Biological valve treatment method and biological valve dry piece

Abstract

The invention relates to the technical field of biomedical treatment, and particularly discloses a biological valve treatment method and a biological valve dry piece. The bioprosthetic valve treatment method provided by the invention comprises the following steps: pretreatment, crosslinking, degreasing, anti-calcification and drying. In the anti-calcification step, an anti-calcification solution is used for treatment; the anti-calcification solution is a 0.08 to 0.12 M PBS (Phosphate Buffer Solution) containing 0.5 to 6 weight percent of ethanolamine and 0.1 to 0.5 weight percent of sodium borohydride; furthermore, the anti-calcification solution is a 0.08 to 0.12 M PBS (Phosphate Buffer Solution) containing 2 to 4 weight percent of ethanolamine and 0.2 to 0.4 weight percent of sodium borohydride; the invention further provides the bioprosthetic valve dry piece obtained through the bioprosthetic valve processing method. According to the bioprosthetic valve treatment method, the anti-calcification performance of the bioprosthetic valve can be improved, and the obtained bioprosthetic valve dry piece has good stability and storage performance.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a method for processing a biological valve and a biological valve dry sheet. Background Art

[0002] Heart valve disease is an important part of cardiovascular disease, and the traditional treatment method is to use mechanical valves or biological valves for replacement surgery. Among them, mechanical valves have good durability and stability, but patients need to take anticoagulants for a long time, and there is a high risk of thrombosis; in contrast, biological valves are more widely used in clinical practice because they are closer to the function and structure of natural valves in the human body. However, biological valves do not perform well in terms of durability and are prone to calcification, which affects their normal function. Therefore, researchers are constantly exploring new treatment methods and technologies to improve the performance and service life of biological valves.

[0003] At present, the main treatment method for biological valves is to reduce the immunogenicity of tissues and enhance the mechanical strength of tissues through cross-linking with glutaraldehyde. However, the above treatment method also has obvious shortcomings; first, glutaraldehyde, as a strong cross-linking agent, will produce a large amount of residual substances inside the tissues. These residues may irritate the human body and even induce inflammatory reactions. Secondly, the aldehyde groups that are not fully involved in cross-linking will gradually oxidize to form carboxyl groups, which are easily combined with calcium ions in the blood, leading to tissue calcification, seriously affecting the long-term stability and functionality of the valve. In addition, biological valves treated with glutaraldehyde usually need to be stored in glutaraldehyde or formaldehyde solutions, which not only increases the cost of storage and transportation, but may also cause further chemical changes, affecting the quality and safety of the valve.

[0004] Therefore, it is particularly urgent to develop a new bioprosthetic valve treatment method that can effectively reduce calcification and facilitate storage. Summary of the invention

[0005] In order to overcome the problems existing in the existing biological valve processing methods, such as easy calcification of the valve, difficulty in storage and transportation, etc., the present application provides a biological valve processing method and a biological valve dry sheet.

[0006] In a first aspect, the present application provides a method for treating a biological valve, which adopts the following technical solution: A method for processing a biological valve comprises the following steps: pretreatment, cross-linking, degreasing, anti-calcification, and drying; The anti-calcification step uses an anti-calcification solution for treatment; the anti-calcification solution is a 0.08-0.12M PBS solution containing 0.5-6wt% ethanolamine and 0.1-0.5wt% sodium borohydride.

[0007] The present application provides a method for treating a biological valve, which, through pretreatment, cross-linking, degreasing and anti-calcification steps, effectively removes residual cells and calcification sites in the tissue while improving the mechanical properties of the biological valve, significantly improves the anti-calcification performance of the biological valve and reduces the risk of immune response. Specifically: The present application cross-links the biological valve to effectively improve the mechanical properties of the biological valve and reduce the risk of damage caused by mechanical stress during its use in the body; then removes the phospholipid calcification sites on the biological valve tissue by degreasing to avoid calcification during use; then uses a specific concentration of ethanolamine and sodium borohydride to treat the degreased biological valve to eliminate the residual free aldehyde groups in the system, on the one hand to reduce cytotoxicity, on the other hand to eliminate aldehyde calcification sites, and reduce the risk of biological valve calcification; finally, after drying, a biological valve dry sheet is obtained, which not only maintains good mechanical properties, but also has better stability and storage, which is very conducive to storage and transportation.

