Cross-linked modified artificial valve as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a cross-linked modified artificial valve and a preparation method and application thereof. The preparation method comprises the following steps: firstly, preparing a novel compound POSS-PEG-NHS, and carrying out esterification reaction on a-NHS group of the compound and a-NH2 group on the surface of the decellularized porcine valve to realize covalent binding; then, an inherent reducing agent glutathione in an organism is added, and the cross-linked modified artificial valve PPN-GSH-AV is obtained. The valve shows excellent cell safety and blood compatibility, and has obvious advantages compared with a traditional glutaraldehyde cross-linked valve. In-vivo experiments show that the PPN-GSH-AV has high biological safety and histocompatibility, and can effectively reduce immunological rejection. In addition, the valve can relieve oxidative stress of macrophages and promote M2 type transformation, so that inflammatory response is relieved. In clinical application, the PPN-GSH-AV shows a good anti-inflammatory effect and excellent anti-calcification performance, in-vitro pulsating flow test results meet international standards, and the PPN-GSH-AV has clinical transformation potential and wide 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] Heart valve disease has become a major disease that threatens the health of Chinese residents, especially among the elderly. With the aging of the population, the prevalence of calcific stenosis of the aortic valve caused by degenerative diseases has reached 2%-7% in people over 65 years old. It is estimated that the number of patients in my country will exceed 40 million by 2025. The disease is mainly manifested as valvular stenosis or insufficiency, which can cause heart failure, arrhythmia and even sudden death in severe cases. Current treatment methods mainly include drug therapy, surgical operations and minimally invasive interventional techniques. Among them, minimally invasive techniques such as transcatheter aortic valve replacement (TAVR) provide new treatment options for elderly and high-risk patients due to their small trauma and fast 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 therapy, 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 inflammatory responses and activate the expression of calcification-related proteins, causing about 60% of patients to develop calcification 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 show 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 biological valves to improve their quality of life. The third-generation biological 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 are still unsolved problems, which has promoted 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 and a preparation method and application thereof. The present invention provides a cross-linked modified artificial valve based on polyhedral oligomeric silsesquioxane-polyethylene glycol-N-hydroxysuccinimide (POSS-PEG-NHS) and a preparation method thereof. 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 the antioxidant property.

[0006] The technical solution of the present invention is achieved in this way:

[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 mercapto 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 2000Da.

[0010] S3, POSS-PEG-OH and succinic anhydride are catalyzed by DMAP under anhydrous conditions to generate POSS-PEG-COOH, which is 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 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 by POSS-PEG-NHS.

[0013] In a second aspect, the present invention provides application of the preparation method in preparing 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, and it has significant cell safety advantages compared to the valves prepared by traditional glutaraldehyde. In the in vivo experiment, 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 beneficial to reduce immune rejection 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 helped 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 shows 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] Although the tensile strength and Young's modulus of the cross-linked modified artificial valve PPN-GSH-AV provided by the present invention are slightly lower than those of the valve cross-linked by glutaraldehyde, it can fully meet the mechanical requirements of normal valves. In the in vitro pulsating flow experiment, the test results of the valve prepared by this material fully meet the requirements of international standards, indicating that it has sufficient strength and stability to withstand the mechanical load during the heart pumping blood.

[0021] The present invention uses pig valves as raw materials. Pigs are the main source of meat products in my country. Compared with the common bovine pericardium, pig valves are abundant in source, simple to obtain, and affordable. They can effectively reduce the cost of obtaining raw materials, reduce the burden of medical expenses for patients, and improve clinical benefits. In addition, the pig valve itself is cross-linked and modified, and its structure and functional state are more in line with the native valve under the hemodynamic environment. 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 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 spectrum) diagram of the synthesis reaction of POSS-PEG-NHS; 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 embodiments 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 a picture 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 embodiments of the present invention are as follows: Figure 3A shows the live and 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 statistical results of HUVECs live and dead cells; Figure 3 C shows the results of live and dead cell staining (CalceinAM 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 result 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 test results of blood compatibility of different valve materials in the embodiments 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 results of HE, Masson and Von kossa staining 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 subcutaneous embedding of different valve materials for 30 days in the embodiment of the present invention;

