Preparation method and application of bioprosthetic valve material with anti-calcification, anti-thrombus and immune safety

Abstract

The invention relates to a preparation method and application of a bioprosthetic valve material with anti-calcification, anti-thrombus and immune safety at the same time, and the preparation method mainly comprises the following steps: firstly, carrying out decellularization treatment on a pericardium material, placing the pericardium material in a glutaraldehyde solution to carry out crosslinking treatment after the treatment is completed, and after the crosslinking is completed, carrying out freeze drying to obtain the bioprosthetic valve material with anti-calcification, anti-thrombus and immune safety. And transferring the crosslinked pericardium material into a sodium thiosulfate solution for modification treatment to obtain the bioprosthetic valve material. According to the preparation method, residual aldehyde groups after glutaraldehyde crosslinking can be eliminated, the biocompatibility and immune safety of the valve material are improved, and the mechanical stability of the valve is not influenced. Materials tests and cell experiments prove that the bioprosthetic valve material has good biocompatibility and anti-calcification performance, promotes cell adhesion, proliferation and cell endothelialization effects, and shows application potential in the field of tissue regeneration.

Description

Technical Field

[0001] The present invention discloses a preparation method and application of a biological valve material with anti-calcification, anti-thrombosis and immune safety at the same time, belonging to the technical field of medical devices. Background Art

[0002] Calcific aortic valve disease (CAVD) is an important cause leading to valvular stenosis and heart failure in patients. It is reported that more than 65% of the population over 40 years old has valvular calcification lesions, and it has gradually become an important factor inducing cardiogenic death. At present, there is no treatment method to reverse valvular calcification, but mechanical or biological valve implants can be used for surgical replacement of the dysfunctional calcified valve. Compared with mechanical heart valves (MHV), biological heart valves (BHV) have excellent hemodynamic performance, do not require lifelong anticoagulants, biocompatibility and rapid postoperative recovery. However, recent clinical studies have shown that bioprosthetic valves may calcify, significantly increasing the risk of death, especially for relieving the symptoms of end-stage diseases, further posing challenges to valve modification for anti-calcification.

[0003] Biological valve calcification is usually caused by the continuous deposition and crystallization of calcium salts in the valve tissue, and this deposition may ultimately lead to valvular stenosis. At present, the interventional biological valves used clinically generally adopt glutaraldehyde-crosslinked biological valve materials. Studies have shown the aldehyde group residues after glutaraldehyde crosslinking of valve materials and their associated cytotoxicity and immune inflammation. The bioprosthetic leaflet tissue mainly includes components such as collagen. Glutaraldehyde binds to collagen in the form of Schiff base to improve the durability of the valve. However, with the prolongation of the implantation time, the residual aldehyde group residues in the valve will reduce the biological safety of the material, such as increasing the risk of platelet adsorption and inducing thrombosis, and at the same time will inhibit the endothelialization effect of valve cells and is prone to induce immune inflammation (such as macrophage and T cell infiltration), resulting in protein degradation in the valve, further affecting the valve structure and then promoting the calcification of the implanted valve, significantly shortening the service life of the valve and threatening the patient's life safety for the second time. Therefore, eliminating the residual aldehyde group has become an important challenge in clinical valve use. Studies have shown that amino acids, sodium borohydride, etc. have the potential to promote the elimination of aldehyde groups (Journal of Materials Chemistry B, 2022, 10, 5571–5581; The Journal of heart valve disease, 2004, 13(3): 487). However, the current methods are complex and difficult to overcome the problems of thrombosis formation, low mechanical and structural stability, and easy calcification of the valve.

[0004] In summary, developing a biological valve material with anticoagulation, anti-calcification, endothelialization promotion, biocompatibility and immune safety at the same time is of great significance for the clinical use of interventional biological heart valves and improving the quality of life of patients. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the present invention provides a preparation method and application of a biological valve material that simultaneously has anti-calcification, anti-thrombosis, and immune safety. The present invention crosslinks the pericardial material with glutaraldehyde to improve the durability of the biological valve material. On this basis, the reducing property of sodium thiosulfate is used to completely eliminate the residual aldehyde groups after glutaraldehyde crosslinking, thereby achieving the purpose of anti-calcification.

[0006] To achieve the above object, the following technical solutions are provided herein:

[0007] The present invention first provides a preparation method of a biological valve material that simultaneously has anti-calcification, anti-thrombosis, and immune safety, which includes the following steps:

[0008] 1) Perform decellularization treatment on the pericardial material. After the treatment is completed, place the pericardial material in a glutaraldehyde solution for crosslinking;

[0009] 2) After crosslinking is completed, transfer the crosslinked pericardial material to a sodium thiosulfate solution for modification treatment to obtain a biological valve material that simultaneously has anti-calcification, anti-thrombosis, and immune safety.