[0008] In the anti-calcification solution of the present application, ethanolamine can cap unreacted aldehyde and acid groups, improve the biocompatibility and durability of tissues, and reduce the risk of calcification and other adverse reactions. Sodium borohydride can reduce the aldehyde groups remaining in the cross-linked tissue to alcohol groups, cap calcification active sites such as aldehyde groups, and improve the stability and durability of biological valves.

[0009] Optionally, the anti-calcification solution is a 0.08-0.12 M PBS solution containing 2-4 wt % ethanolamine and 0.2-0.4 wt % sodium borohydride.

[0010] In some embodiments, the content of ethanolamine in the anti-calcification solution can be 0.5-2wt%, 0.5-3wt%, 0.5-4wt%, 0.5-6wt%, 2-3wt%, 2-4wt%, 2-6wt%, 3-4wt%, 3-6wt% or 4-6wt%.

[0011] In a specific embodiment, the content of ethanolamine in the anti-calcification solution may also be 0.5wt%, 2wt%, 3wt%, 4wt% or 6wt%.

[0012] In some embodiments, the content of sodium borohydride in the anti-calcification solution can be 0.1-0.2wt%, 0.1-0.3wt%, 0.1-0.4wt%, 0.1-0.5wt%, 0.2-0.3wt%, 0.2-0.4wt%, 0.2-0.5wt%, 0.3-0.4wt%, 0.3-0.5wt% or 0.4-0.5wt%.

[0013] In a specific embodiment, the content of sodium borohydride in the anti-calcification solution may also be 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt% or 0.5 wt%.

[0014] Optionally, the anti-calcification solution is a 0.1 M PBS solution containing 3 wt % ethanolamine and 0.3 wt % sodium borohydride.

[0015] Optionally, the specific step of anti-calcification is: washing the cross-linked pericardial tissue with saline, and then placing it in an anti-calcification solution for 1-10 hours to obtain a biological valve.

[0016] Optionally, the specific steps of defatting are: firstly immersing the cross-linked pericardial tissue in a 0.08-0.12M PBS solution containing 0.1-1wt% SDS and 0.1-1wt% glutaraldehyde for 1-2 days, and then immersing it in a 0.08-0.12M PBS solution containing ≤80wt% alcohol reagent for 1-3 days to obtain the defatted pericardial tissue.

[0017] In the present application, since the pericardial tissue contains a small amount of phospholipids, the phospholipids will combine with the calcium ions in the tissue through electrostatic action, thereby causing tissue calcification; and the present application can effectively remove the phospholipid components in the pericardial tissue by degreasing the cross-linked pericardial tissue, reduce the phospholipid calcification sites, and improve the anti-calcification performance of the biological valve. Specifically: isopropyl alcohol and ethanol can destroy the cell membrane structure and fully release the phospholipids into the solution; in addition, alcohol reagents can also reduce the dielectric constant of water and reduce the electrostatic repulsion between phospholipid molecules, making them easier to aggregate and be separated and cleaned, thereby achieving the purpose of complete removal.

[0018] Optionally, the alcohol reagent includes ethanol and isopropanol, the content of the isopropanol is 10-50wt%, and the content of the ethanol is 30-70wt%.

[0019] In some embodiments, the isopropanol content may be 10-20 wt%, 10-30 wt%, 10-40 wt%, 10-50 wt%, 20-30 wt%, 20-40 wt%, 20-50 wt%, 30-40 wt%, 30-50 wt% or 40-50 wt%.

[0020] In a specific embodiment, the content of isopropanol can also be 10 wt%, 20 wt%, 30 wt%, 40 wt% or 50 wt%.

