Non-glutaraldehyde cross-linked biological heart valve as well as preparation method and application thereof

Abstract

The invention discloses a non-glutaraldehyde cross-linked biological heart valve as well as a preparation method and application thereof, and belongs to the field of biomedical materials. The functional non-glutaraldehyde cross-linked BHV material (PT-BS-PP) with enhanced mechanical properties and excellent anticoagulation and anti-calcification properties is successfully prepared by adopting a synergistic cross-linking modification strategy of stabilizing a pig pericardium (PP) by using pentaerythritol acrylate and modifying and cross-linking the surface of the pig pericardium by using 4-vinyl benzene sulfonate in situ ATRP (Atom Transfer Radical Polymerization). In addition to the mechanical property similar to that of glutaraldehyde cross-linked PP (G-PP), PT-BS-PP also has better biocompatibility, stronger anticoagulation and anti-calcification capabilities and stronger endothelialization potential, and is a potential candidate material for clinical application of BHV.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a non-glutaraldehyde cross-linked biological heart valve and a preparation method and application thereof. Background Art

[0002] Valvular heart disease (VHD) is a major health burden affecting millions of people worldwide. The aging population has also led to an increase in the incidence of VHD. For these patients with severe VHD, heart valve replacement is inevitable when medications cannot control the disease. Traditional open-chest surgical heart valve replacement has high risks and a long recovery time, making it unsuitable for elderly patients. In recent years, minimally invasive transcatheter heart valve replacement (THVR) has become the cornerstone of treatment for patients with severe VHD, with more than 300,000 cases performed each year, due to its minimal trauma and rapid recovery. Compared with mechanical heart valves (MHVs), in addition to being compressible, bioprosthetic heart valves (BHVs) do not require long-term anticoagulation therapy and have better hemodynamics, making them the first choice for heart valve replacement, especially in the clinical application of THVR.

[0003] Most commercial BHVs use glutaraldehyde-crosslinked pericardium to improve the mechanical properties of the biomaterial for long-term implantation. However, glutaraldehyde-crosslinked BHVs are cytotoxic due to the presence of aldehyde residues, which can lead to cell apoptosis and further hinder the endothelialization process. Phospholipids released from apoptotic cell fragments bind to free calcium ions and attach to the BHV surface, ultimately accelerating the calcification of the BHV. In addition, during the decellularization process, the highly thrombogenic collagen structure is exposed, which can lead to platelet adhesion and activation, resulting in thrombus formation on the BHV surface. Due to these defects, the service life of glutaraldehyde-crosslinked BHVs is limited to only 10-15 years. Therefore, developing a glutaraldehyde-free crosslinking method to improve the anti-thrombotic properties and anti-calcification function of BHV is an attractive BHV therapeutic strategy.

[0004] Some non-glutaraldehyde cross-linking strategies have been reported to optimize the performance of BHV, such as proanthocyanidins, carbodiimides and polyepoxides. Unfortunately, BHV treated with these cross-linking agents still have disadvantages such as poor mechanical properties, low cross-linking degree, and fewer active functional groups that can be further modified, which limits its further clinical application. The patent application with publication number CN116173302A discloses a non-glutaraldehyde valve cross-linking agent OX-Br, and then the porcine pericardium is cross-linked with OX-Br to obtain the heart valve material OX-PP. On the basis of the heart valve material OX-PP, methyl methacrylate sulfonate betaine was further introduced through an in-situ polymerization reaction to obtain a non-glutaraldehyde cross-linked multifunctional heart valve material SA@OX-PP. However, the enzymatic degradation rate of the BHV material and the calcium ion content inside the valve still need to be reduced.

[0005] Therefore, developing an innovative cross-linking process to effectively reduce the enzymatic degradation rate of BHV and improve the anti-calcification ability of BHV has important research value and broad application prospects. Summary of the invention

[0006] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a non-glutaraldehyde cross-linked biological heart valve and a preparation method and use thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The invention provides a biological valve material, which is a product obtained by ATRP reaction of pericardium grafted with halogenated carboxylic acid, unsaturated polyol ester and benzene sulfonate containing unsaturated substituents.