[0010] Preferably, during decellularization treatment, first treat with sodium dodecyl sulfate and Triton X-100 solution, and after washing with PBS, then treat with deoxyribonuclease and ribonuclease; wherein, the mass concentration of the used sodium dodecyl sulfate and Triton X-100 solution is 0.5%-2%, the deoxyribonuclease concentration is 0-10U / mL, and the ribonuclease concentration is 0-100mg / mL.

[0011] Preferably, the pericardial material is selected from one or more of porcine pericardium, bovine pericardium, equine pericardium, ovine pericardium, ostrich pericardium, etc.

[0012] Preferably, the mass percentage concentration of the glutaraldehyde solution is 0.5-2%, and the pH of the glutaraldehyde solution is 6.0-7.0. Further preferably, the crosslinked pericardial material obtained in step 1) can be stored for standby in a low-concentration glutaraldehyde solution, and the mass percentage concentration of the low-concentration glutaraldehyde solution is 0.1-0.3%.

[0013] In the crosslinking conditions, the thickness of the pericardial material is 0.1-5mm, the ratio of the material volume to the solution volume is 1:2 to 1:300, or the ratio of the material mass (g) to the solution input (mL) is 1:10 to 1:1000. Preferably, the thickness of the pericardial material is 0.1-2mm, the ratio of the material volume to the solution volume is 1:2 to 1:100, or the ratio of the material mass (g) to the solution input (mL) is 1:20 to 1:200.

[0014] Preferably, the concentration of sodium thiosulfate is 50 - 200 mM, and the time for the modification treatment is 1 - 3 days.

[0015] During the modification treatment with sodium thiosulfate, the thickness of the pericardial material is 0.1 - 5 mm, the ratio of the material volume to the solution volume is 1:2 to 1:500, or the ratio of the material mass (g) to the solution dosage (mL) is 1:10 to 1:1000. Preferably, the thickness of the membranous material is 0.1 - 2 mm, the ratio of the material volume to the solution volume is 1:5 to 1:50, or the ratio of the material mass (g) to the solution dosage (mL) is 1:20 to 1:300.

[0016] Preferably, after decellularization treatment, crosslinking treatment, and modification treatment, the obtained material is rinsed with sterile normal saline.

[0017] Preferably, according to the preferred embodiment of the present invention, after obtaining the biological valve material modified with sodium thiosulfate and rinsing it, it is transferred to a sterile PBS buffer solution for storage for later use, and the pH of the sterile PBS buffer solution is 7.2 - 7.4.

[0018] Another object of the present invention is to provide a biological valve material having anti - calcification, anti - thrombosis, and immune safety simultaneously, which is prepared by the foregoing preparation method.

[0019] The present invention also provides the application of the foregoing biological valve material in being used as or preparing artificial biological valves and cardiovascular tissue repair materials.

[0020] Preferably, the artificial biological valve is a mitral valve leaf or a tricuspid valve leaf. The biological valve material obtained by the method of the present invention has low immunogenicity, does not cause a large immune response, and has good anticoagulant performance, which is beneficial to inhibiting the formation of thromboembolism. Therefore, it not only has good biocompatibility and anti - calcification performance, but also can promote cell adhesion and proliferation, showing application potential in the field of tissue regeneration, and is particularly suitable for preparing mitral valve leaf, tricuspid valve leaf, and cardiovascular tissue repair materials.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The preparation method of a biological valve material having anti - calcification, anti - thrombosis, and immune safety simultaneously according to the present invention uses sodium thiosulfate to eliminate the residual aldehyde groups after glutaraldehyde crosslinking. Compared with sodium borohydride, it improves the structural stability and mechanical stability of the pericardial material, and at the same time improves the biocompatibility of the valve material, realizes the immune safety of the material, and can significantly improve the endothelialization effect and anti - calcification performance when it is used as the heart valve leaf material. Description of the Drawings

[0023] Figure 1 Characterization diagrams of the modified crosslinked bioprosthetic valve materials in Example 1 and Example 2.

[0024] Figure 2 Mechanical property diagrams of SPP and the control group in Example 3.

[0025] Figure 3 Stability diagrams of SPP and the control group in Example 3.

