A cross-linked modified artificial valve and its preparation method and application

Abstract

The present invention relates to the field of biomedical technology, and in particular to a cross-linked modified artificial valve, a preparation method and an application thereof. The present invention first prepares a new compound POSS-PEG-NHS, which undergoes an esterification reaction with the NH2 group on the surface of a decellularized porcine valve through its ‑NHS group to achieve covalent bonding. Subsequently, glutathione, a reducing agent inherent in the organism, is added to obtain a cross-linked modified artificial valve PPN-GSH-AV. The valve exhibits excellent cell safety and blood compatibility, and has obvious advantages over traditional glutaraldehyde cross-linked valves. In vivo experiments have shown that PPN-GSH-AV has high biosafety and tissue compatibility, and can effectively reduce immune rejection reactions. In addition, the valve can also reduce the oxidative stress of macrophages, promote M2 transformation, and thus reduce inflammatory reactions. In clinical applications, PPN-GSH-AV exhibits good anti-inflammatory effects and excellent anti-calcification properties, and the in vitro pulsatile flow test results meet international standards. It has the potential for clinical transformation and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a cross-linked modified artificial valve and a preparation method and application thereof. Background Art

[0002] Valvular heart disease has become a major health threat to Chinese residents, with a particularly significant increase in incidence among the elderly. With the aging population, the prevalence of calcific aortic valve stenosis, caused by degenerative disease, has reached 2%-7% in people over 65 years old, and the number of patients in my country is expected to exceed 40 million by 2025. The disease primarily manifests as valvular stenosis or regurgitation, which in severe cases can cause heart failure, arrhythmias, and even sudden death. Current treatment options primarily include medication, surgery, and minimally invasive interventional techniques. Minimally invasive techniques, such as transcatheter aortic valve replacement (TAVR), offer new treatment options for elderly, high-risk patients due to their minimal trauma and rapid recovery.

[0003] The development of artificial valve materials faces significant challenges. Although traditional mechanical valves have a durability of more than 50 years, they require lifelong anticoagulation treatment, which increases the risk of bleeding. Although biological valves avoid the need for anticoagulation, glutaraldehyde-crosslinked bovine pericardial materials have inherent defects - residual aldehyde groups continue to trigger an inflammatory response, activating the expression of calcification-related proteins, causing about 60% of patients to develop calcification and decay 10-15 years after implantation. Existing improvement strategies such as aldehyde neutralization and plant polyphenol cross-linking can slow the calcification process, but there are problems such as insufficient cross-linking efficiency and decreased mechanical properties. Although high-molecular polymer materials such as polyurethane have shown anti-calcification potential, there are bottlenecks in hemodynamic performance and processing technology.

[0004] In current clinical practice, valve selection needs to comprehensively consider factors such as the patient's age and health status. Mechanical valves are often recommended for young patients to avoid the risk of secondary surgery, while people over 60 years old tend to choose bioprosthetic valves to improve their quality of life. The third-generation bioprosthetic valve has extended its service life to 15-20 years through fully enclosed anti-calcification treatment and dry storage technology. However, promoting valve endothelialization and completely blocking the calcification process remain unsolved problems, which has driven the exploration of cutting-edge technologies such as the development of new cross-linking agents and functional modification of endothelial cells. Summary of the Invention

[0005] In view of this, the present invention proposes a cross-linked modified artificial valve, its preparation method, and application. The present invention provides a cross-linked modified artificial valve based on polyhedral oligomeric silsesquioxane-polyethylene glycol-N-hydroxysuccinimide (POSS-PEG-NHS) and its preparation method. In the present invention, a decellularized porcine valve is covalently cross-linked with POSS-PEG-NHS containing an active ester group under mild conditions, and then reduced glutathione (GSH) is introduced to enhance antioxidant properties.

[0006] The technical solution of the present invention is achieved as follows:

[0007] In a first aspect, the present invention provides a method for preparing a cross-linked modified artificial valve, comprising the following steps:

[0008] S1, using 3-mercaptopropyltrimethoxysilane and concentrated HCl to react in anhydrous methanol, generating POSS-8SH by hydrolysis and condensation, and separating and purifying; the POSS-8SH is a polyhedral oligomeric silsesquioxane containing 8 thiol groups;

[0009] S2. POSS-8SH and Allyl-PEG-OH are subjected to a thiol-ene click chemistry coupling reaction under a photoinitiator and UV irradiation to generate POSS-PEG-OH, which is then separated, purified and dried; the Allyl-PEG-OH is a single thiol-terminated polyethylene glycol with a number average molecular weight of 2000 Da.

