solution for 15 h, and then glutaraldehyde was added to the system at a volume ratio of 1:0.4 between the active microgel solution and the aldehyde crosslinking agent, and co-crosslinking was carried out at 35 °C for 25 h. The active microgel solution was prepared by the following method: gelatin (average molecular weight 8 × 10⁻⁶) was added to the solution. 4 ~10×10 4Da) was added to a dilute hydrochloric acid solution with a concentration of 8wt%, and then N,N-methylenebisacrylamide, potassium permanganate, and acrylamide were added sequentially and dissolved in water. The mixture was reacted at 60℃ for 4 hours. After the reaction was completed, the solution was filtered, and the resulting solution was the active microgel solution. The above gelatin, N,N-methylenebisacrylamide, potassium permanganate, acrylamide, and dilute hydrochloric acid solution were mixed in a mass-volume ratio of 1g:0.04:0.02:0.04:30mL. (3) The above co-crosslinked porcine pericardium was taken out and freeze-dried at -20℃ for 17 hours to obtain a composite crosslinked bio-valve.

[0029] Comparative Example 2: (1) The porcine pericardium was soaked in a cationic self-polymer solution at room temperature for 30 h, and then the solution was heated to 30 °C and sonicated for 35 h for pretreatment. The cationic self-polymer solution was prepared as follows: the propylene-based quaternary ammonium salt cationic monomer dimethyl diallyl ammonium chloride was added to deionized water at a mass-volume ratio of 1 g: 7 mL to prepare an aqueous solution. Nitrogen gas was introduced for protection and the system temperature was adjusted to 8 °C using an ice bath. 0.1 wt% of a water-soluble redox initiator (hydrogen peroxide-ferrous sulfate) was added to carry out a solution self-polymerization reaction for 1.2 h. Then the reaction system was heated to 40 °C and reacted for another 1.2 h. After the reaction was completed, the cationic self-polymer solution was obtained. (2) Glutaraldehyde was added to the above reaction system and co-crosslinked at 35 °C for 25 h. (3) The co-crosslinked porcine pericardium was taken out and freeze-dried at -20 °C for 17 h to obtain a composite crosslinked bio-valve.

[0030] Experimental Example 1: Enzyme Degradation Detection

[0031] Three samples of porcine pericardium from Examples 1-4 and Comparative Examples 1-2 were cut into 1×1cm pieces, for a total of 18 samples. The initial weight (W0) of each sample was recorded on a 0.0001 g balance and placed back into a 48-well plate. 0.5 mL of collagenase I PBS solution was added to each well of the 48-well plate using a pipette, ensuring the bioprosthetic valve sample was completely submerged in the collagenase PBS solution (100 U / mL). The 48-well plate was then transferred to a 37°C incubator for 24 hours. After incubation, the solution in the wells was discarded, and the sample was repeatedly agitated with deionized water using a dropper. After three such agitation cycles, the sample was frozen overnight at -80°C and then lyophilized in a vacuum freeze dryer for 48 hours. The final weight (W0) of each sample after collagenase degradation was recorded on a 0.0001 g balance. t The average value was taken, and the results are shown in Table 1. The formula for calculating the enzyme degradation weight loss rate is as follows:

[0032] Enzyme degradation weight loss rate = (W0 - W) t ) / W0×100%

[0033] Table 1. Statistics on the weight loss rate of enzyme degradation in different samples

[0034]

[0035] As can be seen from the results in Table 1, compared with comparative Examples 1-2, the enzymatic degradation weight loss rate of the composite cross-linked bioprosthetic valves obtained in Examples 1-4 of the present invention is reduced. This indicates that the samples in Examples 1-4 have high enzymatic degradation stability, i.e., high cross-linking efficiency during the preparation process. Therefore, it is evident that simultaneous treatment of biomaterials with the two solutions of the present invention can increase the degree of cross-linking of the biomaterials.

[0036] Experimental Example 2: Tests on lactate dehydrogenase activity, calcium uptake, and hemolysis rate

[0037] Fresh rabbit blood was collected and centrifuged at 1500 rpm for 15 min to obtain platelet-rich plasma. Materials from Examples 1-4 and Comparative Examples 1-2 were cut into 10 mm diameter discs, washed three times with PBS, placed in 48-well plates, and incubated at 37°C for 1 h with 100 μL of platelet-rich plasma. 100 μL of platelet-rich plasma was selected as a positive control for quantitative detection. After incubation, the plates were washed three times with PBS. The relative amount of platelet adhesion was determined using a lactate dehydrogenase assay kit. The absorbance at 490 nm was recorded for each group using a microplate reader, and the relative activity of lactate dehydrogenase for each group was calculated. The relative number of platelets was expressed as the relative activity of lactate dehydrogenase. The results are shown in Table 2.