[0026] Figure 4 SEM diagrams and ICP diagrams after 7-day in vitro calcification of SPP and the control group in Example 4.

[0027] Figure 5 SEM diagrams of SPP and the control group after 15-day and 30-day in vitro calcification in Example 4.

[0028] Figure 6 Alizarin red staining and ICP diagrams of SPP and the control group after 30 days in Example 4.

[0029] Figure 7 Cell compatibility diagrams of SPP and the control group in Example 5.

[0030] Figure 8 Blood compatibility diagrams of SPP and the control group in Example 6.

[0031] Figure 9 Section staining and in vivo calcification diagrams of SPP and the control group in Example 7.

[0032] Figure 10 Immunohistochemical analysis diagrams of SPP and the control group in Example 8. Specific implementation manners

[0033] The present invention will be further specifically described below through examples, but the present invention is not limited to the following examples.

[0034] Example 1: Preparation of a bioprosthetic valve material by modifying with sodium thiosulfate

[0035] (1) Select fresh porcine pericardium, rinse the fresh porcine pericardium 3 times with physiological saline for 5 minutes each time, and gently remove the surface fat.

[0036] (2) Decellularization treatment of porcine pericardium: Treat the porcine pericardium with 1% (w / v) sodium dodecyl sulfate (SDS) for 6 h, and then treat it with 1% (w / v) Triton X-100 for 0.5 h. After washing with PBS, treat it with 2 U / mL deoxyribonuclease and 20 mg / mL ribonuclease at 37 °C for 24 h. The decellularization results are as Figure 1 shown in a. As can be seen from the figure, the cell removal of the porcine pericardium is relatively complete.

[0037] (3) The acellular porcine pericardium DPP after decellularization treatment was placed in a glutaraldehyde solution for preliminary crosslinking. At a temperature of 37 °C, the crosslinking time was 2 days, and the mass concentration of the glutaraldehyde solution was 0.625%.

[0038] (4) After the preliminary crosslinking was completed, the biological valve material GPP in the glutaraldehyde solution was taken out and divided into four groups, which were respectively placed in the prepared 50 mM, 100 mM, 200 mM sodium thiosulfate solutions and 100 mM sodium borohydride solution for further modification experiments; the biological valve materials SPP 50 (50 mM STS), SPP100 (100 mM STS), SPP200 (200 mM STS) modified by sodium thiosulfate and the biological valve material BPP modified by sodium borohydride were obtained respectively. Among them, SPP 50, SPP100, and SPP200 were all materials prepared by the method of the present invention, which simultaneously had the characteristics of anti-calcification, anti-thrombosis, and immune safety. Such materials were also referred to as SPP hereinafter. The biological valve material BPP modified by sodium borohydride was used as a control in subsequent experiments.

[0039] Example 2: Characterization of biological valve materials prepared by sodium thiosulfate modification

[0040] Residual aldehyde group detection test: The DPP, GPP, SPP50, SPP100, and SPP200 pericardium samples were all cut into 1 cm × 1 cm pericardium samples, and then washed 3 times with deionized water. After washing, the pericardium samples were immersed in 5 mL of Schiff reagent and left standing at room temperature for 10 minutes. After the reaction, the Schiff reagent reacted with the aldehyde group to form a red or purple-red compound, and the samples were evaluated by visual observation. The results are as Figure 1 shown in b. It can be seen from the figure that after SPP100 treatment, compared with SPP50 and SPP200, it showed a lighter purple color, and SPP100 completely eliminated the residual aldehyde group after glutaraldehyde crosslinked the pericardium.

[0041] Scanning electron microscope (SEM): The DPP, GPP, SPP50, SPP100, and SPP200 pericardium samples were thoroughly rinsed with deionized water, flattened, and freeze-dried. Take a small amount of the freeze-dried pericardium samples, lay their fiber surfaces upward on the conductive adhesive, fix them on the sample stage, and then gold-plate the pericardium samples. Use a vacuum pump to pump out the air in the SEM sample chamber until the required vacuum degree is reached. Turn on the electron gun of the SEM and perform preheating treatment to ensure the normal operation of the electron gun. Finally, set appropriate parameters such as the acceleration voltage of the electron beam, scanning speed, working distance, etc. Through the detection of the scanning electron microscope (SEM), the tightness and integrity of the five groups of pericardium materials in the organizational structure were compared. The results are as Figure 1As shown in c, it shows that STS can successfully eliminate the residual aldehyde groups in GPP without affecting the microstructure of porcine pericardium.