[0021] In some embodiments, the ethanol content may be 30-40wt%, 30-50wt%, 30-60wt%, 30-70wt%, 40-50wt%, 40-60wt%, 40-70wt%, 50-60wt%, 50-70wt% or 60-70wt%.

[0022] In a specific embodiment, the ethanol content may also be 30 wt%, 40 wt%, 50 wt%, 60 wt% or 70 wt%.

[0023] Optionally, the content of isopropanol is 20-40wt%, and the content of ethanol is 40-60wt%.

[0024] Optionally, the specific step of cross-linking is: cutting the pretreated pericardial tissue into flat sheets and fixing them, and then placing them in a 0.08-0.12M PBS solution containing 0.3-1wt% glutaraldehyde for cross-linking for 1-6 days to obtain cross-linked pericardial tissue.

[0025] Optionally, the specific step of drying is: soaking the biological valve after anti-calcification treatment in a glycerol-ethanol mixture for 4-48 hours, and obtaining a biological valve dry sheet after drying; the volume ratio of the glycerol-ethanol mixture is (50-75): (25-50).

[0026] In a specific embodiment, the volume ratio of the glycerol-ethanol mixture is 70:30.

[0027] Optionally, the specific steps of the pretreatment are: obtaining fresh animal pericardial tissue, removing fat, washing it with physiological saline, and storing it in saline at 4-10°C.

[0028] In a second aspect, the present application provides a biological valve dry sheet obtained by processing using the biological valve processing method.

[0029] In summary, this application has the following beneficial effects: 1. The present application provides a method for processing a biological valve, which pre-treats, cross-links, degreases and anti-calcifies fresh animal pericardial tissue in sequence. This method can improve the mechanical properties of the biological valve while effectively removing residual cells and calcification sites in the tissue, thereby significantly improving the anti-calcification performance of the biological valve and reducing the risk of immune response.

[0030] 2. The present application further controls the contents of ethanolamine and sodium borohydride in the anti-calcification solution within the following range: 2-4wt% ethanolamine, 0.2-0.4wt% sodium borohydride. The obtained anti-calcification solution has fewer residual aldehyde groups in the pericardial tissue after treatment, and the obtained biological valve has a better anti-calcification effect.

[0031] 3. The present application uses alcohol reagents to degrease the pericardial tissue, which can effectively remove the phospholipids in the potential calcification sites in the pericardial tissue, thereby improving the anti-calcification effect of the biological valve; through experimental research, it was found that the present application further controls the content of isopropanol and ethanol in the degreasing step within the following range: 20-40wt% isopropanol, 40-60wt% ethanol, which can reduce the phospholipid content in the pericardial tissue and improve the anti-calcification effect of the resulting biological valve. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a flow chart of the method for processing a biological valve provided in this application. DETAILED DESCRIPTION

[0033] The present application provides a method for treating a biological valve, comprising the following steps: (1) Pretreatment: obtain fresh animal pericardial tissue, remove fat, wash with physiological saline, remove residual blood, and store in saline at 4-10°C; (2) Cross-linking: The pre-treated bovine pericardial tissue is cut into flat sheets and fixed, and then placed in a 0.08-0.12 M PBS solution containing 0.3-1 wt % glutaraldehyde for cross-linking for 1-6 days to obtain cross-linked pericardial tissue; (3) Degreasing: the cross-linked pericardial tissue is first immersed in a 0.08-0.12M PBS solution containing 0.1-1wt% SDS and 0.1-1wt% glutaraldehyde for 1-2 days, and then immersed in a 0.08-0.12M PBS solution containing ≤80wt% alcohol reagent for 1-3 days to obtain the defatted pericardial tissue; wherein the alcohol reagent includes ethanol and isopropanol, the content of the isopropanol is 10-50wt%, and the content of the ethanol is 30-70wt%; further, the content of the isopropanol is 20-40wt%, and the content of the ethanol is 40-60wt%; (4) Anti-calcification: The cross-linked pericardial tissue is washed with saline, and then placed in an anti-calcification solution for 1-10 hours to obtain a biological valve; the anti-calcification solution is a 0.08-0.12M PBS solution containing 0.5-6wt% ethanolamine and 0.1-0.5wt% sodium borohydride; further, the anti-calcification solution is a 0.08-0.12M PBS solution containing 2-4wt% ethanolamine and 0.2-0.4wt% sodium borohydride; (5) Drying: The bioprosthesis after anti-calcification treatment is immersed in a glycerol-ethanol mixture with a volume ratio of (50-75): (25-50) for 4-48 hours, and then dried to obtain a bioprosthesis dry sheet.