[0009] Furthermore, the halogenated carboxylic acid is 2-bromo-2-methylpropionic acid; the unsaturated polyol ester is pentaerythritol acrylate; and the benzene sulfonate containing an unsaturated substituent is a benzene sulfonate containing a vinyl group, preferably sodium 4-vinylbenzene sulfonate.

[0010] Furthermore, the pericardium is decellularized pericardium, preferably decellularized porcine pericardium.

[0011] Furthermore, the halogenated carboxylic acid grafted pericardium is prepared by the following method: 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid are mixed and reacted, the pH is adjusted to obtain a mixed solution, and then the pericardium is soaked in the mixed solution to react, thereby obtaining the halogenated carboxylic acid grafted pericardium.

[0012] Furthermore, the reaction time after mixing the 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid is 0.5 to 5 hours, and the pH is adjusted to 7 to 10; the reaction time of the pericardium immersed in the mixed solution is 1 to 5 days; the temperature of the pericardium immersed in the mixed solution is 30 to 45°C; the molar ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid is 1:0.1 to 3:1 to 10.

[0013] Furthermore, the reaction time after mixing the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid is 2 hours, and the pH is adjusted to 8; the reaction time of the pericardium immersed in the mixed solution is 2 days; the temperature of the pericardium immersed in the mixed solution is 37°C; the molar ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid is 1:1:3.

[0014] The present invention also provides a method for preparing the above-mentioned biological valve material, comprising the following steps:

[0015] (1) immersing the pericardium grafted with halogenated carboxylic acid in an organic solvent, removing oxygen, adding unsaturated polyol ester and a catalyst, and performing an atom transfer radical polymerization reaction to obtain an intermediate product PT-PP;

[0016] (2) The intermediate product PT-PP is immersed in water to remove oxygen, and a benzenesulfonate containing an unsaturated substituent and a catalyst are added to carry out an atom transfer radical polymerization reaction. The reaction product is then washed with a ligand to obtain a biological valve material PT-BS-PP.

[0017] Furthermore, in step (1), the organic solvent is anhydrous ethanol; the method for removing oxygen is ultrasonic and argon flushing; the time for removing oxygen is 1 to 5 hours; the catalyst is 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl; the unsaturated polyol ester,

[0018] The molar ratio of 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl is 0.01-0.1:0.1-1:0.01-0.1:0.01-1; the temperature of the atom transfer radical polymerization reaction is 10-40° C., and the time is 10-40 hours;

[0019] In step (2), the method for removing oxygen is ultrasonic wave and argon flushing; the time for removing oxygen is 1 to 5 hours; the catalyst is 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl; the molar ratio of the benzenesulfonate containing unsaturated substituents, 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl is 0.01 to 0.1: 0.1 to 1: 0.01 to 0.1: 0.01 to 1; the temperature of the atom transfer radical polymerization reaction is 10 to 40° C., and the time is 10 to 40 hours; and the ligand is EDTA.

[0020] Furthermore, in step (1), the time for removing oxygen is 2 hours; the molar ratio of the unsaturated polyol ester, 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl is 0.028:0.2:0.02:0.1; the temperature of the atom transfer radical polymerization reaction is 25°C and the time is 24 hours;

[0021] In step (2), the oxygen removal time is 2 hours; the molar ratio of the benzenesulfonate containing unsaturated substituents, 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl is 0.048:0.2:0.02:0.1; the temperature of the atom transfer radical polymerization reaction is 25°C and the time is 24 hours.

[0022] The present invention also provides the use of the above-mentioned biological valve material in preparing an artificial biological valve.

[0023] Furthermore, the artificial bio-valve is an artificial heart valve, an artificial pulmonary valve or an artificial venous valve.