[0042] Determination of amino content: Cut the DPP, GPP, SPP50, SPP100, and SPP200 pericardial samples into pieces of 5 mm × 5 mm, and then put them into centrifuge tubes. Add a total of 2 mL of ninhydrin solution (1 mL of 1% w / v ninhydrin, 1 mL of 0.1 M sodium citrate, pH = 5.4) to each tube. Then heat the test tubes in an oil bath at 100 °C. After incubation for 20 minutes, cool the test tubes to room temperature and collect the supernatant. Subsequently, measure the absorbance (OD) at 570 nm using a multifunctional microplate reader. Calculate the relative amino content according to the following formula: Relative amino content = (OD s - OD0) / (OD p - OD0) × 100%.

[0043] Using fresh pericardium as a control sample, normalize the percentage of amine content to the percentage of amine content. ODp is the absorbance of the reaction between fresh pericardium and ninhydrin, OD s is the absorbance of the remaining samples, and OD0 is the absorbance of the blank control group. The results are as Figure 1 shown in d. The amino content in the GPP and SPP groups decreased significantly, indicating that the glutaraldehyde cross-linking treatment was relatively successful.

[0044] Fourier transform infrared (FTIR) spectroscopy test: Thoroughly rinse the DPP, GPP, SPP50, SPP100, and SPP200 pericardial materials with deionized water and freeze-dry them, and then use a Fourier transform infrared spectrometer (FTIR) to confirm the complete elimination of residual aldehyde groups. Record the spectra in the wavelength range of 4000 - 400 cm-1 using the attenuated total reflection mode. Identify the functional groups on the surface of the pericardial samples by analyzing the characteristic absorption peaks in the infrared spectra. The results are as Figure 1 shown in e. The disappearance of the peak at 1744 cm -1 in SPP100 further confirmed the successful reduction of the residual aldehyde groups in GPP, which is consistent with the Figure 1 results in b.

[0045] Example 3: Stability test of the bio-valve material prepared by sodium thiosulfate modification

[0046] Uniaxial tensile test: To evaluate the mechanical properties of crosslinked PP, samples in the BPP, GPP, and SPP groups were cut into rectangles (1 cm × 5 cm), soaked in PBS to align the fiber orientation with the loading direction, the thickness (δ) was measured with a thickness gauge, and the samples were placed in a uniaxial tensile testing machine. To prevent the samples from slipping, the two ends of the samples were fixed with sandpaper. The initial distance between the two clamps was set to 20 mm, and the samples were loaded at a constant rate of 10 mm / min until the samples were pulled out with a uniaxial tensile tester. The results are as Figure 2 shown. After glutaraldehyde crosslinking, the ultimate tensile strength and elastic modulus of GPP and SPP were higher than those of BPP.

[0047] In vitro collagenase degradation test: The pericardial samples before and after sodium thiosulfate modification were cut into 1 cm × 1 cm sizes, thoroughly rinsed with deionized water, freeze-dried, and weighed. The initial weight of each sample was recorded as W0. The samples were placed in 1.5 mL of Tris buffer (0.05 M Tris-HCl, 1.5 mM CaCl2, pH = 7.4) containing collagenase (150 U mL -1 ) and incubated at 37 °C for 24 h. After incubation, the samples were rinsed with deionized water, freeze-dried, and weighed (recorded as W t ). The weight loss fraction was calculated using the following formula: Weight loss ratio (%) = (W0 - W t ) / W0 × 100%. As Figure 3 shown in a, b, the results showed that the collagen loss rate of DPP was significantly higher than that of the GPP and SPP groups, indicating that GPP and SPP had higher collagen stability.

[0048] Thermal stability analysis: To investigate the thermal stability of crosslinked modified pericardium, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used to perform thermal analysis on the freeze-dried samples. The specific operation steps were as follows: Accurately weigh 5 - 10 mg of the sodium thiosulfate-modified sample of the present invention and place it in a standard aluminum sealed crucible, equilibrate at 30 °C, and then evaluate it by DSC at a heating rate of 10 °C / min in the range of 30 °C - 110 °C under a nitrogen atmosphere, and evaluate it by TGA at a heating rate of 20 °C / min in the range of 30 °C - 800 °C under a nitrogen atmosphere. Record the thermal denaturation temperature and thermogravimetric data of the first heating curve of the sample. The results are as Figure 3 shown in c, d. The average shrinkage temperature of SPP (87 °C) was close to that of GPP (86 °C), which was significantly higher than the shrinkage temperature of DPP (69 °C).