[0034] The animal pericardial tissue used in the examples of the present application is bovine pericardium, and the raw materials, reagents, solvents, etc. used in the present application can all be obtained commercially.

[0035] The present application is further described in detail below in combination with embodiments, performance testing and accompanying drawings.

[0036] Example 1 Embodiment 1 provides a method for treating a biological valve, comprising the following steps: (1) Pretreatment: Obtain fresh bovine pericardium from a slaughterhouse, remove the fat, wash it with saline, remove residual blood, and store it in saline at 4-10°C; (2) Cross-linking: The pretreated bovine pericardial tissue was cut into flat slices and fixed, and then placed in a 0.1 M PBS solution containing 1 wt % glutaraldehyde for cross-linking for 3 days to obtain cross-linked pericardial tissue; (3) Degreasing: The cross-linked pericardial tissue was first immersed in a 0.1 M PBS solution containing 1 wt% SDS and 1 wt% glutaraldehyde for 1 day, and then immersed in a 0.1 M PBS solution containing 30 wt% isopropanol and 40 wt% ethanol for 2 days to obtain the defatted pericardial tissue; (4) Anti-calcification: The defatted pericardial tissue was washed with saline, then placed in an anti-calcification solution for 8 hours to obtain a bioprosthesis; the anti-calcification solution was a 0.1 M PBS solution containing 3 wt% ethanolamine and 0.3 wt% sodium borohydride; (5) Drying: The bioprosthesis after anti-calcification treatment is immersed in a glycerol-ethanol mixture with a volume ratio of 70:30 for 24 hours, and then dried to obtain a bioprosthesis dry sheet.

[0037] Embodiment 2-9 Embodiments 2-9 each provide a method for treating a biological valve.

[0038] The difference between the above embodiment and embodiment 1 is that the concentrations of ethanolamine and sodium borohydride in the anti-calcification solution in step (4) are specifically shown in Table 1 below.

[0039] Table 1 Concentrations of ethanolamine and sodium borohydride in the anti-calcification solution in step (4) of Example 2-9 Examples 10-17 Embodiments 10-17 each provide a method for treating a biological valve.

[0040] The difference between the above embodiment and embodiment 1 is that the concentrations of isopropanol and ethanol in step (3) are specifically shown in Table 2 below.

[0041] Table 2 Concentrations of isopropanol and ethanol in step (3) of Examples 10-17 Comparative Example 1 Comparative Example 1 provides a method for treating a biological valve.

[0042] The difference between the comparative example and Example 1 is that: (4) the anti-calcification step is as follows: (4) Anti-calcification: The cross-linked pericardial tissue was washed with saline and then placed in an anti-calcification solution for 8 hours to obtain a biological valve; the anti-calcification solution was a 0.1 M PBS solution containing 3 wt % ethanolamine.

[0043] Comparative Example 2 Comparative Example 2 provides a method for treating a biological valve.

[0044] The difference between the comparative example and Example 1 is that: (4) the anti-calcification step is as follows: (4) Anti-calcification: The cross-linked pericardial tissue was washed with saline and then placed in an anti-calcification solution for 8 hours to obtain a biological valve; the anti-calcification solution was a 0.1 M PBS solution containing 0.3 wt % sodium borohydride.

