A functional cross-linked biological valve and its preparation method and use

Abstract

The present invention provides a functional cross-linked bioprosthetic valve, its preparation method, and use, belonging to the field of bioprosthetic valve materials. The present invention develops a bifunctional cross-linking agent: carboxylated oxazolidine (OX-CO). Porcine pericardium is then treated with OX-CO for further modification to produce a functional cross-linked bioprosthetic valve. This bioprosthetic valve exhibits excellent mechanical properties, thermal stability, biocompatibility, anti-inflammatory, anti-thrombotic, anti-calcification, and endothelialization-promoting properties, and has broad application prospects.

Description

Technical Field

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

[0002] Cardiovascular disease is a serious threat to human health and life. Valvular heart disease, as a cardiovascular disease, causes great physical and mental suffering to patients. Due to the special status of heart valves, there are currently no effective treatments. Heart valve replacement surgery has become the first choice for these patients with critical valvular heart disease. Currently, there are two most common types of artificial heart valves: mechanical heart valves and bioprosthetic heart valves. Compared with mechanical valves, bioprosthetic heart valves have a better anticoagulant effect, eliminating the need for patients to take anticoagulant medication throughout their lives, and are becoming increasingly popular among patients.

[0003] Currently, commercial bioprosthetic heart valves are typically made of porcine pericardium (PP) or bovine pericardium (BP) cross-linked with glutaraldehyde (Glut). Glutaraldehyde is used to give bioprosthetic heart valves sufficient mechanical properties to support hundreds of billions of opening and closing cycles without deformation during use. However, due to the residual aldehyde groups after cross-linking, glutaraldehyde-cross-linked valves have defects such as calcification and inflammation after implantation. Many researchers have tried to use some non-aldehyde cross-linking agents to replace glutaraldehyde, but most of them are limited in application due to insufficient mechanical properties or easy calcification.

[0004] The literature (T.Yu et al; Nonglutaraldehyde treated porcine pericardium with good biocompatibility, reduced calcification and improved Anti-coagulation for bioprosthetic heart valve applications; Chemical Engineering Journal 414(2021)128900) reported a non-glutaraldehyde heart valve cross-linker: hydroxymethylbicyclic oxazolidine (OX-OH), and used OX-OH and porcine pericardium as raw materials to prepare an OX-OH cross-linked biological heart valve (OX-OH-PP). Compared with the biological heart valve cross-linked by glutaraldehyde, OX-OH-PP not only maintains similar mechanical properties, but also shows better biocompatibility and resistance to calcification. However, in order to meet clinical needs, the anti-inflammatory and anticoagulant properties of OX-OH-PP still need to be further optimized to reduce the inflammatory response in the early stage of implantation and expand its application in biological valves with higher anticoagulation requirements such as pulmonary valves and venous valves. Summary of the Invention

[0005] The purpose of the present invention is to provide a functional cross-linked biological valve and its preparation method and use.

[0006] The present invention provides a biovalve material, which is prepared using pericardium and OX-CO as raw materials, wherein the structure of OX-CO is as follows:

[0007]

[0008] Furthermore, the pericardium is decellularized pericardium, preferably decellularized porcine pericardium.

[0009] The present invention also provides a method for preparing the above-mentioned biological valve material, which comprises the following steps: immersing the pericardium in an OX-CO solution and reacting at 35-40°C for 60-80 hours to obtain the biological valve material; the concentration of the OX-VI solution is 0.1-0.3M, preferably 0.2M.

[0010] The present invention also provides a modified biological valve material, which is prepared using the above-mentioned biological valve material and 3-aminophenylboronic acid as raw materials.

[0011] The present invention also provides a method for preparing the modified biovalvular material, comprising the following steps: immersing the biovalvular material in a solution containing 3-aminophenylboronic acid, reacting at 35-40° C. for 20-30 hours to obtain the modified biovalvular material;

[0012] Preferably, the solution containing 3-aminophenylboronic acid further comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the concentration of 3-aminophenylboronic acid is 0.05-0.2M, preferably 0.1M, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.05-0.2M, preferably 0.1M, and the concentration of N-hydroxysuccinimide is 0.05-0.2M, preferably 0.1M.