[0049] Example 4: In vitro calcification test

[0050] Take the 1 cm × 1 cm sodium thiosulfate-modified pericardial slices processed in the above Example 1, immerse the DPP, GPP, BPP, and SPP samples into the prepared calcification solution respectively, and perform a water bath at 100 rpm for 72 h in a 37 °C constant temperature water bath. After 72 h, change the calcification solution and continue the reaction according to the above reaction conditions to allow the pericardial slices to react with calcium ions in the solution. Samples are taken on the 7th, 15th, and 30th days, slowly rinsed with deionized water on the surface and fixed with 4% paraformaldehyde for 24 h, and then freeze-dried. In order to visually observe the calcification morphology on the surface of the pericardium, a scanning electron microscope (SEM) is used to observe the microscopic morphology of the pericardium. In addition, in order to further analyze the elemental composition of the calcified substances, we combine an energy dispersive spectrometer (EDS) to preliminarily analyze the Ca element content distribution in the samples. As Figure 4 shown in a, b, the in vitro calcification results at 7 days show that the calcification degree of BPP is significantly higher than that of SPP. As Figure 5 、 Figure 6 shown, it is found that a relatively serious calcification phenomenon appears in the GPP cross-linked group, while there is no obvious calcified area on the surface of the SPP group, indicating that the SPP cross-linked group has a better anti-calcification effect.

[0051] Example 5: Evaluation of the cytocompatibility of the biological valve material prepared by sodium thiosulfate modification

[0052] (1) Sterilization treatment of the biological valve material: The valve material sample obtained in Example 1 is sterilized in PBS containing 2% double antibiotics for 24 hours and rinsed 3 times with sterile PBS.

[0053] (2) Preparation of the extract of the biological valve material: Cut the sterilized valve material sample into a 2 cm × 2 cm square and soak it in 5 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Place the treated valve material sample in an incubator and incubate at 37 °C for 24 hours.

[0054] (3) Cell culture and treatment: Seed HUVEC cells on a 96-well tissue culture plate (TCP) at a seeding density of 1×10 5 cells / mL. Incubate at 37 °C and 5% CO2 for 24 hours to promote cell attachment. After 24 hours, replace the original medium with 100 μL of fresh medium of the extract of the biological valve material before and after modification and continue to incubate for 24 hours.

[0055] (4) Determination of cell viability by CCK-8 method: Add 10 μL of CCK-8 solution to each well, and then incubate for 1 hour in the dark. Measure the absorbance at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader to evaluate cell viability and the cytotoxicity of the biological valve samples.

[0056] (5) Cell morphology analysis: To more intuitively observe cell viability and growth status, HUVECs were evenly seeded onto the surface of sterile samples at a density of 1×105 cells / plate and cultured for 24 hours and 72 hours respectively. The live and dead states of cells were identified by fluorescence staining using a calcein-AM / PI kit. In addition, to further observe the cell growth status, HUVECs were seeded onto the sample surface, and the cell nuclei were stained with DAPI and the cytoskeleton was stained with Actin-Tracker Red-Rhodamine to clearly show the cell morphology and growth condition. As Figure 7 shown, the results indicate that the cell survival rate of SPP for HUVECs can be higher than 95%. Further cell morphology analysis shows that HUVECs on the pericardium in the SPP group can adhere and extend to form obvious filopodia structures, and the cytoskeleton structure is clear, indicating that SPP has good biocompatibility and endothelialization promotion effect.

[0057] Example 6: Blood compatibility evaluation of the bio-valve material prepared by sodium thiosulfate modification

[0058] (1) Collection of red blood cells: Fresh rabbit blood anticoagulated with sodium citrate was centrifuged at 3,000 rpm for 5 minutes to collect red blood cells (RBC). The red blood cells were diluted with PBS at a ratio of 1:10.

[0059] (2) Treatment of bio-valve materials: Three groups of samples were cut into squares (1 cm×1 cm, n = 6), placed in 2 mL centrifuge tubes, rinsed 3 times with sterile PBS, and 200 μL of the diluted blood solution and 1 mL of PBS were added to each tube.

[0060] (3) Hemolysis test: The DPP, GPP, BPP, and SPP samples were incubated at 37 °C for 1 h, and then the supernatant was gently added to 1.5 mL centrifuge tubes and centrifuged at 3,000 rpm for 5 min. Among them, deionized water was used as the positive control and PBS was used as the negative control. The absorbance of hemoglobin at 545 nm was measured using a microplate reader.