[0045] Comparative Example 3 Comparative Example 3 provides a method for treating a biological valve.

[0046] The difference between the comparative example and Example 1 is that the bioprosthetic valve treatment method does not include a degreasing step, which is as follows: (1) Pretreatment: Obtain fresh bovine pericardium from a slaughterhouse, remove the fat, wash it with saline, remove residual blood, and store it in saline at 4-10°C; (2) Cross-linking: The pretreated bovine pericardial tissue was cut into flat slices and fixed, and then placed in a 0.1 M PBS solution containing 1 wt % glutaraldehyde for cross-linking for 3 days to obtain cross-linked pericardial tissue; (3) Anti-calcification: The cross-linked pericardial tissue was washed with saline, then placed in an anti-calcification solution for 8 hours to obtain a biological valve; the anti-calcification solution was a 0.1 M PBS solution containing 3 wt% ethanolamine and 0.3 wt% sodium borohydride; (4) Drying: The bioprosthesis after anti-calcification treatment is immersed in a glycerol-ethanol mixture with a volume ratio of 70:30 for 24 hours, and then dried to obtain a bioprosthesis dry sheet.

[0047] Comparative Example 4 Comparative Example 4 provides a method for treating a biological valve.

[0048] The difference between the comparative example and Example 1 is that the method for treating the biological valve does not include an anti-calcification step, which is as follows: (1) Pretreatment: Fresh bovine pericardial valves were obtained from a slaughterhouse, the fat was removed, and the valves were washed with saline to remove residual blood, and then stored in saline at 4-10°C; (2) Cross-linking: The pretreated bovine pericardial tissue was cut into flat slices and fixed, and then placed in a 0.1 M PBS solution containing 1 wt % glutaraldehyde for cross-linking for 3 days to obtain cross-linked pericardial tissue; (3) Degreasing: The cross-linked pericardial tissue was first immersed in a 0.1 M PBS solution containing 1 wt% SDS and 1 wt% glutaraldehyde for 1 day, and then immersed in a 0.1 M PBS solution containing 30 wt% isopropanol and 40 wt% ethanol for 2 days to obtain the defatted pericardial tissue; (4) Drying: The defatted bioprosthesis is immersed in a glycerol-ethanol mixture with a volume ratio of 70:30 for 24 hours, and then dried to obtain a bioprosthesis dry sheet.

[0049] Comparative Example 5 Comparative Example 5 provides a method for treating a biological valve.

[0050] The difference between the comparative example and Example 1 is that the bioprosthetic valve treatment method does not include the defatting and anti-calcification steps, which are as follows: (1) Pretreatment: Fresh bovine pericardial valves were obtained from a slaughterhouse, the fat was removed, and the valves were washed with saline to remove residual blood, and then stored in saline at 4-10°C; (2) Cross-linking: The pretreated bovine pericardial tissue was cut into flat slices and fixed, and then placed in a 0.1 M PBS solution containing 1 wt % glutaraldehyde for cross-linking for 3 days to obtain cross-linked pericardial tissue; (3) Drying: The cross-linked pericardial tissue is immersed in a glycerol-ethanol mixture with a volume ratio of 70:30 for 24 hours, and then dried to obtain a biological valve dry sheet.

[0051] Performance testing The results of the performance testing of the biological valve dry sheets obtained in Examples 1-17 and Comparative Examples 1-5 are shown in Table 3 below.

[0052] (1) Evaluation of anti-calcification effect: According to the rat subcutaneous implantation test for anti-calcification evaluation of animal-derived cardiovascular implants in accordance with YY / T 1859-2022, the anti-calcification effect of the biological valve stem sheets provided in Examples 1-17 and Comparative Examples 1-5 was tested, and the untreated bovine pericardial valve was used as a blank control; the calcium content of each biological valve stem sheet after the rat subcutaneous implantation test was detected, and the calcification rate of the biological valve stem sheets provided in Examples 1-17 and Comparative Examples 1-5 was calculated; the calculation method of the calcification rate is as follows: Calcification rate = calcium content of biological valve stem after implantation / calcium content of untreated bovine pericardial valve after implantation × 100%.