[0013] The present invention also provides a functional cross-linked biological valve material, which is prepared using the modified biological valve material and chlorogenic acid as raw materials.

[0014] The present invention also provides a method for preparing the above-mentioned functional cross-linked biological valve material, which comprises the following steps: immersing the above-mentioned modified biological valve material in a chlorogenic acid solution, reacting at room temperature for 20-30 hours, and obtaining a functional cross-linked biological valve material.

[0015] Furthermore, in the chlorogenic acid solution, the concentration of chlorogenic acid is 0.1-0.3M, preferably 0.2M.

[0016] Furthermore, the solvent in the chlorogenic acid solution is an ethanol aqueous solution, preferably an ethanol aqueous solution with a volume concentration of 10%.

[0017] The present invention also provides the use of the above-mentioned biological valve material, the above-mentioned modified biological valve material, and the above-mentioned functional biological valve material in the preparation of artificial biological valves; the artificial biological valve is preferably an artificial heart valve, an artificial pulmonary valve, or an artificial venous valve.

[0018] The present invention develops a bifunctional crosslinking agent: carboxylated oxazolidine (OX-CO). Then, porcine pericardium treated with OX-CO is reacted with 3-aminophenylboronic acid to prepare a biological heart valve XO-PB-PP. XO-PB-PP is further modified with chlorogenic acid to obtain a functional cross-linked biological heart valve OX-CA-PP.

[0019] The bioprosthetic valve material provided by the present invention has the following beneficial effects:

[0020] 1. On the one hand, by introducing a boronate bond that responds to reactive oxygen species (ROS), the chlorogenic acid in the bioprosthetic heart valve material of the present invention can be intelligently released; on the other hand, once acute inflammation occurs after bioprosthetic valve implantation due to intraoperative infection and other postoperative complications, the sudden increase in ROS will lead to the release of chlorogenic acid, alleviating the inflammatory response.

[0021] 2. In the initial stage of contact between blood and biological valves, chlorogenic acid can inhibit platelet adhesion and activation and reduce the risk of valve thrombosis; at the same time, chlorogenic acid can promote endothelial cell proliferation, which is conducive to the formation of a long-term interface with good blood compatibility.

[0022] 3. Leaflet thrombosis, the high cytotoxicity of G-PP, and inflammatory reactions all increase the risk of calcification. Once calcification occurs, the valve leaflet material structure may deteriorate, which is fatal to the bioprosthetic heart valve and seriously reduces its service life. However, the functional cross-linked bioprosthetic heart valve provided by the present invention significantly reduces calcification and prolongs the valve's service life.

[0023] 4. The bioprosthetic valve material provided by the present invention has excellent mechanical properties, thermal stability, biocompatibility, anti-inflammatory, anti-thrombotic, anti-calcification and endothelialization-promoting properties, and has broad application prospects.

[0024] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0025] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Schematic diagram of the preparation and working principle of the functional cross-linked valve.

[0027] Figure 2 : OX-OH and OX-CO 1 H NMR spectroscopy.

[0028] Figure 3 : Amino group conversion rate of cross-linked valve materials.

[0029] Figure 4 :.(A) Wide-scan XPS spectra of cross-linked valve materials before and after modification; (B) Ultimate tensile strength and tangent modulus of D-PP, G-PP, OX-CO-PP, OX-PB-PP and OX-CA-PP (n=5); (C) Thermal shrinkage temperature of D-PP, G-PP, OX-CO-PP, OX-PB-PP and OX-CA-PP (n=3, *p<0.05, **p<0.01); (D) Cumulative release of chlorogenic acid from OX-CA-PP within 32 h (n=3). Figure 5 : Microscopic morphology of valve material observed by SEM (bar=50μm).

[0030] Figure 6 :(A) Relative weight loss of D-PP, G-PP, OX-CO-PP, OX-PB-PP and OX-CA-PP after collagenase digestion (n=6); (B) Relative weight loss of each sample after elastase digestion (n=6); (C) Survival rate of L929 cells after incubation with the extracted valve material extract for 24 hours or 48 hours (n=6); (D) Fluorescence staining to detect the cytoskeleton (FITC-phalloidin, green) and cell nucleus (DAPI, blue) of HUVES cells grown on valve materials for 24 hours or 48 hours (*p<0.05, **p<0.01, ***p<0.001).