[0061] As Figure 8 shown, the hemolysis rate of SPP is lower than 5%. Based on its good blood compatibility, the safety of the SPP pericardium material in blood contact applications is ensured. The lower platelet adhesion results further indicate that SPP has good potential for anticoagulant effects.

[0062] Example 7: Tissue remodeling and in vivo calcification test

[0063] (1) The DPP, GPP, and SPP were cut into squares (1 cm×1 cm) and thoroughly washed with PBS before implantation.

[0064] (2) Male Sprague Dawley rats (body weight = 200 g) were anesthetized by injecting 3% sodium pentobarbital at a dose of 10 mL / kg. -1 The anesthesia was performed at a dose of 10 mL / kg.

[0065] (3) Three 1-cm incisions were created on each side of the back of the rats, and the patches were implanted subcutaneously into the rats, and finally the incisions were sutured.

[0066] (4) The rats were sacrificed 15 days and 30 days after implantation, and specimens were collected. Samples fixed with tissue fixative were used for HE, Masson staining, alizarin red section staining for qualitative analysis, and ICP experiments to test the calcium ion content in the samples.

[0067] As Figure 9 shown, the HE staining results showed good cell proliferation, and the Masson staining results showed collagen degradation and microstructural damage in the GPP group within 30 days of implantation; in vivo calcification tests showed that at 15 days after implantation, the degree of calcification of the SPP sample was reduced by 79.88% compared with the GPP sample; at 30 days after implantation, the degree of calcification of the SPP sample was reduced by 76.21% compared with the GPP sample.

[0068] Example 8: Immunohistological test

[0069] (1) To evaluate the in vivo inflammatory response, immunohistochemistry (IHC) staining was performed on the samples in Example 4. (2) Immunofluorescence staining of CD3 for T lymphocytes and CD68 for all macrophages was performed to visualize the inflammatory response of the scaffold after implantation.

[0070] (3) Immunohistochemical staining of the PP samples with antibodies against pro-inflammatory factors TNF-α and IL-6 was performed to further study the degree of immune response in the PP samples.

[0071] As Figure 10 shown, the analysis results of immunohistochemical staining sections showed that GPP induced a severe inflammatory response after implantation, and the inflammatory cytokines in the SPP group were significantly reduced at 15 days and 30 days after implantation, indicating that treatment with STS could inhibit the inflammatory response of porcine pericardium materials, further explaining the immune-inflammatory exemption effect formed by SPP.

[0072] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the appended claims.

Claims

1. A preparation method of a biological valve material with anti-calcification, anti-thrombosis and immune safety at the same time, characterized in that, The method sequentially includes the following steps: 1) Decellularize the pericardial material, and after the treatment is completed, place the pericardial material in a glutaraldehyde solution for crosslinking; 2) After the crosslinking is completed, transfer the crosslinked pericardial material to a sodium thiosulfate solution for modification treatment to obtain a biological valve material with anti-calcification, anti-thrombosis and immune safety.

2. The preparation method according to claim 1, wherein, The pericardial material is selected from one or more of porcine pericardium, bovine pericardium, equine pericardium, ovine pericardium, and ostrich pericardium.

3. The method according to claim 1, wherein The mass percentage concentration of the glutaraldehyde solution is 0.5-5%, and the pH of the glutaraldehyde solution is 5.0-7.

0.

4. The preparation method according to claim 1, characterized in that, The crosslinking in step 1) is carried out at 4-40 °C, and the crosslinking time is 1-7 days.

5. The preparation method according to claim 1, characterized in that, The sodium thiosulfate in step 2) is anhydrous sodium thiosulfate, sodium thiosulfate pentahydrate, etc., the concentration of the prepared solution is 50-500 mM, and the modification treatment time is 1-7 days.

6. The preparation method according to claim 1, wherein, After the decellularization treatment, crosslinking and modification treatment, the obtained material is rinsed with sterile normal saline.

7. The preparation method according to claim 6, characterized in that The method further includes: transferring the biological valve material after the sodium thiosulfate modification treatment to a sterile PBS buffer solution for storage and standby after rinsing, and the pH of the sterile PBS buffer solution is 7.2-7.

4.

8. A biological valve material having anti-calcification, anti-thrombosis and immune safety prepared by the method according to any one of claims 1-7.

9. Use of the biological valve material according to claim 8 as or in the preparation of an artificial biological valve or a cardiovascular repair material.

10. The application according to claim 9, characterized in that, The artificial biological valve is an aortic valve leaflet, a mitral valve leaflet, a tricuspid valve leaflet, or a pulmonary valve leaflet.

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