A self-repairing biological heart valve and a preparation method thereof

Abstract

The application relates to the field of biomimetic materials and discloses a self-repairing biological heart valve and a preparation method thereof. The biological heart valve comprises a tannic acid cross-linked pericardium modified by a polydopamine nanoparticle-loaded polyhydroxy hydrogel protective layer; the polydopamine nanoparticles are distributed on the surface of the tannic acid cross-linked pericardium and / or in the polyhydroxy hydrogel protective layer. The biological heart valve has good mechanical properties and can realize in-situ damage self-repairing under near-infrared light, which helps to improve the durability of the biological heart valve.

Description

Technical Field

[0001] The present invention relates to the field of bionic materials, and in particular to a self-repairing biological heart valve and a preparation method thereof. Background Art

[0002] Valvular calcific heart disease is a global, elderly disease. If treatment measures are not taken in time, it will lead to adverse cardiovascular and cerebrovascular events. In recent years, transcatheter heart valve replacement (THVR) has become a common treatment for patients with severe valvular stenosis due to its advantages such as less trauma and no need for lifelong anticoagulant medication. Glutaraldehyde-cross-linked pig or bovine pericardium is commercially used as a biomaterial for THVR, but this type of biological heart valve often faces the problem of thrombosis, and due to multifactorial processes such as allograft-related immune inflammatory response, calcification and surface damage (tear or damage), the biological valve eventually fails. Because biological valves cannot regenerate and remodel the extracellular matrix, any changes in the collagen network (such as stratification, structural rearrangement and destruction) are irreversible and difficult to intervene, which ultimately affects the durability of the biological valve. Therefore, for patients with biological valve failure who have a longer life expectancy, a secondary, high-risk valve replacement is usually required.

[0003] The human body's natural aortic valve is mainly composed of a cell protection barrier formed by valve endothelial cells and valve interstitial cells embedded in the extracellular matrix. Under physiological conditions, endothelial cells inhibit the adhesion and aggregation of platelets, immune inflammatory cells, etc. by releasing important signaling molecules such as nitric oxide and prostacyclin, and play an important role in maintaining the homeostasis of heart valves. Therefore, the endothelial cell layer simulation strategy can be used as an ideal modification method for blood-contact materials. In recent years, endothelial biomimetic strategies have been widely used in various biomaterials such as vascular stents, wound dressings, and tumors. For example, patent CN201810732906.0 discloses an anticoagulant hydrogel coating that can catalyze the release of nitric oxide and its preparation method. By applying this hydrogel coating to the surface of the heart valve, it can be used to catalyze the release of NO, thereby exerting an anticoagulant effect and avoiding the formation of thrombus. However, the above method can only give the heart valve better biological functions, but cannot give it better mechanical properties and self-repair functions. Due to the special hemodynamic environment of the valve, the valve endothelial cells are exposed to more complex shear stress and blood flow patterns. Therefore, valve biomaterials not only need to have good biological functions, but also the improvement of mechanical properties and post-injury repair are also the key to solving the failure of biological valves. Summary of the Invention

[0004] To address the technical issues of existing biorepairable heart valves, which suffer from poor mechanical properties and an inability to self-repair, the present invention provides a self-repairing biorepairable heart valve and its preparation method. The biorepairable heart valve exhibits superior mechanical properties and can self-repair in situ under near-infrared light, thereby improving the durability of the biorepairable heart valve.

[0005] The specific technical solutions of the present invention are:

[0006] In a first aspect, the present invention provides a self-repairing biological heart valve; the self-repairing biological heart valve comprises a tannic acid cross-linked pericardium modified with a polyhydroxy hydrogel protective layer loaded with polydopamine nanoparticles; the polydopamine nanoparticles are distributed on the surface of the tannic acid cross-linked pericardium and / or in the polyhydroxy hydrogel protective layer.

[0007] Tannic acid molecules contain 25 phenolic hydroxyl groups, which not only cross-link with protein chains in the pericardium but also form strong hydrogen bonds with the polyhydroxy hydrogel protective layer and polydopamine nanoparticles, ensuring a secure bond between the hydrogel protective layer and the pericardium. Simultaneously, the polydopamine nanoparticles form strong hydrogen bonds with the polyhydroxy hydrogel protective layer and act as a cross-linker within the polyhydroxy hydrogel protective layer. This approach provides superior mechanical support for the entire bioprosthetic heart valve, imparting superior mechanical properties and improving its durability.