[0024] The present invention has achieved the following beneficial effects:

[0025] The present invention adopts a synergistic cross-linking modification strategy of tetraacrylate pentaerythritol stabilizing porcine pericardium (PP) and sodium 4-vinylbenzene sulfonate in situ ATRP modification to cross-link the surface of porcine pericardium, and successfully prepares a functional non-glutaraldehyde cross-linked BHV material (PT-BS-PP) with enhanced mechanical properties, excellent anti-coagulation and anti-calcification properties. In addition to having similar mechanical properties to glutaraldehyde cross-linked PP (G-PP), the material also has better biocompatibility, stronger anti-coagulation and anti-calcification capabilities and stronger endothelialization potential, and is a highly potential candidate material for the clinical application of BHV.

[0026] Compared with the non-glutaraldehyde cross-linked multifunctional heart valve material disclosed in the patent application with publication number CN116173302A, the enzyme degradation rate and calcium ion content inside the valve of the BHV material of the present invention are significantly reduced, and the biological stability and anti-calcification ability are significantly improved; at the same time, it has excellent mechanical properties.

[0027] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0028] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the preparation of the non-glutaraldehyde cross-linked BHV material PT-BS-PP of the present invention.

[0030] Figure 2 is the weight loss rate of D-PP, G-PP, PT-PP and PT-BS-PP.

[0031] Figure 3 (A) Ultimate tensile strength and (B) elongation of D-PP, PT-PP and PT-BS-PP.

[0032] Figure 4 (A) L929 cell cytotoxicity and (B) L929 cell viability staining (green) results of G-PP, PT-PP and PT-BS-PP.

[0033] Figure 5 Live fluorescence staining images of HUVECs on G-PP, PT-PP and PT-BS-PP after 1 day and 3 days of culture, as well as fluorescence staining images of the cytoskeleton (red) and nucleus (blue) of HUVECs on the membrane after 1 day of culture.

[0034] Figure 6 (A) Schematic diagram of the arteriovenous shunt method for determining the in vitro blood compatibility of PPs; (B) photos of G-PP, PT-PP and PT-BS-PP before and after the test; (C) quantitative statistics of thrombi attached to the surfaces of G-PP, PT-PP and PT-BS-PP after the in vitro shunt test and (D) SEM images of the surfaces.

[0035] Figure 7 (A) Alizarin red staining results and (B) calcification quantification results of G-PP, PT-PP and PT-BS-PP in rats after subcutaneous implantation for 30 days and 60 days. DETAILED DESCRIPTION

[0036] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0037] The following experiments without any explanation of temperature are reactions under normal temperature conditions, where normal temperature is room temperature, which is 25±5℃.

[0038] Example 1: Preparation of non-glutaraldehyde cross-linked BHV PT-BS-PP of the present invention

[0039] (1) Preparation of decellularized porcine pericardium

[0040] An aqueous solution containing 0.5% w / w sodium dodecyl sulfate and 0.5% w / w sodium deoxycholate was prepared as a decellularization solution, and fresh porcine pericardium (PP) was immersed in it and shaken on a shaker for 12 hours. The porcine pericardium was then taken out and washed repeatedly 5 times with deionized water to remove the decellularization solution. The obtained decellularized porcine pericardium (D-PP) was stored in glycerol for later use.

[0041] (2) Preparation of PT-BS-PP cross-linked valve

[0042] Prepare 50mg / ml 2-bromo-2-methylpropionic acid solution, add EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide) with a final concentration of 0.1mol / L to activate the carboxyl group. After reacting for 2h, adjust the pH of the reaction system to 8 with triethylamine. Fix D-PP with a frame, immerse it in the reaction mixture, and react at 37°C for 48h to successfully obtain Br-PP material.

[0043] Br-PP was placed in a reaction vessel containing 200 mL of anhydrous ethanol, and the oxygen in the solution was removed by ultrasonic and argon flushing for 2 h. Pentaerythritol acrylate (10.0 g, 0.028 mol) was added as a cross-linking monomer, and HEMETA (1,1,4,7,10,10-hexamethyltriethylenetetramine, 0.046 g, 0.2 mmol), CuCl2 (2.5 mg, 0.02 mmol) and CuCl (9.9 mg, 0.1 mmol) were added to carry out ATRP reaction for 24 h. After the reaction was completed, the obtained sample (PT-PP material) was washed with ethanol and deionized water to remove unreacted substances.