[0031] Figure 7 : Hemolysis rate (A) and hemolysis phenomenon (B) of the cross-linked valve material; (C) APTT and PT of plasma after 90 minutes of incubation with the cross-linked valve material; (D) Plasma C3a content after treatment with the cross-linked valve material; (E) Permeability of the catheter after 2 hours of arteriovenous blood circulation; Thrombus on the surface of the valve material after 2 hours of arteriovenous blood circulation (F) and SEM image (G).

[0032] Figure 8 (A) After incubation with the extracted valve material extract, TNF-α in the supernatant was detected by ELISA kit; (B) After incubation with the valve material extract, IL-6 in the supernatant was detected by ELISA kit; (C) Immunochemical staining with CD3 and CD68 markers 7 days after subcutaneous implantation (bar = 100 μm); (D) Image J counting of the number of CD3-positive and CD68-positive cells in the periphery of the specimen 7 days after implantation (n = 3). (*p < 0.05, **p < 0.01, ***p < 0.001).

[0033] Figure 9 : (A) Alizarin red staining of valve material 30 and 60 days after subcutaneous implantation to assess calcification (bar = 200 μm). (B) ICP-AES analysis of calcium content in valve material 30 and 60 days after subcutaneous implantation (n = 6). DETAILED DESCRIPTION

[0034] Unless otherwise specified, the raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercial products.

[0035] Unless otherwise specified, the operating temperature in the examples is room temperature (i.e., 25±5°C).

[0036] Unless otherwise specified, the solutions used in the examples are aqueous solutions.

[0037] OX-OH was prepared according to the method described in the literature (T. Yu et al; Nonglutaraldehyde treated porcinepericardium with good biocompatibility, reduced calcification and improved Anti-coagulation for bioprosthetic heart valve applications; Chemical Engineering Journal 414 (2021) 128900).

[0038]

[0039] Example 1: Preparation of a functional cross-linked bioresective heart valve

[0040] according to Figure 1 The process shown is to prepare a functional cross-linked bioremediation heart valve. The specific steps are as follows:

[0041] 1. Synthesis of crosslinker OX-CO

[0042]

[0043] Trimethylaminomethane (36.00 g, 0.297 mol) and paraformaldehyde (20.52 g, 0.684 mol) were dissolved in toluene (300 mL) in a 500 mL three-necked flask and heated to reflux for 8 h. After the reaction, toluene was removed by rotary evaporation, and the crude product was recrystallized from ethyl acetate on ice to obtain hydroxymethyloxazolidine (OX-OH).

[0044] OX-OH (48.00 g, 0.331 mol) and succinic anhydride (36.2 g, 0.360 mol) were dissolved in dry tetrahydrofuran (200 mL). After refluxing at 130° C. for 8 hours, white crystals were obtained by rotary evaporation, and the crude product was recrystallized from ethyl acetate on ice to obtain carboxymethyloxazolidine (OX-CO).

[0045] OX-OH and OX-CO were successfully prepared with yields of 86% and 61% respectively. Figure 2 As shown: 1 H NMR (400 MHz, CDCl 3 ) δ = 4.46 (dd, J = 19.0, 5.5, 4H), 3.78 (q, J = 8.8, 4H”), 3.59 (d, J = 13.5, 2H), 2.93 (s, 1H). 1 H NMR (400 MHz, CDCl3) δ = 7.27 (s, 11H), 4.55–4.33 (m, 59H), 4.19 (s, 26H), 3.89–3.74 (m, 58H), 3.62 (s, 2H), 3.22 (s, 1H), 2.77–2.49 (m, 57H), -0.00 (s, 7H).