[0008] Furthermore, in the present invention, polydopamine nanoparticles can serve as a crosslinker and photothermal agent for the polyhydroxy hydrogel protective layer, exhibiting a strong photothermal absorption effect under near-infrared light. This can impart enhanced self-repair capabilities to the physically crosslinked polyhydroxy hydrogel protective layer, driving the self-repair of the bioprosthetic heart valve. Therefore, when a bioprosthetic heart valve of the present invention is damaged, it can be repaired in situ under near-infrared light, resulting in improved durability and eliminating the need for secondary, high-risk valve replacement surgery after injury.

[0009] Preferably, the polydopamine nanoparticles are compounded with an amino-containing NO catalytic releaser.

[0010] Amino-containing NO catalytic releasers can be stably fixed in polydopamine nanoparticles through covalent bonding of amino groups and catechol groups. After contacting with NO donors in the blood (such as S-nitrosoglutathione), they can promote NO production, thereby exerting the effects of anti-platelet aggregation, anti-thrombotic adhesion, anti-inflammation and promoting endothelialization.

[0011] The present invention adds an amino-containing NO catalyst-releasing agent to polydopamine nanoparticles, which has the following advantages: if the amino-containing NO catalyst-releasing agent is added to the hydrogel protective layer, the bonding strength between the NO catalyst-releasing agent and the hydrogel protective layer is relatively weak, and it may fall off during long-term use, resulting in reduced function; if the amino-containing NO catalyst-releasing agent is bound to tannic acid, although the NO catalyst-releasing agent can be stably fixed, it is present at the interface between the pericardium and the hydrogel protective layer and is not easily contacted with NO donors in the blood; in contrast, by adding the amino-containing NO catalyst-releasing agent to the polydopamine nanoparticles, the NO catalyst-releasing agent can be stably fixed in the biological heart valve. At the same time, because the polydopamine nanoparticles are larger than tannic acid molecules, they can penetrate into the interior of the polyhydroxy hydrogel protective layer and even be exposed on its surface, which is conducive to the contact between the NO catalyst-releasing agent and the NO donors in the blood, thereby promoting NO production.

[0012] Furthermore, the amino-containing NO catalytic releaser is selenocystamine.

[0013] Selenocystamine is a common NO catalytic releaser that can produce nitric oxide by contacting S-nitrosoglutathione in the blood, and the amino groups it contains can enable it to be stably fixed in polydopamine nanoparticles.

[0014] Preferably, antiplatelet drugs are dispersed in the polyhydroxy hydrogel protective layer.

[0015] By dispersing antiplatelet drugs in the polyhydroxy hydrogel protective layer, the biological heart valve can quickly release antiplatelet drugs in the early stage of transplantation, thereby exerting an antiplatelet viscosity effect and avoiding thrombosis.

[0016] In addition, the NO catalytic releaser complexed in polydopamine nanoparticles and the antiplatelet drug dispersed in the polyhydroxy hydrogel protective layer can be combined to quickly release the antiplatelet drug in the early stage of transplantation and gradually release the antiplatelet drug and NO in the later stage of transplantation, thereby having better anti-thrombotic adhesion function in both the early and late stages.

[0017] Furthermore, the antiplatelet drug is tirofiban.

[0018] Preferably, part or all of the polydopamine nanoparticles are modified on the surface of the tannic acid cross-linked pericardium.

[0019] In polyhydroxy polymer hydrogels, the binding stability of polydopamine nanoparticles is relatively low; however, on tannic acid-cross-linked pericardium, the action of tannic acid can improve the binding efficiency and binding stability of polydopamine nanoparticles, thereby enabling the biological heart valve to maintain good self-repair ability during long-term use. Moreover, when the polydopamine nanoparticles are compounded with NO catalytic releasers, by improving the binding stability of the polydopamine nanoparticles, it also helps the biological heart valve maintain good NO release function over a long period of time.

[0020] Preferably, the pericardium is porcine pericardium or bovine pericardium.

[0021] Preferably, the matrix material of the polyhydroxy hydrogel protective layer is polyvinyl alcohol.

[0022] Polyvinyl alcohol hydrogel can give the surface of biological heart valves better biocompatibility.

[0023] In a second aspect, the present invention provides a method for preparing the self-repairable biological heart valve, comprising the following steps:

[0024] (1) Immersing the pericardium in a tannic acid solution for cross-linking to obtain a tannic acid-cross-linked pericardium;

[0025] (2) adding the tannic acid-crosslinked pericardium to a dopamine precursor solution to perform a polymerization reaction, removing the pericardium to obtain the tannic acid-crosslinked pericardium with polydopamine nanoparticles modified on its surface;

[0026] (3) Tannic acid-cross-linked pericardium modified with polydopamine nanoparticles on its surface was added to a polyhydroxy polymer solution and then transferred to a mold for freeze-thaw cycles to obtain a self-repairing biological heart valve.