[0044] The cleaned PT-PP was placed in 200 mL of deionized water and rinsed with ultrasound and Ar2 for 2 h to remove O2. Sodium 4-vinylbenzenesulfonate (10.0 g, 0.048 mmol) was added as a functional monomer, and HEMETA (0.046 g, 0.2 mmol), CuCl2 (2.5 mg, 0.02 mmol) and CuCl (9.9 mg, 0.1 mmol) were added to the mixed solution for ATRP reaction for 24 h. After the reaction was completed, the reaction sample was washed with EDTA (1%, wt%) to obtain the PT-BS-PP material.

[0045] Comparative Example 1: Preparation of glutaraldehyde cross-linked BHV material G-PP

[0046] (1) Preparation of decellularized porcine pericardium

[0047] The operation is the same as step (1) of Example 1.

[0048] (2) Preparation of G-PP cross-linked valve

[0049] Decellularized porcine pericardium (D-PP) was fixed with a frame and immersed in a 0.625% w / w glutaraldehyde solution for 72 hours of shaking cross-linking. After cross-linking, the obtained glutaraldehyde cross-linked valve (G-PP) was stored in a 0.5% glutaraldehyde solution at 4°C.

[0050] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0051] Experimental Example 1: Degradation resistance test of the non-glutaraldehyde cross-linked BHV material PT-BS-PP of the present invention

[0052] The main component of porcine pericardium is collagen. The cross-linking efficiency based on ATRP reaction can be further characterized by the degradation of cross-linked PP by collagenase I.

[0053] Use PBS buffer solution with pH = 7.4 to prepare a collagenase I solution with a concentration of 1 mg / mL (125U / mL). Prepare D-PP, G-PP, PT-PP and PT-BS-PP into a size of 1cm×1cm and wash them three times with deionized water, with 6 parallel samples set up in each group. Freeze-dry each group of samples to constant weight, and weigh the mass of the sample with a one-hundred-thousandth balance, recorded as W0. Incubate each sample with 1mL of collagenase I solution for 24 hours, then wash it thoroughly with deionized water, freeze-dry it and weigh it, recorded as W1. The weight loss rate is used to evaluate the sample's ability to resist protease degradation, and the weight loss rate is calculated according to the following formula:

[0054]

[0055] The results are as follows Figure 2 As shown, the weight loss rates of PT-PP and PT-BS-PP are similar to those of G-PP, and the weight loss rates of PT-PP, PT-BS-PP and G-PP are 3.53±1.56%, 3.50±1.21% and 3.25±1.17%, respectively, which proves that the cross-linking strategy based on ATRP reaction and secondary ATRP modification of the present invention has a strong cross-linking efficiency, and the obtained non-glutaraldehyde cross-linked BHV material PT-BS-PP has excellent anti-degradation performance.

[0056] Experimental Example 2: Mechanical Strength Test of the Non-Glutaraldehyde Cross-linked BHV Material PT-BS-PP of the Present Invention

[0057] Cross-linking of the pericardium can provide sufficient mechanical properties for the bioprosthetic valve material to support the bioprosthetic valve in the process of opening and closing hundreds of millions or even billions of times without deformation and damage. Previous literature reports and clinical applications have proved that the valve material cross-linked with glutaraldehyde has excellent mechanical properties and can meet the requirements of long-term use of bioprosthetic valves.

[0058] D-PP, G-PP, PT-PP and PT-BS-PP were prepared into 40mm×10mm size and soaked in physiological saline overnight, with 6 parallel samples in each group. The thickness of each sample was measured and recorded using a thickness gauge. The tensile properties of the samples were determined using a uniaxial tensile tester (Bio-Tester 5000, Cellscale). All samples were kept wet during the test, and the stretching rate was set to 12.5mm / min until they were stretched to break. The stress-strain curve was obtained from the Cauchy stress and Green strain, and the ultimate tensile strength and elongation of each group of samples were obtained from the final stress-strain curve, and the tangent modulus and elongation were calculated from the slope of the linear region of the stress-strain curve.