[0046] 2. Preparation of Functional Cross-linked Bioprosthetic Heart Valves

[0047] Fresh porcine pericardium was immersed in a PBS buffer solution containing 0.5% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, and 1% penicillin-streptomycin under vigorous stirring for 12 h and then rinsed five times with deionized water to obtain decellularized porcine pericardium (D-PP) and stored in an ethanol / glycerol solution.

[0048] The obtained D-PP was washed twice with deionized water and immersed in an OX-CO solution (0.2 M) for 72 h with stirring at 37° C. The supernatant was then discarded and ultrasonically cleaned with deionized water five times to obtain OX-CO cross-linked PP (OX-CO-PP).

[0049] OX-CO-PP was soaked in 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.1 M N-hydroxysuccinimide (NHS) for 2 h, and then 0.1 M 3-aminophenylboronic acid was added. After stirring at 34°C for 24 h, the mixture was taken out and rinsed with deionized water five times to obtain OX-PB-PP.

[0050] OX-PB-PP was immersed in a 10% (v / v) ethanol aqueous solution containing 0.2 M chlorogenic acid for 24 hours, taken out and washed with ethanol five times to obtain OX-CA-PP.

[0051] Comparative Example 1: Preparation of G-PP

[0052] A 1.5% (w / v) glutaraldehyde solution was prepared, to which 6% (w / v) NaCl was added, and the pH was adjusted to 5 with 1M hydrochloric acid. The fixed decellularized porcine pericardium (D-PP) was then placed vertically in the glutaraldehyde solution and shaken at room temperature for 2 hours to allow the glutaraldehyde solution to penetrate as much of the pericardium as possible. Finally, the pH was slowly adjusted to neutral using a saturated sodium carbonate solution. The solution was then shaken at 120 rpm for 24 hours at room temperature for cross-linking. After removal, the solution was ultrasonically cleaned five times to obtain the glutaraldehyde-cross-linked bioprosthetic heart valve: Glut-PP.

[0053] The following experimental examples demonstrate the beneficial effects of the cross-linked valve material prepared by the present invention.

[0054] Experimental Example 1: Physical and Chemical Performance Test

[0055] 1. Experimental methods

[0056] The cross-linked valve materials were characterized by X-ray photoelectron spectroscopy (XPS). The surface morphology and amine content of the cross-linked valve materials were observed by scanning electron microscopy and analyzed by the ninhydrin method. The elemental content was determined by elemental analysis (Vario EL cube, Elementar), and the C, N, H, and B contents of OX-PB-PP and OX-CA-PP were determined by inductively coupled plasma spectroscopy (ICP-AES) (VG PQExCell, TJA, USA).

[0057] Assuming that the nitrogen molar amount between OX-PB-PP and OX-CA-PP is equal, the drug loading capacity of chlorogenic acid can be calculated by the following formula:

[0058] N P ×M P =N C ×(M P +M C ),

[0059] Drug loading = M C / (M P +M C )=(N P -N C ) / N P ×100%,

[0060] where N P is the mass fraction of N in OX-PB-PP, M P is the mass of OX-PB-PP, N C is the mass fraction of N in OX-CA-PP, M C is the mass of chlorogenic acid grafted on OX-PB-PP.

[0061] Uniaxial tensile test: Cross-linked PP was cut into rectangles (10 mm × 40 mm), and the thickness was measured and recorded using a thickness gauge. The ultimate tensile strength was evaluated using a uniaxial tensile testing machine, and the tangent modulus was obtained from the stress-strain curve.

[0062] After weighing, the sample was placed in a sealed aluminum crucible and then heated from 40°C to 110°C at a rate of 10°C / min under N2 atmosphere using a differential scanning calorimeter (DSC, TA Instruments, USA).