[0027] Preferably, in step (2), the dopamine precursor solution contains an amino-containing NO catalytic releaser.

[0028] Compared with first synthesizing polydopamine nanoparticles and then grafting amino-containing NO catalyst releasers onto them, adding the NO catalyst releaser to the dopamine precursor solution can ensure the stability of the covalent bonding between polydopamine and the NO catalyst releaser, which is beneficial to maintain the release of NO and exert its biological function during long-term use and self-repair.

[0029] Preferably, in step (2), in the dopamine precursor solution, the molar ratio of dopamine to the amino-containing NO catalytic releaser is 9 to 12:1.

[0030] Preferably, in step (2), the method for preparing the dopamine precursor solution comprises the following steps: preparing dopamine into an alkaline solution, adding an amino group-containing NO catalyst releaser solution thereto, and mixing to obtain a dopamine precursor solution.

[0031] Preferably, the volume ratio between the amino-containing NO catalyst releaser solution and the alkaline solution is 1:15-20.

[0032] Preferably, the concentration of the amino-containing NO catalyst releaser solution is 0.8-1.3 mg / mL.

[0033] Preferably, in step (3), the method for preparing the polyhydroxy polymer solution comprises the following steps: taking the remaining liquid after removing the pericardium in step (2), and dissolving the polyhydroxy polymer therein.

[0034] The full dissolution of polyhydroxy polymers often requires heating, and the temperature tolerance of the pericardium must not exceed 50°C. Therefore, in order to ensure that the heating and dissolution process of the polyhydroxy polymers does not affect the performance of the pericardium, the present invention adopts the method of first removing the pericardium and then dissolving the polyhydroxy polymers into the remaining liquid, rather than directly dissolving the polyhydroxy polymers into the liquid containing the pericardium.

[0035] Preferably, in step (3), the polyhydroxy polymer solution contains an antiplatelet drug.

[0036] Preferably, in step (3), the concentration of the polyhydroxy polymer in the polyhydroxy polymer solution is 25 to 30 wt%.

[0037] Preferably, in step (3), in the polyhydroxy polymer solution, the mass ratio of the polyhydroxy polymer to the antiplatelet drug is 1:0.015-0.025.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] (1) In the biological heart valve of the present invention, the coordinated effect between tannic acid, polydopamine nanoparticles and the polyhydroxy hydrogel protective layer present in specific positions can give the biological heart valve better mechanical properties, making it less likely to be damaged during use, thereby improving its durability.

[0040] (2) In the biological heart valve of the present invention, polydopamine nanoparticles distributed on the surface of the tannic acid cross-linked pericardium and / or in the polyhydroxy hydrogel protective layer can give the polyhydroxy hydrogel protective layer self-repair ability, so that the biological heart valve can be repaired in situ under near-infrared conditions, and can achieve both structural repair and functional (anti-thrombotic, anti-calcification, endothelialization) recovery, so that the biological heart valve does not need to undergo a secondary, high-risk valve replacement surgery after damage.

[0041] (3) In the biological heart valve of the present invention, the use of a polyhydroxy hydrogel protective layer, a NO catalytic releaser compounded in polydopamine nanoparticles, and an antiplatelet drug dispersed in the polyhydroxy hydrogel protective layer can give the biological heart valve better biological functions, enabling it to achieve passive and active anti-thrombotic adhesion effects in the early and late stages of transplantation, and effectively prevent the immune inflammatory activation state in the early stage of transplantation, promote endothelial cell adhesion and proliferation, and at the same time give it better anti-calcification ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The figure shows the structural and functional characterization results of the biological heart valve. Figure 1 A is a scanning electron microscope image of the surface and cross section of a bioprosthetic heart valve; Figure 1 B is an enlarged image of polydopamine-selenocystamine composite nanoparticles (i.e., the product prepared in step (4) of Example 1) fixed on a biological valve; Figure 1 C is the EDS scanning and analysis of the surface elements of the heart bioprosthetic valve; Figure 1 D is ATR-FTIR chemical bond analysis (n=5); Figure 1 E is the result of hydrophilicity test (water contact angle); Figure 1 F is a representative fluorescence image of the FITC-BSA protein adhesion experiment; Figure 1 G is the statistical analysis of water contact angle (n=3); Figure 1 H is Figure 1 Statistics of fluorescence intensity in F (n = 5); Figure 1 I is the NO production determined by Griess method (n=3). Figure 1 In D, the five curves from top to bottom are GLUT, TA, PVA@TA, PVA@PDA-Se@TA, and Ti-PVA@PDA-Se@TA.