[0010] S3, POSS-PEG-OH and succinic anhydride were catalyzed by DMAP under anhydrous conditions to generate POSS-PEG-COOH, which was then separated, purified and dried;

[0011] S4, using EDCI and NHS to activate the carboxylic acid group of POSS-PEG-COOH to generate POSS-PEG-NHS, which is then separated, purified and dried;

[0012] S5. Prepare 0.2 g / L POSS-PEG-NHS solution, put in the decellularized porcine valve, soak and react for 18-36 hours to obtain PPN-AV, take out the valve and soak it in 0.02 g / mL L-reduced glutathione solution for 18-36 hours to obtain PPN-GSH-AV, which is the cross-linked modified artificial valve; the PPN-AV is a decellularized porcine valve cross-linked with POSS-PEG-NHS.

[0013] In a second aspect, the present invention provides the application of the preparation method in the preparation of biomedical materials.

[0014] In a third aspect, the present invention provides a cross-linked modified artificial valve prepared according to the preparation method.

[0015] In a fourth aspect, the present invention provides the use of the cross-linked modified artificial valve in the preparation of biomedical materials.

[0016] The beneficial effects of the present invention include at least the following:

[0017] The cross-linked modified artificial valve provided by the present invention exhibits good cell safety. As time goes by, cells can proliferate normally. Compared with the traditional valve prepared by glutaraldehyde, it has significant cell safety advantages. In in vivo experiments, after the material was implanted subcutaneously in rats, there was obvious cell infiltration and growth, and no obvious increase in inflammatory indicators was observed, reflecting high biosafety and tissue compatibility, which is conducive to reducing immune rejection reactions after implantation. PPN-GSH-AV achieved good results in platelet adhesion, anti-thrombosis, and hemolysis experiments with rat blood, indicating that it has good blood compatibility and can effectively reduce the risk of blood-related complications such as thrombosis.

[0018] In addition, co-culture of PPN-GSH-AV with macrophages and subcutaneous embedding experiments showed that it exhibited less reactive oxygen species (ROS) generation, higher arginase-1 (Arg-1) expression and lower inducible nitric oxide synthase (iNOS) expression, indicating that the oxidative stress of macrophages was reduced and they transformed into the M2 type that inhibits inflammation and promotes repair, which helps to reduce the inflammatory response and tissue damage after implantation.

[0019] In clinical applications, the cross-linked modified artificial valve material provided by the present invention has shown good anti-inflammatory effects at the cellular level and in rats' subcutaneous tissue, and can effectively reduce the immune response after foreign body implantation. After 30 days of subcutaneous embedding in rats, no obvious calcification was observed in Von Kossa staining, and the anti-calcification performance was good, which can avoid short-term calcification decay after implantation and reduce the risk of reoperation in patients. The material was prepared into a biological valve for in vitro pulsatile flow testing. When it was opened, its effective opening area reached the international standard, and when it was closed, the reflux volume ratio was less than 15% of the international standard requirement. The transvalvular pressure difference was within the physiological range, and it has the potential for further clinical transformation.

[0020] While the tensile strength and Young's modulus of the cross-linked PPN-GSH-AV artificial valve provided by this invention are slightly lower than those of glutaraldehyde-cross-linked valves, they fully meet the mechanical requirements of normal valves. In in vitro pulsatile flow tests, valves made with this material fully met international standards, demonstrating sufficient strength and stability to withstand the mechanical loads of the heart during pumping.

[0021] This invention uses porcine valves as raw material. Porcine is the primary source of meat products in my country. Compared to the more common bovine pericardium, porcine valves are more abundant, easier to obtain, and more affordable. This effectively reduces raw material acquisition costs, alleviating the burden of medical expenses on patients and improving clinical outcomes. Furthermore, the porcine valve itself is cross-linked and modified, resulting in a structure and function that more closely resembles native valves under hemodynamic conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 They are as follows: Figure 1 A is a schematic diagram of the process of POSS-PEG-NHS synthesis reaction; Figure 1 B is the FTIR (Fourier transform infrared spectroscopy) diagram of the POSS-PEG-NHS synthesis reaction; the four curves in the figure represent the infrared spectra of POSS, POSS-PEG-OH, POSS-PEG-COOH and POSS-PEG-NHS, respectively; the positions of the characteristic absorption peaks and the corresponding functional groups are as follows: Si-O-Si: corresponding to the characteristic absorption peak of the POSS cage structure; C=O: corresponding to the characteristic absorption peak of the ester group or the carboxylic acid group; COC: corresponding to the characteristic absorption peak of the ether bond; NH: corresponding to the characteristic absorption peak of the amide bond; Figure 1 C is the H NMR spectrum of the POSS-PEG-NHS synthesis reaction ( 1 H NMR) diagram; the positions a, b, c, d, e, f, g, h, i, j, k, etc. marked in the figure correspond to different chemical shifts;