[0053] (2) Tensile strength: Samples were randomly cut from the bioprosthetic valve stems, each with a length of 50 mm and a width of 10 mm. The test gauge length was set to 25 mm, and the tensile strength of each bioprosthetic valve stem was tested using a constant rate elongation tester.

[0054] Table 3 Performance test results of biological valve stem sheets obtained in Examples 1-17 and Comparative Examples 1-5 / Calcification rate (%) Tensile strength(MPa) Example 1 1.05 19.7 Example 2 4.25 20.2 Example 3 2.38 21.3 Example 4 1.96 20.5 Example 5 3.61 22.3 Example 6 3.85 20.4 Example 7 1.57 21.5 Example 8 2.22 20.6 Example 9 3.08 22.1 Example 10 2.65 21.7 Embodiment 11 1.74 21.2 Example 12 1.28 20.5 Embodiment 13 1.22 22.0 Embodiment 14 1.57 19.8 Embodiment 15 2.38 21.7 Example 16 1.87 22.0 Embodiment 17 2.41 21.5 Comparative Example 1 20.63 20.8 Comparative Example 2 15.25 22.0 Comparative Example 3 8.46 21.0 Comparative Example 4 28.81 20.8 Comparative Example 5 36.77 19.9 According to the test results in Table 3, the calcification rate of the biological valve dry sheets provided in Examples 1-17 of the present application is only 1.05-4.25%, and the tensile strength is 19.7-22.3MPa, indicating that the present application can effectively reduce the calcification sites in the tissue and improve the anti-calcification performance of the biological valve by degreasing the cross-linked pericardial tissue and using 0.08-0.12M PBS solution containing 0.5-6wt% ethanolamine and 0.1-0.5wt% sodium borohydride to perform anti-calcification treatment on the biological valve.

[0055] Comparative Examples 1-2 use a 0.1M PBS solution containing only 3wt% ethanolamine or a 0.1M PBS solution containing only 0.3wt% sodium borohydride as an anti-calcification solution. The calcification rate of the obtained biological valve dry sheet is as high as 15.25-20.63%, indicating that the anti-calcification effect of treating the biological valve with only a PBS solution containing ethanolamine or sodium borohydride as an anti-calcification solution is not good. A large number of calcification sites still remain in the treated tissue, and the anti-calcification effect of the obtained biological valve is not good.

[0056] The calcification rate of the biological valve dry sheet obtained in Comparative Example 3 without degreasing treatment is as high as 8.46%; this is because the method provided in Comparative Example 3 will cause a small amount of phospholipid calcification sites to remain in the tissue, thereby making the anti-calcification effect of the obtained biological valve slightly poor.

[0057] The calcification rate of the biological valve dry sheet obtained in Comparative Example 4 without anti-calcification treatment is as high as 28.81%; this is because the method provided in Comparative Example 4 will cause a large number of free aldehyde groups to remain in the tissue, and the aldehyde groups are easily oxidized into carboxyl groups, which then combine with calcium ions in the tissue to cause tissue calcification, making the anti-calcification effect of the biological valve poor.

[0058] The calcification rate of the biological valve dry sheet obtained in Comparative Example 5 without anti-calcification and degreasing treatment was as high as 36.77%; this was because the method provided in Comparative Example 5 did not remove the free aldehyde groups and phospholipids remaining in the tissue after cross-linking, and the tissue contained a large number of calcification sites, resulting in poor anti-calcification effect of the biological valve.

[0059] The test results of Examples 1-9 show that the calcification rate of the biological valve dry pieces obtained in Examples 2, 5-6, and 9 is 3.08-4.25% (>3%); while the calcification rate of the biological valve dry pieces obtained in Examples 1, 3-4, and 7-8 is only 1.05-2.38% (≤3%). Therefore, it is explained that the present application further controls the content of ethanolamine and sodium borohydride in the anti-calcification solution within the following range: 2-4wt% ethanolamine, 0.2-0.4wt% sodium borohydride, and the obtained anti-calcification solution has less residual aldehyde groups in the pericardial tissue after treatment, and the obtained biological valve has a better anti-calcification effect.