[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] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in 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 flow chart as follows Figure 1 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 kept 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. Dichloromethane (DCM) was used to dissolve the oily product, 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] Take POSS-8SH (0.15g, 0.24mmol), Allyl-PEG-OH (2.4g, 1.2mmol, 2000Da, purchased from Adamas life) and photoinitiator 2,2-dimethoxy-2-phenylacetophenone (0.051g, DMPA accounts for 2% of the total mass of the reaction system, CAS: 24650-42-8, purchased from Sigma-Aldrich) and dissolve in 40mL ultra-dry DCM. Stir the reaction for 12h under 365nm UV light. After the reaction is completed, 10mL of water is added to the reaction system and the aqueous phase is separated. The aqueous phase is extracted with DCM three times, and the DCM phases are combined and washed with saturated NaCl solution, and then anhydrous MgSO 4After drying, most of the solvent was removed by vacuum distillation after filtration. Recrystallization was performed with frozen MTBE (methyl tert-butyl ether), suction filtration, and freeze drying to obtain a white solid powder POSS-PEG-OH (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 frozen MTBE, filtered, and freeze-dried to finally obtain white solid powder POSS-PEG-COOH.

[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, and then directly washed with brine (3×100 mL). The organic layer was collected, dried over anhydrous sodium sulfate, concentrated in vacuo, recrystallized with frozen MTBE, filtered, and freeze-dried to finally obtain white solid powder POSS-PEG-NHS. In the above reaction process for preparing POSS-PEG-NHS, POSS-PEG-COOH and NHS are esterified to modify the terminal carboxyl group to an NHS group.

[0041] 2. Preparation of PPN-AV

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

[0043] A. Fresh porcine valves were obtained from the slaughterhouse in Jiangxia District, Wuhan City, and were kept on ice and brought back to the laboratory. Excess fat and blood on the surface were removed and washed 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. Preparation of M3 system: CHAPS (10 g) was completely dissolved in 1M Tris-HCl pH=7.8 (20 mL), TnBP (272 μL) was added, and the volume was adjusted to 500 mL with sterile water. The clean decellularized porcine valve was placed in the M3 system, incubated at 37°C and 110 rpm for 24 h.

[0045] C. Preparation of M4 system: CHAPS (10 g), SB3-10 (10 g), ASB-14 (5 g) were completely dissolved in 1M Tris-HCl pH = 7.8 (20 mL), TnBP (272 μL) was added, and the volume was adjusted to 500 mL with sterile water. The valves in the M3 system were taken out and placed in the M4 system, and the reaction was carried out for 24 hours in a constant temperature shaker at 37°C and 110 rpm.

[0046] D. Nuclease system: MgCl (95 mg) and NaCl (584 mg) were dissolved in 1M Tris-HCl pH=7.8 (25 mL), Benonase (200 μL) was added, and the volume was adjusted to 500 mL with sterile water. The valves in the M4 system were cleaned with sterile PBS and placed in the nuclease system, in a constant temperature shaker at 37°C, 110 rpm, for 24 hours to remove the residual nucleic acid components on the surface.

[0047] E. Take out the prepared DPAV from the nuclease system, wash it with sterile PBS, and then soak it in PBS and store it in a 4°C refrigerator.

[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 of about 1.5 cm*3 cm in size 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 taken out, 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 24 h to obtain GAV.

[0056] Example 2 Characterization Test

[0057] 1. Methods

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

[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 first treated with gold spraying at 40 mA for 60 seconds before SEM observation. The three-dimensional morphology of the sample surface was reconstructed based on the AFM collected data, and then the 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 is at 1730 cm -1 There is a strong absorption peak at 1110cm -1 The COC symmetric vibration peak of the PEG segment appears at 2870-3000cm -1 The methylene groups (-CH 2 -) stretching vibration peak, confirming that PEG was successfully grafted onto the POSS surface and NHS active groups were introduced through amidation reaction. Figure 1 As shown in C, 1 In the H NMR spectrum, the methylene group (-CH 2 -) proton signal; 2.5-2.6ppm shows NHS group and methylene (-CH 2 -) proton characteristic peak; no residual signal of free carboxylic acid or unreacted raw materials was detected; thus, it can be seen that the amidation reaction was completely carried out and POSS-PEG-NHS was successfully synthesized. In addition, the NHS ester group, as an "active ester", is essentially a reversible reaction intermediate formed by NHS activation of carboxylic acid. When this intermediate reacts with amino (-NH 2 ) will spontaneously undergo an ester exchange reaction to form a stable amide bond. Therefore, by introducing -NHS to activate the -COOH group, it can react with -NH at room temperature. 2 The reaction occurs to form amide bonds, achieving covalent cross-linking 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 2H) It can be further understood that the DPAV surface is relatively flat with a small range of height variation, while the PPN-AV and PPN-GSH-AV surfaces present more uneven structures, which increases the complexity of the surface and may be more conducive to cell adhesion. The GAV surface also has obvious height differences. The above surface characteristics will also affect its interaction 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, higher than PPN-AV; GAV has the highest surface roughness; the relatively high roughness characteristics of PPN-AV and PPN-GSH-AV may give them advantages in promoting cell adhesion. Generally speaking, the complexity of the microstructure of the material surface has a significant effect on the type and amount of protein adsorption. Rough surfaces or surfaces with special textures are often more likely to adsorb proteins that promote cell adhesion, which in turn has a profound effect on subsequent cell behavior. Therefore, it can be inferred that PPN-AV and PPN-GSH-AV may show more outstanding performance advantages in terms of cell behavior regulation and tissue regeneration.