[0024] Figure 2 The test results of different valve materials in the embodiment of the present invention are as follows: Figure 2 A is the elongation at break; Figure 2 B is Young's modulus; Figure 2 C is the ultimate tensile strength; Figure 2 D is the surface and cross section photographed by SEM (scanning electron microscope); Figure 2 E is the statistical result of water contact angle; Figure 2 F is the water contact angle picture; Figure 2 G is the statistical result of surface roughness; Figure 2 H is an image of surface roughness measured by atomic force microscopy (AFM); DPAV, PPN-AV, PPN-GSH-AV, and GAV represent different experimental groups; data are expressed as SD ± mean, ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; (this description will not be repeated in the following text);

[0025] Figure 3 The test results of cell compatibility of different valve materials in the embodiment of the present invention are as follows: Figure 3A shows the live-dead cell staining results of HUVECs (human umbilical vein endothelial cells) inoculated with different valve materials on the 4th day after culture; Figure 3 B is the HUVECs live and dead cell statistics; Figure 3 C shows the results of live and dead cell staining (Calcein AM and PI staining) of Raw264.7 (macrophages) inoculated with different valve materials on the 4th day after culture; Figure 3 D is the statistical result of Raw264.7 live and dead cells; Figure 3 E is the statistical results of cell activity of HUVECs cultured for 48 h; Figure 3 F is the statistical result of the cell activity of Raw264.7 cultured for 48 h; Figure 3 G is the proliferation curve of HUVECs;

[0026] Figure 4 The blood compatibility test results of different valve materials in the embodiment of the present invention are as follows: Figure 4 A is a SEM observation of platelet (PLTs) adhesion experiment; Figure 4 B is a diagram of hemolysis experiment; Figure 4 C is a diagram of the thrombosis experiment; Figure 4 D is the statistical result of platelet adhesion; Figure 4 E is the statistical result of hemolysis rate; Figure 4 F is the statistical result of thrombosis;

[0027] Figure 5 The following are the HE, Masson, and Von Kossa staining results of different valve materials after subcutaneous embedding for 30 days in the embodiment of the present invention;

[0028] Figure 6 The results of CD3, CD68, CD206, Arg-1 / iNOS, and ROS staining after 30 days of subcutaneous embedding of different valve materials in the examples of the present invention are shown;

[0029] Figure 7 They are as follows: Figure 7 A is a picture of the interventional flap prepared by PPN-GSH-AV; Figure 7 B shows the opening and closing of the interventional valve prepared by PPN-GSH-AV in the pulsatile flow system; Figure 7 C is the reflux volume ratio; Figure 7 D is the effective opening area (EOA) of the valve; Note: Figure 7 In CD, the blue dotted line represents the artificial valve related parameter standard (ISO5840) developed by the International Organization for Standardization (ISO). Since the sizes of the prepared interventional valves are inconsistent, the corresponding EOA international standard values ​​will also be different. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the embodiments described are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0031] Example 1 Preparation of cross-linked modified artificial valve (PPN-GSH-AV)

[0032] 1. Preparation of POSS-PEG-NHS (reaction process as shown in Figure 1 (As shown in A)

[0033] (1) Preparation of polyhedral oligomeric silsesquioxane containing 8 thiol groups (POSS-8SH)

[0034] 10mL of 3-mercaptopropyltrimethoxysilane (CAS: 4420-74-0, purchased from Sigma-Aldrich) was added to 250mL of anhydrous methanol, and then 20mL of 37% (w / w) concentrated HCl was slowly added dropwise to the reaction system. After the addition was complete, the reaction mixture was heated to reflux and the reaction was maintained for 24h. After the reaction was completed, the reaction solution was cooled to room temperature and placed in a refrigerator to freeze overnight. Subsequently, the methanol in the supernatant was poured out, the oily product was washed three times with ice methanol, and then the ice methanol was poured out. The oily product was dissolved in dichloromethane (DCM), and ice methanol was added to induce precipitation. The mixture was frozen overnight, and then the supernatant was poured out to obtain the product POSS-8SH (5.0g, 73% w / w).