[0059] The results are as follows Figure 3 As shown in Figure A, the ultimate tensile strengths of PT-PP, PT-BS-PP, and G-PP were 20.43 ± 1.29, 19.07 ± 1.41, and 19.26 ± 2.01 MPa, respectively, indicating that PT-PP and PT-BS-PP24 have good resistance potential to high cardiac load in vivo. The elongation of PT-P and PT-BS-PP was not significantly different from that of G-PP, as shown in Figure 2. Figure 3 As shown in B, the damage to the heart valve caused by material relaxation due to valve opening and closing hundreds of millions of times after implantation can be avoided. This experiment proves that the PT-BS-PP obtained by ATRP reaction has excellent mechanical properties.

[0060] Experimental Example 3: Cytotoxicity test of the non-glutaraldehyde cross-linked BHV material PT-BS-PP of the present invention Cytotoxicity is a basic indicator for testing the biocompatibility of biomaterials.

[0061] The preparation method of PPs extract is as follows:

[0062] G-PP, PT-PP and PT-BS-PP were prepared into 20 mm × 30 mm size, washed with PBS three times and then sterilized by soaking in 75% (v / v) ethanol for 12 h. The sterilized samples were washed with PBS five times to remove the residual ethanol solution. 2 The samples were immersed in DMEM complete medium (10% fetal bovine serum, 90% DMEM medium, 1% penicillin-streptomycin solution) containing 10% serum and 1% penicillin-streptomycin solution at a ratio of / mL, and extracted in a sterile constant temperature incubator at 37°C for 72h. The obtained extracts were the extracts of different PPs. The culture medium without sample addition was set as the control group.

[0063] L929 fibroblasts in good growth state were digested with trypsin, and counted after being fully blown to a single cell state. The cell concentration was adjusted to 25,000 / mL with DMEM complete medium, and the cells were planted on a 96-well plate at a density of 5,000 / well, and incubated at 37°C, 5% CO2 for 24 hours. The old culture medium was discarded, and 200 μL of material extract was added to each well, and the incubation continued for 24 hours to 72 hours. Six parallel samples were set up for each group, and 200 μL of DMEM complete medium was used as a control. After the extract and cells were incubated to the specified time point, the old culture medium was discarded, and each well was washed three times with sterile PBS solution. 200 μL of 10% CCK-8 reagent was added to each well to detect cell viability, and the cells were incubated in a 37°C constant temperature incubator for 1 hour. After the incubation was completed, the 96-well plate was taken out and wrapped with tin foil to avoid light. The OD value of each well at 450 nm was measured on a microplate reader (Synergy, Bio-Tek), and the cytotoxicity of each group of samples was calculated based on the OD value.

[0064] L929 cells were cultured with different PPs extracts, and the cell viability of each group was detected using the CCK-8 kit after 24h and 72h of incubation. Figure 4 As shown in A, after being cultured with PT-PP and PT-BS-PP extracts for 72 h, the viability of L929 cells was higher than 95%, but after being cultured with G-PP extract for 72 h, the cell viability of L929 was lower than 40%.

[0065] After culturing L929 cells with different PPs extracts for 1 day (i.e. 24 hours) and 3 days (i.e. 72 hours), FDA (fluorescein diacetate) dye was used to stain for 5 minutes in the dark to observe the status of L929 cells. Figure 4 As shown in B, there were more active cells in the PT-PP and PT-BS-PP groups compared with the G-PP group, which was consistent with the results of the cell viability assay.

[0066] The above experiments all show that PT-BS-PP has good cell compatibility.