[0066] Example 3 Mechanical properties

[0067] The preparation process of the five heart valve material samples of DPAV, PAV, PPN-AV, PPN-GSH-AV and GAV provided in Example 1 strictly follows GB / T 16777-2008 "Biological Evaluation of Medical Devices" and ISO 10993 series standards, and the uniformity of key performance parameters such as material microstructure and crosslinking density is ensured through standardized production processes. No less than 10 independent samples are prepared for each material, and the sample size error is controlled within the range of ±0.02mm, of which 5 are used for mechanical property testing, 3 are used for microscopic characterization, and 2 are used as spare samples.

[0068] 1. Methods

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

[0070] The samples to be tested were 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. Thereafter, both ends of each sample were firmly clamped into the clamps of the tensile tester. 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 tester until it broke. During this process, the force-displacement curve was recorded for further analysis. The load was continued until the specimen broke completely, and the maximum load value was taken to calculate the tensile strength. Three parallel tests were set 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 (10mm*10mm) and rinsed 3 times with deionized water. The material was then placed between two glass slides and freeze-dried. After drying, the sample was flattened on a smooth glass slide and the contact angle of water 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 B, the PPN-AV and PPN-GSH-AV valve materials of the present invention exhibit a significantly higher Young's modulus than DPAV, which indicates that these two valve materials have a stronger ability to resist tensile deformation than DPAV. At the same time, compared with PAV, the Young's modulus of PPN-AV and PPN-GSH-AV also shows a higher level, which shows 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, which means that these two materials can withstand greater tensile forces and have stronger resistance to tensile failure. In comparison with PAV, the tensile strength of PPN-AV and PPN-GSH-AV also shows advantages. Therefore, it can be clearly concluded that the tensile strength performance of PPN-AV and PPN-GSH-AV is significantly better than that of DPAV and PAV.

[0077] There was no significant difference in elongation at break among 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 Cell compatibility test

[0081] 1. Methods

[0082] The prepared valve material was cut into small discs of d = 6 mm and sterilized and plated in a 96-well plate. Then, human umbilical vein endothelial cells (HUVECs) and macrophages (Raw264.7) were plated on the surface of the material at 5000 / well and 100000 / well, respectively, and cultured in a 37°C incubator for 48 hours. The OD was detected by CCK8 kit. 450nm , and the cytotoxicity of the material was obtained. CalceinAM / PI staining was used to observe the live and dead cells on the 4th day of culture on different valve materials and the statistics were counted. Continuous culture was performed on different valve materials, and CCK8 was used to determine the amount of cells attached to the stent at different time points to draw the HUVECs proliferation curve

[0083] 2. Results

[0084] like Figure 3 As shown, for HUVECs culture, the cell viability of the PPN-GSH-AV group was significantly higher than that of the GAV group after 48 hours of culture. The DPAV and PPN-AV groups also showed relatively high cell viability. This shows that the GAV group has relatively significant cytotoxicity, while the cytotoxicity of the PPN-GSH-AV, DPAV and PPN-AV groups is relatively low. 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 a 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 rule, 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 The value was higher than that of other groups. This indicates that PPN-GSH-AV not only supports the adhesion of HUVECs, but also promotes their proliferation more effectively, and performs 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. Methods

[0088] The prepared valve material was cut into small discs of d = 6 mm 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 good anti-platelet adhesion, anti-thrombosis and non-hemolytic properties of valve materials. Use SEM to observe and count the adhesion of platelets on the surface of different valve materials; conduct hemolysis experiments and thrombosis experiments, observe and count the hemolysis rate and thrombosis.