[0035] (2) Preparation of POSS-PEG-OH

[0036] POSS-8SH (0.15 g, 0.24 mmol), Allyl-PEG-OH (2.4 g, 1.2 mmol, 2000 Da, purchased from Adamas Life), and the photoinitiator 2,2-dimethoxy-2-phenylacetophenone (0.051 g, DMPA represents 2% of the total reaction mass, CAS: 24650-42-8, purchased from Sigma-Aldrich) were dissolved in 40 mL of ultra-dry DCM. The reaction was stirred under 365 nm UV light for 12 h. After completion of the reaction, 10 mL of water was added to the reaction system, and the aqueous phase was separated. The aqueous phase was extracted three times with DCM. The combined DCM phases were washed with saturated NaCl solution and dried over anhydrous MgSO₄. After filtration, the majority of the solvent was removed by distillation under reduced pressure. POSS-PEG-OH was recrystallized from chilled MTBE (methyl tert-butyl ether), filtered, and freeze-dried to obtain a white solid powder (1.91 g, 75% w / w).

[0037] (3) Preparation of POSS-PEG-COOH

[0038] POSS-PEG-OH (2 g, 0.12 mmol), succinic anhydride (0.384 g, 3.84 mmol, CAS: 108-30-5, purchased from Adamas) and DMAP (4-dimethylaminopyridine, 0.234 g, 1.92 mmol, CAS: 1122-58-3, purchased from Sigma-Aldrich) were reacted in anhydrous DCM (200 mL) for 24 h, and then the solution was washed with brine (3×40 mL, 1% w / v sodium chloride solution). The organic layer was collected, dried over anhydrous sodium sulfate, concentrated in vacuo, recrystallized with chilled MTBE, filtered, and freeze-dried to obtain POSS-PEG-COOH as a white solid powder.

[0039] (4) Preparation of POSS-PEG-NHS

[0040] POSS-PEG-COOH (2 g, 0.12 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.368 g, 2.4 mmol, CAS: 25952-53-8, purchased from tansoole), and N-hydroxysuccinimide (NHS, 0.441 g, 3.84 mmol, CAS: 6066-82-6, purchased from tansoole) were dissolved in anhydrous DCM (200 mL) and reacted for 24 h. The mixture was then washed directly with brine (3 × 100 mL). The organic layer was collected, dried over anhydrous sodium sulfate, concentrated in vacuo, recrystallized from chilled MTBE, filtered, and freeze-dried to obtain POSS-PEG-NHS as a white solid powder. During the reaction to prepare POSS-PEG-NHS, the terminal carboxyl group of POSS-PEG-COOH was modified with NHS through an esterification reaction.

[0041] 2. Preparation of PPN-AV

[0042] 1) Preparation of decellularized porcine valve (DPAV)

[0043] A. Fresh porcine valves were obtained from a slaughterhouse in Jiangxia District, Wuhan City, stored on ice, and brought back to the laboratory. Excess fat and blood were removed from the surface and the valves were cleaned with sterile sodium heparin saline. Valve decellularization was performed according to the method provided in the reference (Qiao WH, Liu P, Hu D, et al. Sequential hydrophile and lipophile solubilization as an efficient method for decellularization of porcine aortic valve leaflets: Structure, mechanical property, and biocompatibility study [J]. Journal of tissue engineering and regenerative medicine, 2018, 12(2): e828-e840. DOI: 10.1002 / term.2388.).

[0044] B. M3 system preparation: After CHAPS (10 g) was completely dissolved in 1 M Tris-HCl (pH 7.8) (20 mL), TnBP (272 μL) was added and the volume was adjusted to 500 mL with sterile water. A clean, decellularized porcine valve was placed in the M3 system and shaken at 37°C, 110 rpm, for 24 hours.

[0045] C. M4 system preparation: Completely dissolve CHAPS (10 g), SB3-10 (10 g), and ASB-14 (5 g) in 1 M Tris-HCl (pH 7.8) (20 mL). Add TnBP (272 μL) and dilute to 500 mL with sterile water. Remove the valve from the M3 system and place it in the M4 system. Incubate at 37°C on a shaker at 110 rpm for 24 h.

[0046] D. Nuclease System: Dissolve MgCl (95 mg) and NaCl (584 mg) in 1M Tris-HCl (pH 7.8) (25 mL), add Benonase (200 μL), and dilute to 500 mL with sterile water. Rinse the valves in the M4 system with sterile PBS and place in the nuclease system in a 37°C shaker at 110 rpm for 24 hours to remove any residual nucleic acids.

[0047] E. Remove the prepared DPAV from the nuclease system, wash it with sterile PBS, and store it in PBS at 4°C.

[0048] 2) POSS-PEG-NHS modified decellularized porcine valve

[0049] POSS-PEG-NHS solid was dissolved in deionized water to prepare a 0.2 g / L liquid. Four decellularized porcine valves (approximately 1.5 cm*3 cm) were soaked in 5 mL of the liquid and placed in a shaker at 37°C and 110 rpm for 24 h to obtain PPN-AV.