[0067] Experimental Example 4: Test on the endothelial adhesion and proliferation performance of the non-glutaraldehyde cross-linked BHV material PT-BS-PP of the present invention

[0068] Prepare 10 mm diameter G-PP, PT-PP and PT-BS-PP sheet samples, sterilize with 75% (v / v) ethanol, wash 3 times with PBS, and place at the bottom of a 48-well plate. 4 Human umbilical vein endothelial cells (HUVECs) were seeded at a density of 10 cells.

[0069] ① After the cells have grown on the sample surface for 1 or 3 days, live cells were stained with FDA (fluorescein diacetate) solution for 5 minutes in the dark. The adhesion of live endothelial cells on the sample surface was photographed using a laser confocal microscope (Zeiss, LSM 800) in a layer scanning manner.

[0070] ② After the cells have grown on the sample surface for 1 day, a portion of the sample was fixed with 4% paraformaldehyde fixative, and then the cytoskeleton and nucleus of the cells on the sample surface were labeled with FITC-phalloidin and DAPI, respectively. The cell morphology on the sample surface was observed by laser confocal microscopy.

[0071] The results are as follows Figure 5 As shown in the figure, after 1 and 3 days of culture, only sparse endothelial cells were visible on the G-PP surface, and the staining results of the nucleus and cytoskeleton also showed that HUVECs on the G-PP surface were abnormally round, which may be due to the cytotoxicity caused by the residual aldehyde groups in G-PP. On the PT-PP and PT-BS-PP surfaces, more well-growing endothelial cells can be clearly observed, and there are more cells on the PT-BS-PP surface. It proves that the PT-BS-PP material is conducive to the adhesion and proliferation of endothelial cells.

[0072] Experimental Example 5: Anti-thrombotic performance test of the non-glutaraldehyde cross-linked BHV material PT-BS-PP of the present invention

[0073] The in vitro arteriovenous shunt (AV-shunt) experiment was approved by the Medical Ethics Committee of Sichuan University (Ethics Permit No. K2022020). The in vivo anticoagulant ability of the samples was evaluated by extracorporeal circulation in the rabbit carotid artery and vein. G-PP, PT-PP, and PT-BS-PP (10 mm × 15 mm, n = 3) were photographed and curled and attached to the inner wall of the circulation loop. Rabbits were first anesthetized by intravenous injection of sodium pentobarbital (2%, v / v, 30 mg / kg), and then the carotid artery and jugular vein were quickly isolated by local anesthesia with lidocaine solution. Then, the carotid artery and jugular vein were connected to the extracorporeal circulation with an indwelling needle. After interacting with blood for 1 hour, the samples were rinsed with saline, weighed, and photographed for comparison. Finally, the samples were fixed with 2.5% glutaraldehyde overnight. After drying with gradient ethanol dehydration, the surface adhesion of the samples was observed by scanning electron microscopy.

[0074] The schematic diagram of the in vitro arteriovenous shunt experiment is as follows Figure 6 As shown in A, the results are Figure 6 B~6D. Figure 6 It can be seen intuitively in B that after G-PP and PT-PP were in contact with flowing blood for 1 hour, many thrombi attached to the surface of G-PP and PT-PP, while only a thin layer of blood cells attached to the surface of PT-BS-PP. The results of statistical analysis of thrombus formation weight and surface morphology are shown in Figure 2. Figure 6As shown in C, the thrombosis rate of PT-BS-PP was reduced by 80% and 59% compared with G-PP and PT-PP, respectively. Figure 6 As shown in D, abundant thrombi and blood cells were observed on the surfaces of G-PP and PT-PP. In contrast, only a small amount of blood cells were observed on the surface of PT-BS-PP, which proved that the PT-BS-PP material of the present invention has significant anti-thrombotic ability.

[0075] Experimental Example 6: Anti-calcification performance test of the non-glutaraldehyde cross-linked BHV material PT-BS-PP of the present invention

[0076] Calcification is one of the most serious problems currently faced by bioprosthetic valve materials in clinical use. Calcification of bioprosthetic valves can cause tissue hardening, restrict their movement, and eventually cause valve failure. Prolonging the service life of bioprosthetic valves is to reduce the degree of calcification of bioprosthetic valves so that they can better serve young patients with valvular heart disease.