[0063] 2. Experimental results

[0064] Table 1. Elemental analysis results of OX-PB-PP and OX-CA-PP (n=3)

[0065]

[0066] The cross-linking efficiency was quantitatively evaluated by the ninhydrin method. Figure 3 As shown in Figure 2, the cross-linked valve materials OX-CO-PP, OX-PB-PP, and OX-CA-PP showed a high amino conversion rate and had a cross-linking efficiency comparable to that of G-PP. The surface chemical composition of these cross-linked valve materials was analyzed by XPS. Figure 4 As shown in Figure A. In the wide scan spectrum of OX-PB-PP, the boron element signal is attributed to aminophenylboronic acid. Compared with OX-PB-PP, the proportion of C in OX-CA-PP increases and the proportion of N decreases, which is attributed to the grafting of chlorogenic acid. The boron content of OX-PB-PP and OX-CA-PP was determined by ICP-AES to be 2.39±0.03 mg / g, and the aminophenylboronic acid content of OX-PB-PP was calculated to be 30.01±0.32 mmol / g. The C, H, and N contents were detected by elemental analysis to quantify the chlorogenic acid loading capacity. According to the formula in the above experiment, the chlorogenic acid loading was calculated to be 23.74% (M / M).

[0067] The microstructure of the cross-linked valve material was observed by SEM. Figure 5 As shown in the figure, the collagen fibers of D-PP are loose and have many gaps, while after cross-linking of G-PP, OX-CO-PP, OX-PB-PP and OX-CA-PP, the collagen fibers are tightly connected and more dense, which will be beneficial to improve the mechanical properties and thermal stability of valve materials. Figure 4 As shown in Figure 2, the ultimate tensile strength of all cross-linked valves (G-PP: 14.3±1.7 MPa; OX-CO-PP: 15.17±2.02 MPa; OX-PB-PP: 16.56±2.43 MPa; OX-CA-PP: 17.35±1.75 MPa) was significantly higher than that of D-PP (10.3±1.29 MPa), indicating that the cross-linking agent formed strong chemical bonds between the collagen fibers. Furthermore, the tangent modulus of all cross-linked valve materials (G-PP: 81.95±11.9 MPa; OX-CO-PP: 90.47±7.98 MPa; OX-PB-PP: 89.41±5.82 MPa; OX-CA-PP: 87.71±7.93 MPa) was also higher than that of D-PP (54.14±8.52 MPa). There was no significant difference in the ultimate tensile strength and tangent modulus between G-PP and these non-glutaraldehyde cross-linked valve materials, indicating that OX-CO-PP, OX-PB-PP, and OX-CA-PP have good mechanical properties. Figure 4 C It can be seen that the thermal shrinkage temperatures of all cross-linked valve materials are similar (G-PP: 86.61±1.52℃; OX-CO-PP: 86.47±0.89℃; OX-PB-PP: 87.76±0.01℃; OX-CA-PP: 88.46±2.79℃), but are significantly higher than that of D-PP (73.28±1.52℃), further illustrating that OX-CO-PP, OX-PB-PP and OX-CA-PP have a high degree of cross-linking and good thermal stability.

[0068] Experimental Example 2: Chlorogenic acid release assay

[0069] 1. Experimental methods

[0070] Drug release: OX-CA-PP was immersed in 2 mL of 1 mM H2O2 solution and continuously shaken at 37°C. The supernatant was removed at predetermined intervals and refilled with the same volume of fresh culture medium containing 1 mM H2O2. The fluorescence intensity of the extracted supernatant was measured using a fluorescence microplate reader.

[0071] 2. Experimental results

[0072] It is well known to those skilled in the art that, along with the inflammatory process, the proliferation of inflammatory cells will express a large amount of reactive oxygen species (ROS). Figure 4D shows that in response to ROS stimulation, the effective cumulative release of chlorogenic acid for 32 hours was 117.73 ± 20.24 μg / cm 2 Without ROS stimulation, only a small amount of chlorogenic acid leakage occurred within 32 hours (32.40±5.65μg / cm 2 This indicates that when inflammation occurs, the ROS expressed by proliferating inflammatory cells triggers the efficient release of chlorogenic acid from the implanted bioprosthetic valve material, enabling on-demand intelligent drug release and in situ inflammation relief.

[0073] Experimental Example 3: Enzyme degradation experiment

[0074] 1. Experimental methods

[0075] After freeze-drying, the original dry weight of the different samples was recorded. They were then incubated in collagenase or elastase solution for 24 hours. The resulting samples were then freeze-dried again and weighed.