[0050] 3. Preparation of PPN-GSH-AV

[0051] The cross-linked PPN-AV was removed, washed with PBS, and then immersed in a 0.02 g / mL GSH (L-reduced glutathione, CAS No. 70-18-8) solution. The reaction was continued for 24 hours to obtain PPN-GSH-AV.

[0052] 4. Preparation of DPAV, PAV, and GAV (valve materials for the control group)

[0053] (1) The PAV (i.e., porcine valve) material of the negative control group was directly obtained from the slaughterhouse and cleaned; the preparation of the DPAV group was based on the above description.

[0054] (2) The positive control group GAV (glutaraldehyde cross-linked valve) was prepared as follows:

[0055] DPAV was soaked in 0.625% (w / v) glutaraldehyde solution and placed in a shaker at 37° C. and 110 rpm for reaction for 24 h to obtain GAV.

[0056] Example 2 Characterization Test

[0057] 1. Method

[0058] The samples were sequentially immersed in 30%, 50%, 70%, 80%, 90%, 100%, and 100% (v / v) alcohol for 15 min for gradient dehydration. The samples were then freeze-dried using a freeze dryer (Shanghai Lichen Model: LC-10N-60A) and analyzed using Fourier transform infrared spectroscopy (FTIR) and 1 The structures of different samples were characterized by H NMR.

[0059] Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize different valve materials (DPAV, PPN-AV, PPN-GSH-AV, and GAV). Because the samples are not conductive, they were gold-sprayed at 40 mA for 60 seconds before SEM observation. AFM data was used to reconstruct the three-dimensional surface topography of the samples, and roughness parameters were calculated.

[0060] 2. Results

[0061] (1) Synthesis and characterization of POSS-PEG-NHS

[0062] like Figure 1 As shown in B, the FTIR spectrum at 1730 cm -1 A strong absorption peak is shown at 1110 cm -1 The COC symmetrical vibration peak of the PEG segment appears at 2870-3000cm -1 The methylene (-CH2-) stretching vibration peak of the POSS skeleton was observed in the range of 1:1, which confirmed that PEG was successfully grafted onto the POSS surface and NHS active groups were introduced through amidation reaction. Figure 1 As shown in C, 1The H NMR spectrum shows methylene (-CH2-) proton signals of the PEG segment at 3.6-3.8 ppm; characteristic peaks of methylene (-CH2-) protons from the NHS group and succinic anhydride ring-opening are observed at 2.5-2.6 ppm; no residual signals from free carboxylic acid or unreacted starting materials are detected. This indicates that the amidation reaction proceeded completely, and POSS-PEG-NHS was successfully synthesized. Furthermore, the NHS ester group, as an "active ester," is essentially a reversible reaction intermediate formed by NHS activation of a carboxylic acid. When this intermediate comes into contact with an amino group (-NH2), a transesterification reaction spontaneously occurs to form a stable amide bond. Therefore, by introducing an -NHS-activated -COOH group, it reacts with -NH2 at room temperature to form an amide bond, achieving covalent crosslinking with the decellularized porcine valve.

[0063] (2) Surface microscopic characteristics

[0064] From the SEM results ( Figure 2 D) It can be observed that the surface of PPN-AV shows some texture changes, which may appear as fine grooves or protrusions, while the texture of the surface of PPN-GSH-AV is more complex. This complexity may be related to the modification of glutathione (GSH), which increases the diversity of its surface microstructure. These differences in surface structure will affect the initial contact of the material with blood, cells, etc., and thus affect its biocompatibility. From the AFM results ( Figure 2 H) It can be further seen that the DPAV surface is relatively flat, with a narrow range of height variations, while the PPN-AV and PPN-GSH-AV surfaces exhibit a more complex and uneven structure, which increases surface complexity and may be more conducive to cell adhesion. The GAV surface also has significant height variations. These surface characteristics also affect their interactions with biomolecules.

[0065] from Figure 2 G shows that DPAV has the lowest surface roughness, while PPN-AV has a higher surface roughness than DPAV; the surface roughness of PPN-GSH-AV is further increased, exceeding that of PPN-AV; and GAV has the highest surface roughness. The relatively high roughness characteristics of PPN-AV and PPN-GSH-AV may give them an advantage in promoting cell adhesion. Generally speaking, the complexity of the microstructure of the material surface has a significant impact on the type and amount of protein adsorption. Rough surfaces or surfaces with special textures are often more likely to adsorb proteins that can promote cell adhesion, which in turn has a profound impact on subsequent cell behavior. Therefore, it can be speculated that PPN-AV and PPN-GSH-AV may exhibit more outstanding performance advantages in aspects such as cell behavior regulation and tissue regeneration.