[0077] The use of experimental animals and experimental protocols were approved by the Medical Ethics Committee of Sichuan University. Each group of cross-linked valves was punched into a 1 cm × 1 cm square with a hole punch, sterilized with 75% ethanol, and then washed three times with sterile PBS buffer. SD rats (100 g ± 10 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (dose of 40 mg / kg). The hair on the back of the rat was shaved to expose the skin, and it was wiped and disinfected with iodine cotton balls. An incision of about 1 cm long was made in the upper and lower parts of the midline of the back, and the skin was expanded to the left and right to form a pocket shape. The cross-linked valves were placed in each pocket and sutured. Wait for the specified time to remove the sample for analysis.

[0078] The samples were taken out on the 30th and 60th day after the implantation. The samples were divided into two parts. One part was used for qualitative analysis of calcification. This part of the sample was taken out together with the surrounding tissue and fixed with paraformaldehyde for 24 hours. After being embedded in paraffin, it was prepared into sections and stained with alizarin red dye. The other part of the sample was used for quantitative analysis of calcification. The membrane samples of this part of the sample were washed three times with deionized water and freeze-dried. Each sample was weighed separately using a one-hundred-thousandth balance. The weighed samples were transferred to a 15mL centrifuge tube, 5mL of concentrated nitric acid was added to each centrifuge tube, and acid hydrolyzed at 95℃ for 2h. The sample solution after acid hydrolysis was made up to 10mL with deionized water, and the calcium concentration was measured by inductively coupled plasma spectrometer (ICP).

[0079] The specimens of G-PP, PT-PP and PT-BS-PP were taken out after 30 and 60 days of subcutaneous implantation, respectively, and calcification analysis was performed. The calcification of cross-linked PPs was detected by Alizarin red staining. Figure 7As shown in A. G-PP showed obvious small red spots on the slice image 30 days after implantation, and as the implantation time increased, the calcification deposits on G-PP became more dense, and the entire material turned red. However, no red spots appeared in the slice images of PT-PP and PT-BS-PP. Quantitative analysis of calcium content in ICP is shown in Figure 7 As shown in B, the calcium content of G-PP after 30 days and 60 days of implantation was 75.24±2.5μg / mg and 155.16±14.39μg / mg, respectively. Compared with G-PP with high calcium content, the calcium content of PT-PP was 2.88±1.61μg / mg and 4.92±0.51μg / mg after 30 days and 60 days of implantation, respectively; the calcium content of PT-BS-PP was 2.91±1.86 and 4.29±2.8μg / mg, respectively. The above results prove that the cross-linked modified PPs based on ATRP of the present invention have good anti-calcification ability and are expected to become BHV materials that improve the service life of materials to a certain extent.

[0080] In summary, the present invention adopts a synergistic cross-linking modification strategy of pentaerythritol tetraacrylate stabilizing porcine pericardium (PP) and sodium 4-vinylbenzene sulfonate in situ ATRP modification of the cross-linked porcine pericardium surface, and successfully prepares a functional non-glutaraldehyde cross-linked BHV material with enhanced mechanical properties, excellent anti-coagulation and anti-calcification properties. First, 2-bromo-2-methylpropionic acid is grafted onto decellularized porcine pericardium (D-PP) by EDC / NHS activation to obtain Br-PP. Then, pentaerythritol acrylate is selected as the monomer for the primary reaction of ATRP to prepare cross-linked polypropylene (PT-PP). Sodium 4-vinylbenzene sulfonate is used as a subsequent polymerization monomer to play a role in surface modification, giving the functional cross-linked PP (PT-BS-PP) anti-coagulation and anti-calcification biological activities. In addition to having similar mechanical properties to glutaraldehyde-cross-linked PP (G-PP), PT-BS-PP also has better biocompatibility, stronger anticoagulation and anticalcification abilities, and stronger endothelialization potential, demonstrating that PT-BS-PP is a highly promising candidate material for the clinical application of BHV.