[0089] 2. Results

[0090] Figure 4 The results showed that the PPN-GSH-AV valve material had good anti-platelet adhesion, non-hemolytic and anti-thrombotic 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 good at resisting platelet adhesion. Hemolysis test results ( Figure 4 B 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 in the DPAV, PPN-AV and PPN-GSH-AV groups was significantly lower than that in the GAV group ( Figure 4 C and 4F), the degree of thrombosis in the PPN-GSH-AV group was relatively low, which indicates that PPN-GSH-AV also has relatively superior anti-thrombotic properties.

[0091] Example 6 Histology and Immunofluorescence Analysis

[0092] 1. Methods

[0093] Different valve materials were cut into 1cm*1cm pieces and embedded in the subcutaneous tissue of 8-week-old SD rats. The samples were collected 30 days later and fixed with 4% formaldehyde solution to preserve their structure. Subsequently, the samples were dehydrated, embedded in paraffin, and cut into 4mm thick sections. These sections were then subjected to various staining schemes to show different components: hematoxylin-eosin (HE) staining was used to observe cell morphology and overall tissue condition, Masson staining was used to evaluate collagen fiber status, and Vonkossa staining was used to detect calcium salt deposition.

[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 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 showed that the cell morphology of the PPN-GSH-AV group was relatively regular and the tissue integrity was good; Masson staining showed that its collagen fiber preservation was better than that of the DPAV, PPN-AV and GAV groups, indicating that the material performed outstandingly in maintaining the structural integrity of collagen fibers; Vonkossa staining results showed that the calcium salt deposition in the PPN-GSH-AV group was significantly less than that in other groups, indicating that it had a strong anti-calcification ability. Based on these results, PPN-GSH-AV not only showed good biocompatibility, but also had significant advantages in anti-calcification and maintaining histological structural stability.

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

[0099] The histological characteristics of different valve materials after subcutaneous embedding were different. Compared with DPAV and GAV, the number of CD3-positive T cells and CD68-positive macrophages around PPN-GSH-AV and PPN-AV was relatively small, indicating that the immune response they triggered was weak and the immunogenicity was low. In addition, from the perspective of CD206, Arg-1 / iNOS, and ROS markers, PPN-GSH-AV and PPN-AV had high expression of Arg-1, a representative protein of M2 macrophages that mainly promoted repair, and low expression of iNOS, a representative protein of M1 macrophages that mainly promoted inflammation, indicating that PPN-GSH-AV and PPN-AV may also have advantages in regulating related cell functions and can induce macrophage M2 polarization, further explaining their good performance in reducing immune rejection reactions. These results mean that PPN-GSH-AV has excellent pro-repair and recellularization capabilities when used in vivo, which can reduce the adverse effects caused by immune responses.

[0100] Example 7 Interventional valve performance test

[0101] 1. Methods

[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 with a cardiac output of 5L / min (close to the cardiac output range of most adults), and 10 cardiac cycles are tested. The opening and closing state of the valve leaflets is photographed by a high-speed camera to evaluate whether there is curling, deformation, insufficiency, etc. The test results are used to evaluate the opening and closing conditions 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 interventional valve prepared by PPN-GSH-AV performed well in opening and closing, RF and EOA measurements, indicating that the material can meet the basic functional requirements of heart valves under simulated physiological conditions. PPN-GSH-AV can meet international standards in important parameters such as effective opening area, transvalvular pressure gradient, and regurgitation fraction. This shows that after modification, the decellularized porcine valve can achieve the functional state of the native valve, meet the basic opening and closing under physiological conditions, and have no obvious leaflet deformation, curling or stenosis, or insufficiency.

[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 protection scope 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 mercapto groups; S2, POSS-8SH and Allyl-PEG-OH were subjected to thiol-ene click chemistry coupling reaction under photoinitiator and UV irradiation. POSS-PEG-OH is generated, and then separated, purified and dried; the Allyl-PEG-OH is a single thiol-terminated polyethylene glycol with a number average molecular weight of 2000Da. S3, POSS-PEG-OH and succinic anhydride are catalyzed by DMAP under anhydrous conditions to generate POSS-PEG-COOH, which is 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 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 by POSS-PEG-NHS.

2. The preparation method according to claim 1, characterized in that In the 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 the step S2, 365 nm wavelength UV irradiation 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 110 rpm shaking.

7. The preparation method according to claim 1, characterized in that: In the 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 to reflux in an oil bath at 60-70° C. for reaction for 18-36 hours; the volume ratio of the 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.

Images