[0076] 2. Experimental results

[0077] The stability of the cross-linked valve material was further evaluated by measuring its resistance to enzymatic degradation. Figure 6 As can be seen in Figures A and B, compared with D-PP, all other cross-linked valve materials exhibited good resistance to enzymatic degradation. Under collagenase or elastase degradation, OX-CO-PP and OX-PB-PP both exhibited similar weight loss rates to G-PP, while OX-CA-PP exhibited a relatively higher weight loss rate than the other cross-linked valve materials. This may be due to the instability of the boronate bond and the release of chlorogenic acid during enzymatic degradation.

[0078] Experimental Example 4: Cytotoxicity Experiment

[0079] 1. Experimental methods

[0080] The cytotoxicity of the cross-linked valve material was evaluated according to ISO 109993-5. After sterilization, the cross-linked valve material was placed in DMEM complete medium (6 cm 2 The cells were then immersed in a 5% PBS (surface area / volume) solution for 72 hours. L929 cells were trypsinized. After uniform dispersion, the cells were seeded into a 96-well plate at a density of 5,000 cells. After 24 hours of culture, the culture medium was replaced with the above-mentioned extraction medium. After an additional 24 or 48 hours of culture, cell viability was determined using a CCK-8 assay.

[0081] 2. Experimental results

[0082] like Figure 6As shown in Figure C, after 24 or 48 hours, the cell viability of OX-CO-PP, OX-PB-PP, and OX-CA-PP was high, and the cell proliferation of OX-CA-PP was significant, indicating that these cross-linked valve materials have good cytocompatibility. In contrast, the viability of L929 cells incubated with G-PP extract was only 45% after 24 hours, and further decreased to 38% after 48 hours, indicating that G-PP is relatively toxic.

[0083] Experimental Example 5: Endothelial Adhesion Assay

[0084] 1. Experimental methods

[0085] After sterilization in 75% ethanol for 24 hours and thorough rinsing with PBS, samples (10 mm diameter discs) were placed in 48-well plates. Human vascular endothelial cells (HUVECs) were seeded on the sample surfaces at a density of 2 × 104 cells. After 24 or 48 hours of culture, the samples were slowly removed and fixed with 2.5% glutaraldehyde. The cytoskeleton and nuclei were then labeled with TRITC phalloidin and DAPI, respectively. The stained cells were observed under a laser confocal microscope (Zeiss LSM800, DE).

[0086] 2. Experimental results

[0087] like Figure 6 As shown in D, endothelial cells grown in G-PP were round and fewer in number at 24 or 48 h, which may be due to its high toxicity and inhibitory effect. Compared with G-PP, a large number of endothelial cells with normal stretching and spindle-shaped morphology were found in the OX-CO-PP, OX-PB-PP, and OX-CA-PP groups. Among them, OX-CA-PP showed the best endothelial cell proliferation.

[0088] Experimental Example 6: Blood Compatibility Test

[0089] 1. Experimental methods

[0090] Red blood cells were collected from rabbit blood centrifuged at 1000 rpm for 15 min and then diluted 10-fold with PBS. Each sample, 10 mm in diameter, was immersed in 800 mL of PBS solution and 200 mL of RBCs were added. Deionized water and PBS were used as positive and negative controls, respectively. After incubation at 37°C for 3 hours, the sample was removed and centrifuged at 3000 rpm for 15 minutes. The absorbance of the supernatant was measured at 545 nm using a microplate reader.

[0091] Platelet-poor plasma (PPP) was obtained from rabbit blood centrifuged (3000 rpm, 15 minutes). Each sample (10 mm diameter) was immersed in 500 μL PPP at 37°C for 90 minutes. The activated partial thromboplastin time (APTT) and prothrombin time (PT) of the obtained plasma were then analyzed using a semi-automated hematology analyzer. C3a levels were measured using a C3a ELISA kit.

[0092] 2. Experimental results

[0093] As a blood contact material, blood compatibility is very important. The hemolysis rate of cross-linked valve materials is as follows: Figure 7 As shown in A, the hemolysis rate of all cross-linked valve materials is less than 1%, which meets the safety standard of less than 5%, indicating good blood safety. Figure 7 As shown in B, no obvious hemolysis was observed in each cross-linked valve material group.