[0066] Example 3 Mechanical properties

[0067] The five heart valve material samples provided in Example 1—DPAV, PAV, PPN-AV, PPN-GSH-AV, and GAV—were prepared in strict accordance with GB / T 16777-2008, "Biological Evaluation of Medical Devices," and the ISO 10993 series of standards. Standardized production processes ensured uniformity in key performance parameters, such as material microstructure and crosslink density. At least 10 independent samples were prepared for each material, with a dimensional tolerance within ±0.02 mm. Five samples were used for mechanical property testing, three for microscopic characterization, and two as backup samples.

[0068] 1. Method

[0069] (1) Elongation at break, Young's modulus and tensile strength tests

[0070] The sample to be tested was cut into rectangles (30mm*10mm) and soaked in PBS before testing. When measuring the thickness of the sample, it was recorded at three different points using a vernier caliper. After that, both ends of each sample were firmly clamped into the clamps of the tensile testing machine. The initial length (L0) of the sample between the clamps was recorded for later analysis. The sample was stretched at a constant speed of 20mm / min in the tensile testing machine until it broke. During this process, the force-displacement curve was recorded for further analysis. The load was continued until the specimen was completely broken, and the maximum load value was taken to calculate the tensile strength. Three parallel tests were set up for each group of materials, and the results were presented as the arithmetic mean ± standard deviation.

[0071] (2) Water Contact Angle (WCA) test

[0072] The sample to be tested was cut into squares (10 mm x 10 mm) and rinsed three times with deionized water. The material was then placed between two glass slides and freeze-dried. After drying, the sample was flattened onto a smooth glass slide and the water contact angle was measured using a tensiometer.

[0073] 2. Results

[0074] (1) Elongation at break, Young's modulus and tensile strength tests

[0075] like Figure 2 As shown in Figure 2B, the PPN-AV and PPN-GSH-AV valve materials of the present invention exhibit significantly higher Young's moduli than DPAV, indicating that these two valve materials are more resistant to tensile deformation than DPAV. Furthermore, the Young's moduli of PPN-AV and PPN-GSH-AV are also higher than those of PAV, demonstrating that PPN-AV and PPN-GSH-AV are significantly superior to PAV in terms of rigidity.

[0076] Furthermore, if Figure 2 As shown in Figure C, the tensile strength of PPN-AV and PPN-GSH-AV is also significantly higher than that of DPAV, indicating that these two materials can withstand greater tensile forces and have a stronger ability to resist tensile failure. In comparison with PAV, the tensile strength of PPN-AV and PPN-GSH-AV also demonstrates an advantage. Therefore, it can be clearly concluded that the tensile strength performance of PPN-AV and PPN-GSH-AV is significantly superior to that of DPAV and PAV.

[0077] There was no significant difference in elongation at break between the DPAV, PAV, PPN-AV, and PPN-GSH-AV groups, indicating that there was no significant difference in the compliance between the valve stent and the natural valve in the PPN-GSH-AV group ( Figure 2 A).

[0078] (2)WCA test

[0079] WCA test results are as follows Figure 2 As shown in E and 2F, the water contact angles of different materials are different, reflecting the difference in surface hydrophilicity and hydrophobicity. PPN-AV and PPN-GSH-AV show stronger hydrophilicity than DPAV or GAV.

[0080] Example 4 Cytocompatibility test

[0081] 1. Method

[0082] The prepared valve material was cut into small discs of 6 mm in diameter and sterilized and plated in a 96-well plate. Human umbilical vein endothelial cells (HUVECs) and macrophages (Raw264.7) were then plated on the surface of the material at 5,000 cells / well and 100,000 cells / well, respectively. After incubation at 37°C for 48 hours, the OD values ​​were measured using a CCK8 assay. 450nm The cytotoxicity of the materials was determined. Calcein AM / PI staining was used to observe the live and dead cells on the 4th day of culture on different valve materials and the numbers were counted. Continuous culture was performed on different valve materials, and CCK8 was used to measure the amount of cells attached to the scaffold at different time points to draw the HUVECs proliferation curve.

[0083] 2. Results

[0084] like Figure 3As shown, for HUVECs culture, the PPN-GSH-AV group had significantly higher cell viability after 48 hours of culture compared to the GAV group. The DPAV and PPN-AV groups also showed relatively high cell viability. This indicates that the GAV group has relatively significant cytotoxicity, while the PPN-GSH-AV, DPAV, and PPN-AV groups have relatively low cytotoxicity. For macrophages, the cell viability of the DPAV, PPN-AV, and PPN-GSH-AV groups was significantly higher than that of the GAV group, indicating that GAV has greater cytotoxicity to macrophages, while the other three materials have relatively little effect on macrophage activity. The proportion of live cells also showed the same pattern, which further verified that PPN-GSH-AV, DPAV, and PPN-AV are significantly superior to GAV in terms of cell safety.