[0060] The test results of Example 1 and Examples 10-16 show that the present application uses a 0.08-0.12M PBS solution with an isopropanol content of 10-50wt% and an ethanol content of 30-70wt% to degrease the pericardial tissue after crosslinking, which can effectively remove the phospholipids in the pericardial tissue and improve the anti-calcification effect of the biological valve. Further comparison found that the calcification rate of the biological valve dry sheet obtained in Example 10, Example 15, and Example 17 was 2.38-2.65% (>2%); while the calcification rate of the biological valve dry sheet obtained in Example 1, Examples 11-14, and Example 16 was only 1.05-1.87% (≤2%). Therefore, it is explained that the present application further controls the content of isopropanol and ethanol in the degreasing step within the following range: 20-40wt% isopropanol, 40-60wt% ethanol, which can make the phospholipids in the pericardial tissue after treatment less, and the anti-calcification effect of the obtained biological valve is better.

[0061] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for treating a biological valve, characterized in that: The following steps are involved: Pretreatment, cross-linking, degreasing, anti-calcification, drying; The anti-calcification step uses an anti-calcification solution for treatment; the anti-calcification solution is a 0.08-0.12M PBS solution containing 0.5-6wt% ethanolamine and 0.1-0.5wt% sodium borohydride.

2. The method for treating a biological valve according to claim 1, characterized in that: The anti-calcification solution is a 0.08-0.12 M PBS solution containing 2-4 wt % ethanolamine and 0.2-0.4 wt % sodium borohydride.

3. The method for treating a biological valve according to claim 1, characterized in that: The anti-calcification solution is a 0.1 M PBS solution containing 3 wt % ethanolamine and 0.3 wt % sodium borohydride.

4. The method for treating a biological valve according to claim 1, characterized in that: The specific steps of anti-calcification are: washing the cross-linked pericardial tissue with saline, and then placing it in an anti-calcification solution for 1-10 hours to obtain a biological valve.

5. The method for treating a biological valve according to claim 1, characterized in that: The specific steps of degreasing are: firstly immersing the cross-linked pericardial tissue in a 0.08-0.12M PBS solution containing 0.1-1wt% SDS and 0.1-1wt% glutaraldehyde for 1-2 days, and then immersing it in a 0.08-0.12M PBS solution containing ≤80wt% alcohol reagent for 1-3 days to obtain the defatted pericardial tissue.

6. The method for treating a biological valve according to claim 5, characterized in that: The alcohol reagent includes ethanol and isopropanol, the content of the isopropanol is 10-50 wt %, and the content of the ethanol is 30-70 wt %.

7. The method for treating a biological valve according to any one of claims 1 to 6, characterized in that: The specific steps of cross-linking are: cutting the pretreated pericardial tissue into flat sheets and fixing them, and then placing them in a 0.08-0.12M PBS solution containing 0.3-1wt% glutaraldehyde for cross-linking for 1-6 days to obtain cross-linked pericardial tissue.

8. The method for treating a biological valve according to any one of claims 1 to 6, characterized in that: The specific steps of drying are: soaking the biological valve after anti-calcification treatment in a glycerol-ethanol mixture for 4-48 hours, and obtaining a biological valve dry sheet after drying; the volume ratio of the glycerol-ethanol mixture is (50-75): (25-50).

9. The method for treating a biological valve according to any one of claims 1 to 6, characterized in that: The specific steps of the pretreatment are: obtaining fresh animal pericardial tissue, removing fat, washing it with physiological saline, and storing it in saline at 4-10°C.

10. A biological valve stem, characterized in that: The bioprosthesis is obtained by the processing method of any one of claims 1 to 9.

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