[0094] The anticoagulant effect was assessed by APTT and PT. Figure 7 As shown in Figure C, there was no significant difference in PT among all groups. The APTT values ​​of G-PP, OX-CO-PP, and OX-PB-PP were similar, 14.97s, 18.8s, and 17.97s, respectively, while the APTT of OX-CA-PP (28.6s) was significantly higher. The interaction between blood and the sample surface may lead to variable activation of the complement immune system, which was assessed by measuring C3a content. Figure 7 As shown in Figure 2D, there was no significant difference in C3a content among all cross-linked valve materials compared with the control group, indicating that these cross-linked valve materials did not cause acute immune system activation.

[0095] Experimental Example 7: Arteriovenous Shunt

[0096] 1. Experimental methods

[0097] All animal experiments were approved by the Animal Care and Use Committee of the National Biomaterials Engineering Research Center. New Zealand white rabbits were anesthetized by injecting sodium pentobarbital (40 mg / kg) into the ear vein. The fat and muscle tissue near the carotid artery and jugular vein were stripped to expose the carotid artery and internal jugular vein (Scheme 2). The catheter containing the sample was then connected to the blood vessel to allow blood to pass through. After circulating for 2 hours, the material was removed, and the blood cells adsorbed on the surface were gently washed with physiological saline, and the thrombus formation was observed, and then fixed with 4% paraformaldehyde. After gradient ethanol dehydration and freeze-drying, the surface of the material was observed by scanning electron microscopy.

[0098] 2. Experimental results

[0099] Extracorporeal arteriovenous shunts allow blood to flow through the material, observing the risk of thrombosis. Figure 7 The results in E show that the catheters loaded with G-PP and OX-CO-PP formed thrombi and blocked, while the catheters loaded with OX-PB-PP and OX-CA-PP remained transparent. After removing these cross-linked valve materials, the thrombi on the surface of the materials were Figure 7 F. There was a lot of thrombus formation in G-PP and some thrombus formation in OX-CO-PP. However, no obvious thrombus formation was found in OX-PB-PP and OX-CA-PP. Scanning electron microscopy results showed ( Figure 7 G-PP and OX-CO-PP showed a large number of thrombus complexes composed of fibrin and blood cells, while OX-PB-PP showed fewer blood cells, and OX-CA-PP showed the least. OX-CA-PP's favorable hematologic safety and superior anticoagulant properties significantly reduce the risk of thrombosis after implantation.

[0100] Experimental Example 8: In vitro anti-inflammatory experiment

[0101] 1. Experimental methods

[0102] RAW 264.7 cells were digested and seeded in a 4 After 24 hours of culture, the cells were pretreated with LPS (10 μg / mL) for 3 hours, and then the original culture medium was replaced with 500 μL of DMEM medium containing 1 mM H2O2 solution. Subsequently, different groups of cross-linked valve materials (10 mm round) were co-incubated with macrophages for 24 hours. TNF-α and IL-6 levels were detected by ELISA kits. The viability of RAW264.7 cells was determined by CCK-8 analysis using the same method as the above L929 cells.

[0103] 2. Experimental results

[0104] The inflammatory response after implantation is one of the key factors in the calcification of bioprosthetic heart valve leaflets. A milder inflammatory response can help reduce the risk of leaflet calcification. This study investigated the inflammatory response of cross-linked valve materials. Figure 8As shown. First, the RAW cell activity results showed that the proliferation of macrophages in the OX-CO-PP, OX-PB-PP and OX-CA-PP groups was not disturbed. However, the survival rate of macrophages in the G-PP group was reduced, mainly due to its high cytotoxicity. Macrophages were pre-activated with LPS (10 μg / mL) to simulate an inflammatory environment. The proinflammatory factors TNF-α and IL-6 secreted by macrophages were quantitatively analyzed using an ELISA kit. Compared with G-PP, OX-CO-PP and OX-PB-PP, which did not inhibit the inflammatory response, OX-CA-PP had a significant anti-inflammatory effect, and the levels of TNF-α and IL-6 were reduced by 48.52% and 49.49%, respectively, compared with the LPS group, indicating that chlorogenic acid released after ROS activation can play an important role in inhibiting the inflammatory response. Macrophages and T lymphocytes were labeled with CD68 and CD3 antibodies, respectively. According to the results of immunohistochemical staining, the antibody expression of G-PP on the 7th day was significantly higher than that of non-glutaraldehyde cross-linked valve materials, while the antibody expression of OX-CA-PP was the lowest after the 7th day, further indicating that OX-CA-PP has better biocompatibility and anti-inflammatory effects.