[0085] In addition, compared with the DPAV, PPN-AV and GAV groups, the cell proliferation curve of the PPN-GSH-AV group showed a more obvious upward trend. 450nm This indicates that PPN-GSH-AV not only supports the adhesion of HUVECs but also promotes their proliferation more effectively, performing best in promoting cell proliferation, while the cell proliferation ability of the GAV group is relatively weak.

[0086] Example 5 Blood compatibility test

[0087] 1. Method

[0088] The prepared valve material was cut into small discs of 6 mm in diameter and co-cultured with platelet-rich plasma (PRP), fresh whole blood, and red blood cell suspension, and the LDH (lactate dehydrogenase) test and OD were measured. 540nm Verify the valve material's good anti-platelet adhesion, anti-thrombosis properties, and non-hemolytic properties. Use SEM to observe and count the platelet adhesion on the surface of different valve materials; conduct hemolysis experiments and thrombosis experiments to observe and count the hemolysis rate and thrombosis.

[0089] 2. Results

[0090] Figure 4 The results showed that the PPN-GSH-AV valve material has good anti-platelet adhesion, non-hemolysis and anti-thrombosis properties, as follows: the platelet adhesion in the GAV group was significantly higher than that in the DPAV, PPN-AV and PPN-GSH-AV groups ( Figure 4 A and 4D), the platelet adhesion in the PPN-GSH-AV group was relatively less. This indicates that the GAV valve material is more likely to adsorb platelets, while PPN-GSH-AV is relatively better at resisting platelet adhesion. Figure 4B and 4E) showed that there was no significant difference in the hemolysis rate of DPAV, PPN-AV, PPN-GSH-AV and GAV, and all were at a low level, indicating that these materials showed good blood compatibility in terms of hemolysis and met the relevant blood contact safety requirements. The thrombosis rate of the DPAV, PPN-AV and PPN-GSH-AV groups was significantly lower than that of the GAV group ( Figure 4 C and 4F), the degree of thrombosis in the PPN-GSH-AV group was relatively low, indicating that PPN-GSH-AV also has relatively superior anti-thrombotic properties.

[0091] Example 6 Histological and immunofluorescence analysis

[0092] 1. Method

[0093] The different valve materials were cut into 1 cm x 1 cm pieces and embedded subcutaneously in 8-week-old SD rats. Thirty days later, the samples were harvested and fixed in 4% formaldehyde to preserve their structure. Subsequently, the samples were dehydrated, embedded in paraffin, and cut into 4 mm thick sections. These sections were then subjected to various staining schemes to visualize different components: hematoxylin and eosin (HE) staining was used to observe cellular morphology and overall tissue structure, Masson staining was used to assess collagen fibrillary status, and Vonkossa staining was used to detect calcium deposits.

[0094] In order to specifically mark immune cells, CD3 antibodies are used to specifically mark T lymphocytes; CD68 antibodies, CD206 antibodies and Arg-1 / iNOS detection methods are used to mark and functionally characterize macrophages; ROS detection reagents are used to detect the reactive oxygen species levels of immune cells to mark the functional status of related immune cells.

[0095] 2. Results

[0096] (1) In vivo calcification and histological evaluation Figure 5 )

[0097] HE staining revealed relatively regular cell morphology and good tissue integrity in the PPN-GSH-AV group. Masson staining demonstrated superior collagen fiber preservation compared to the DPAV, PPN-AV, and GAV groups, suggesting that the material excels in maintaining collagen fiber structural integrity. Vonkossa staining revealed significantly less calcium deposition in the PPN-GSH-AV group than in the other groups, indicating its strong anti-calcification ability. These results suggest that PPN-GSH-AV not only exhibits excellent biocompatibility but also demonstrates significant advantages in anti-calcification and maintaining histological structural stability.

[0098] (2) In vivo inflammation evaluation Figure 6 )

[0099] Different valve materials exhibited distinct histological characteristics after subcutaneous implantation. Compared with DPAV and GAV, PPN-GSH-AV and PPN-AV were surrounded by relatively fewer CD3-positive T cells and CD68-positive macrophages, indicating a weaker immune response and lower immunogenicity. Furthermore, PPN-GSH-AV and PPN-AV showed high expression of Arg-1, a protein representing pro-repair M2 macrophages, and low expression of iNOS, a protein representing pro-inflammatory M1 macrophages, based on CD206, Arg-1 / iNOS, and ROS markers. These findings suggest that PPN-GSH-AV and PPN-AV may also have advantages in regulating relevant cellular functions, inducing M2 macrophage polarization, further demonstrating their superior ability to mitigate immune rejection. These results suggest that PPN-GSH-AV possesses excellent pro-repair and recellularization capabilities when used in vivo, potentially mitigating adverse effects of immune responses.