[0038] Anti-calcification performance test: Samples from both the experimental and control groups were cleaned and cut into 1cm × 1cm pieces. Three male SD rats (45-50g each) were selected. The rats were anesthetized by intraperitoneal injection of 0.1mL of 3% sodium pentobarbital. The fur on the muscles along both sides of the spine was shaved, and the area was disinfected with iodine and alcohol. One sample from the experimental group (Example 1) was subcutaneously implanted on the right back, and one sample from the control group (Comparative Example 1) was subcutaneously implanted on the left back. The skin incision was sutured. Samples from Examples 2-4 and Comparative Example 2 were implanted into other rats using the same method. After 30 days, the grafts were removed. The host tissue on the surface of the grafts was carefully removed, and the grafts were rinsed with physiological saline. After freeze-drying, the dry weight was measured. Then, the grafts were digested with 6N concentrated hydrochloric acid in a 90°C water bath until no visible solid particles were found. Quantitative analysis of calcium was then performed using inductively coupled plasma atomic emission spectrometry (ICP-AES). The results are shown in Table 2.

[0039] Hemolysis rate test: Fresh rabbit blood was collected, centrifuged at 1500 rpm for 15 min, the supernatant was discarded, and red blood cells were collected. Samples from controls 1-2 and examples 1-4 were placed in 2 ml centrifuge tubes, and red blood cells were diluted with PBS (9 / 1, PBS / RBC) and incubated at 37°C for 1 hour. After centrifugation at 3000 rpm for 5 min, the supernatant was transferred to a 96-well plate. The absorbance at 545 nm was recorded using a microplate reader, and the hemolysis rate was calculated. The results are shown in Table 2.

[0040] Table 2. Detection of relative lactate dehydrogenase activity, calcium deposition, and hemolysis rate in different samples.

[0041]

[0042] As can be seen from the results in Table 2, compared with Comparative Examples 1-2, the relative activity of lactine dehydrogenase, calcium adsorption, and hemolysis rate of Examples 1-4 of the present invention were significantly reduced. Therefore, the method of the present invention can improve the hydrophilicity, blood compatibility, and anti-calcification ability of biomaterials, potentially extending their service life.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method of making a composite crosslinked biological dry valve, characterized in that, Comprise; (1) the biomaterial is immersed in a cationic self-polymer solution at room temperature for 24-36 h, and then the solution is warmed to 25-35 DEG C for 30-40 h under ultrasonic pretreatment; wherein the cationic self-polymer solution is prepared by the following method: a propylene quaternary ammonium salt cationic monomer is added to deionized water to configure an aqueous solution, nitrogen is introduced for protection, and the system temperature is adjusted to 5-10 DEG C by ice bath or liquid nitrogen, 0.1wt% water-soluble oxidation-reduction system initiator is added for solution self-polymerization reaction for 1-1.5 h, then the reaction system is warmed to 30-50 DEG C and reacted for another 1-1.5 h, and the cationic self-polymer solution is obtained after the reaction is completed; (2) the pretreated biomaterial is immersed in an active microgel solution for 10-20 h, and then an aldehyde crosslinking agent is added in the system, and co-crosslinking is carried out at 30-40 DEG C for 20-30 h; wherein the active microgel solution is prepared by the following method: gelatin is added to a 5-10wt% dilute acid solution, and then N,N-methylene bisacrylamide, potassium permanganate and acrylamide are sequentially dissolved in water, and reacted at 50-70 DEG C for 3-5 h, after the reaction is completed, the solution is filtered, and the obtained solution is the active microgel solution; (3) the co-crosslinked biomaterial is taken out and freeze-dried to obtain a composite crosslinked valve; In step (2), the gelatin, N,N-methylene bisacrylamide, potassium permanganate, acrylamide and dilute acid solution are mixed in a mass-volume ratio of 1g:0.03-0.05:0.01-0.03:0.03-0.05:20-40 mL.

2. The method of making a composite crosslinked biological dry valve of claim 1, wherein, In step (1), the propylene quaternary ammonium salt cationic monomer is mixed with deionized water in a mass-volume ratio of 1g:5-9 mL.

3. The method for preparing the composite cross-linked bio-valve membrane according to claim 1, characterized in that, In step (1), the propylene quaternary ammonium salt cationic monomer is selected from any one or a mixture of two or more of dimethyldiallylammonium chloride, acryloyloxyethyl trimethyl ammonium chloride and methacryloyloxyethyl trimethyl ammonium chloride.

4. The method of claim 1, wherein the composite crosslinked biologic dry valve is prepared by the steps of: In step (1), the oxidizing agent in the water-soluble oxidation-reduction system initiator includes hydrogen peroxide, persulfate and hydroperoxide, and the reducing agent includes ferrous sulfate and sodium sulfite.

5. The method for preparing the composite cross-linked bio-valve membrane according to claim 1, characterized in that, In step (2), the aldehyde crosslinking agent is glutaraldehyde or formaldehyde.

6. The method of claim 1, wherein the composite crosslinked biologic dry valve is prepared by the steps of: In step (2), the active microgel solution and the aldehyde crosslinking agent are added in a volume ratio of 1:0.2-0.

6.

7. The method of claim 1, wherein the composite crosslinked biologic dry valve is prepared by the steps of: The average molecular weight of the gelatin in step (2) is between 8 x 10 4 ~ 10 x 10 4 Da.

8. The method for preparing the composite cross-linked bio-valve membrane according to claim 1, characterized in that, In step (2), the acid is selected from one of hydrochloric acid, phosphoric acid, acetic acid, citric acid, malic acid, fumaric acid and tartaric acid.

9. The method for preparing the composite cross-linked bio-valve membrane according to claim 1, characterized in that, In step (3), the freeze-drying conditions are: temperature -20 to -15 DEG C, and time 16-18 h.

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