Claims

1. A biological valve material, characterized in that: The product is obtained by ATRP reaction of pericardium grafted with halogenated carboxylic acid with unsaturated polyol ester and benzene sulfonate containing unsaturated substituents.

2. The biological valve material according to claim 1, characterized in that: The halogenated carboxylic acid is 2-bromo-2-methylpropionic acid; the unsaturated polyol ester is pentaerythritol acrylate; the benzene sulfonate containing an unsaturated substituent is a benzene sulfonate containing a vinyl group, preferably sodium 4-vinylbenzene sulfonate.

3. The biological valve material according to claim 1, characterized in that: The pericardium is decellularized pericardium, preferably decellularized porcine pericardium.

4. The biological valve material according to claim 1, characterized in that: The halogenated carboxylic acid grafted pericardium is prepared by the following method: 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid are mixed and reacted, the pH is adjusted to obtain a mixed solution, and then the pericardium is soaked in the mixed solution for reaction, thereby obtaining the halogenated carboxylic acid grafted pericardium.

5. The biological valve material according to claim 4, characterized in that: The reaction time after mixing the 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid is 0.5 to 5 hours, and the pH is adjusted to 7 to 10; the reaction time of the pericardium immersed in the mixed solution is 1 to 5 days; the temperature of the pericardium immersed in the mixed solution is 30 to 45°C; the molar ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid is 1:0.1 to 3:1 to 10.

6. The biological valve material according to claim 5, characterized in that: The reaction time after mixing the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid is 2 hours, and the pH is adjusted to 8; the reaction time of the pericardium immersed in the mixed solution is 2 days; the reaction temperature of the pericardium immersed in the mixed solution is 37°C; the molar ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and halogenated carboxylic acid is 1:1:

3.

7. A method for preparing the biological valve material according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) immersing the pericardium grafted with halogenated carboxylic acid in an organic solvent, removing oxygen, adding unsaturated polyol ester and a catalyst, and performing an atom transfer radical polymerization reaction to obtain an intermediate product PT-PP; (2) The intermediate product PT-PP is immersed in water to remove oxygen, and a benzenesulfonate containing an unsaturated substituent and a catalyst are added to carry out an atom transfer radical polymerization reaction. The reaction product is then washed with a ligand to obtain a biological valve material PT-BS-PP.

8. The method according to claim 7, characterized in that: In step (1), the organic solvent is anhydrous ethanol; the method for removing oxygen is ultrasonic wave and argon flushing; the time for removing oxygen is 1 to 5 hours; the catalyst is 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl; the molar ratio of the unsaturated polyol ester, 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl is 0.01 to 0.1: 0.1 to 1: 0.01 to 0.1: 0.01 to 1; the temperature of the atom transfer radical polymerization reaction is 10 to 40° C., and the time is 10 to 40 hours; In step (2), the method for removing oxygen is ultrasonic wave and argon flushing; the time for removing oxygen is 1 to 5 hours; the catalyst is 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl; the molar ratio of the benzenesulfonate containing unsaturated substituents, 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl is 0.01 to 0.1: 0.1 to 1: 0.01 to 0.1: 0.01 to 1; the temperature of the atom transfer radical polymerization reaction is 10 to 40° C., and the time is 10 to 40 hours; and the ligand is EDTA.

9. The method according to claim 8, characterized in that: In step (1), the oxygen removal time is 2 hours; the molar ratio of the unsaturated polyol ester, 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl is 0.028:0.2:0.02:0.1; the temperature of the atom transfer radical polymerization reaction is 25° C. and the time is 24 hours; In step (2), the oxygen removal time is 2 hours; the molar ratio of the benzenesulfonate containing unsaturated substituents, 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuCl2 and CuCl is 0.048:0.2:0.02:0.1; the temperature of the atom transfer radical polymerization reaction is 25°C and the time is 24 hours.

10. Use of the biological valve material according to any one of claims 1 to 6 in the preparation of an artificial biological valve; preferably, the artificial biological valve is an artificial heart valve, an artificial pulmonary valve or an artificial venous valve.

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