[0100] Example 7 Interventional Valve Performance Test

[0101] 1. Method

[0102] After the PPN-GSH-AV valve material is prepared into a finished valve, an in vitro pulsating flow test is performed to evaluate whether it meets international standards. The valve material is cut and sutured to prepare a TAVR valve, and the sewn valve product is placed in an in vitro pulsating flow system. At a cardiac output of 5L / min (close to the cardiac output range of most adults), 10 cardiac cycles are tested. The opening and closing state of the valve leaflets is filmed with a high-speed camera to evaluate whether there is curling, deformation, insufficiency, etc. The test results are then used to evaluate the opening and closing state in the pulsating flow system, and the regurgitant volume ratio (RF%) and the effective opening area (EOA) of the valve are measured.

[0103] 2. Results Figure 7 )

[0104] The PPN-GSH-AV-fabricated interventional valve performed well in opening and closing, RF, and EOA measurements, demonstrating that the material can meet the basic functional requirements of heart valves under simulated physiological conditions. Key parameters such as effective opening area, transvalvular pressure gradient, and regurgitant fraction met international standards. This demonstrates that the modified decellularized porcine valve can achieve the same functional characteristics as a native valve, meeting basic physiological opening and closing requirements without significant leaflet deformation, curling, stenosis, or regurgitation.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a cross-linked modified artificial valve, characterized in that: The steps include: S1, using 3-mercaptopropyltrimethoxysilane and concentrated HCl to react in anhydrous methanol, generating POSS-8SH by hydrolysis and condensation, and separating and purifying; the POSS-8SH is a polyhedral oligomeric silsesquioxane containing 8 thiol groups; S2, POSS-8SH and Allyl-PEG-OH were subjected to thiol-ene click chemistry coupling reaction under photoinitiator and UV irradiation. Producing POSS-PEG-OH, which is then separated, purified and dried; the Allyl-PEG-OH is a single thiol-terminated polyethylene glycol with a number average molecular weight of 2000 Da; S3, POSS-PEG-OH and succinic anhydride were catalyzed by DMAP under anhydrous conditions to generate POSS-PEG-COOH, which was then separated, purified and dried; S4, using EDCI and NHS to activate the carboxylic acid group of POSS-PEG-COOH to generate POSS-PEG-NHS, which is then separated, purified and dried; S5. Prepare 0.2 g / L POSS-PEG-NHS solution, put in the decellularized porcine valve, soak and react for 18-36 hours to obtain PPN-AV, take out the valve and soak it in 0.02 g / mL L-reduced glutathione solution for 18-36 hours to obtain PPN-GSH-AV, which is the cross-linked modified artificial valve; the PPN-AV is a decellularized porcine valve cross-linked with POSS-PEG-NHS.

2. The preparation method according to claim 1, characterized in that In step S2, the photoinitiator is 2,2-dimethoxy-2-phenylacetophenone, the molar ratio of POSS-8SH to Allyl-PEG-OH is 1:(4-6), and the photoinitiator accounts for 0.5-5% of the total mass of the reaction system.

3. The preparation method according to claim 2, characterized in that In step S2, UV irradiation at a wavelength of 365 nm is used, and the reaction is stirred for 7-14 hours.

4. The preparation method according to claim 1, characterized in that In the step S3, the molar ratio of the POSS-PEG-OH, succinic anhydride and DMAP is 1:(30-34):(14-18).

5. The preparation method according to claim 1, characterized in that In the step S4, the molar ratio of the POSS-PEG-COOH, EDCI and NHS is 1:(18-22):(30-34).

6. The preparation method according to claim 1, characterized in that In step S5, the soaking reaction conditions are 37° C. and a shaking table at 110 rpm.

7. The preparation method according to claim 1, characterized in that In step S1, 3-mercaptopropyltrimethoxysilane and anhydrous methanol are mixed in a volume ratio of 1:25, 37% w / w concentrated hydrochloric acid is added dropwise, and the mixture is heated under reflux in an oil bath at 60-70° C. for 18-36 hours; the volume ratio of concentrated hydrochloric acid to 3-mercaptopropyltrimethoxysilane is 2:

1.

8. Use of the preparation method according to any one of claims 1 to 7 in the preparation of biomedical materials.

9. A cross-linked modified artificial valve, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

10. Use of the cross-linked modified artificial valve according to claim 9 in the preparation of biomedical materials.

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