[0105] Experimental Example 9: Subcutaneous Implantation Experiment

[0106] 1. Experimental methods

[0107] All animal experiments were approved by the Animal Care and Use Committee of the National Biomaterials Engineering Research Center. The cross-linked valve material was sterilized with 75% ethanol solution and then rinsed thoroughly with PBS. SD rats (approximately 50 g) were anesthetized with 40 mg / kg of sodium pentobarbital, and the cross-linked valves were implanted subcutaneously on the back. The specimens and surrounding tissues were placed in sample bottles on days 7, 30, and 60. The calcium content in the cross-linked valves was determined by ICP-AES. Furthermore, the samples were fixed and stained with alizarin red to observe calcium deposition.

[0108] 2. Experimental results

[0109] The calcification of cross-linked valve material Alizarin red can chelate with calcium ions to form a red complex. The darker the red, the more serious the calcification. Figure 9 As shown in A, the calcification of G-PP increased significantly from 30 to 60 days, while the calcification of OX-CO-PP, OX-PB-PP and OX-CA-PP was not obvious at 30 or 60 days. Figure 9B) After 30 days of implantation, the calcium content of G-PP (48.17 mg / g) was significantly higher than that of OX-CO-PP (2.72 mg / g), OX-PB-PP (1.98 mg / g), and OX-CA-PP (2.05 mg / g). After 60 days of implantation, the calcium content of G-PP increased to 129.14 mg / g, whereas the calcium content of OX-CO-PP (2.43 mg / g), OX-PB-PP (3.67 mg / g), and OX-CA-PP (4.04 mg / g) remained at a lower level.

Claims

1. A use of a functional cross-linked bioprosthetic valve material in the preparation of an artificial bioprosthetic valve, characterized in that: The functional cross-linked biovalve material is prepared using a modified biovalve material and chlorogenic acid as raw materials; the modified biovalve material is prepared using a biovalve material and 3-aminophenylboronic acid as raw materials; the biovalve material is prepared using pericardium and OX-CO as raw materials, wherein the pericardium is porcine pericardium after decellularization, wherein the structure of OX-CO is as follows: ； The method for preparing the functional cross-linked bioprosthetic valve material comprises the following steps: immersing the modified bioprosthetic valve material in a chlorogenic acid solution, reacting at room temperature for 20-30 hours to obtain a functional cross-linked bioprosthetic valve material; the chlorogenic acid concentration in the chlorogenic acid solution is 0.2 M; The preparation method of the biological valve material comprises the following steps: immersing the pericardium in an OX-CO solution, reacting at 35-40° C. for 60-80 hours to obtain the biological valve material; the concentration of the OX-CO solution is 0.2 M; The preparation method of the modified bioprosthetic valve material comprises the following steps: immersing the bioprosthetic valve material in a solution containing 3-aminophenylboronic acid, reacting at 35-40° C. for 20-30 hours to obtain the modified bioprosthetic valve material; the solution containing 3-aminophenylboronic acid further comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the concentration of 3-aminophenylboronic acid is 0.1 M, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.1 M, and the concentration of N-hydroxysuccinimide is 0.1 M.

2. The use according to claim 1, characterized in that: The solvent in the chlorogenic acid solution is ethanol aqueous solution.

3. The use according to claim 2, characterized in that: The solvent in the chlorogenic acid solution is an ethanol aqueous solution with a volume concentration of 10%.

4. The use according to claim 1, characterized in that: The artificial biovalve is an artificial heart valve, an artificial pulmonary valve or an artificial venous valve.

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