[0043] Figure 2 This is the result of the near-infrared responsive self-repair function evaluation. Figure 2 A is the functional evaluation of the bioprosthetic heart valve before and after self-repair; Figure 2 B is the surface tomography OCT analysis of the self-healing effect before and after near-infrared treatment; Figure 2 C is the local temperature change during the near-infrared responsive self-healing process; Figure 2 D is a schematic diagram for evaluating the mechanical properties of self-repairing hydrogels; Figure 2 E is the Young's modulus measurement result (n=5); Figure 2 F is the tensile test result (n=5); Figure 2 G is the ultimate tensile strength measurement result (n=5).

[0044] Figure 3 This is a graph showing the evaluation results of the anti-thrombotic ability of bioprosthetic heart valves. Figure 3 A is a general picture of thrombus adhesion on the surface of cardiac bioprosthetic valve after incubation with fresh rat whole blood; Figure 3 B is a scanning electron microscope image of thrombus adhesion on the surface of the heart bioprosthetic valve after incubation with fresh rat whole blood; Figure 3 C is a photograph of the experimental results of the calcified whole blood coagulation test to evaluate coagulation performance; Figure 3 D is Figure 3 Quantitative results of C (n=5); Figure 3 E is the platelet adhesion quantified by the lactate dehydrogenase method in the presence of donors (n=5); Figure 3 F is the quantitative platelet adhesion in the absence of donors by the lactate dehydrogenase method (n=5); Figure 3 G is an endovascular implantation model to evaluate the anti-thrombotic ability of bioprosthetic valves in vivo; Figure 3 H is the scanning electron microscope image of thrombus adhesion on the surface of each group of bioprosthetic valves one week after intravascular implantation. Figure 3 B and Figure 3 In the magnified image (“Magnified”) in H, a color image is used because the red thrombus cannot be shown in the non-color image.

[0045] Figure 4 This is the result of evaluating the anti-calcification performance of bioprosthetic heart valves in vitro. Figure 4 A is a schematic diagram of the in vitro calcification induction experiment; Figure 4 B is the scanning electron microscopy and EDS element scanning images of calcium salt deposition on the surface of each group of bioprosthetic valves in the in vitro calcification induction experiment; Figure 4 C is the result of Alizarin red staining of in vitro calcification induction experiment; Figure 4 D is an in vivo subcutaneous implantation model to evaluate the anti-calcification performance; Figure 4 E is the alizarin red staining results of the bioprosthetic valves in each group 4 weeks after subcutaneous implantation; Figure 4 F is the anti-calcification ability of each group of materials evaluated under hemodynamic environment; Figure 4 G is the result of Alizarin red staining 3 weeks after intravascular implantation.

[0046] Figure 5 This is the result diagram of anti-inflammatory performance evaluation. Figure 5 A is a schematic diagram of the inflammatory response evaluation process; Figure 5 B is the immunofluorescence image of co-staining of iNOS and CD206; Figure 5 C is the TNF-α concentration in the supernatant measured by ELISA (n=3); Figure 5 D is the IL-10 concentration in the supernatant measured by ELISA (n=3); Figure 5 E Fluorescence images of ROS produced by HUVECs adhered to the surface of each material group; Figure 5F is the level of ROS produced by HUVECs adhered to the surface of each material group detected by DCFH-DA probe; Figure 5 G is a representative HE-stained image of subcutaneous implantation for 1 week; Figure 5 H is a representative image of CD68 immunofluorescence staining after 1 week of subcutaneous implantation; Figure 5 I is the statistical data of fibrous capsule thickness after 1 week and 4 weeks of subcutaneous implantation; Figure 5 J is the CD68 positive cell count after 1 week and 4 weeks of subcutaneous implantation. Figure 5 B. Figure 5 E and Figure 5 In H, since it is impossible to distinguish different colors of fluorescence in the non-colored image, a color image was used; Figure 5 I and Figure 5 In J, the five columns from left to right in “1weeks” and “4weeks” represent Ti-PVA@PDA-Se@TA, PVA@PDA-Se@TA, PVA@TA, TA, and GLUT respectively.

[0047] Figure 6 This is a graph showing the evaluation results of the anti-inflammatory ability in the intravascular implantation model. Figure 6 A is a representative image of Masson staining; Figure 6 B is the statistical analysis of the thickness of the fibrous capsule formed around the material 3 weeks after intravascular transplantation (n=5); Figure 6 C is the CD86 staining statistics 3 weeks after intravascular implantation; Figure 6 E is the fluorescence image of CD206 3 weeks after intravascular implantation; Figure 6 F is the CD206 staining statistics 3 weeks after intravascular implantation. Figure 6 C and Figure 6 In E, since it is impossible to distinguish different colors of fluorescence in the non-colored image, a color image was used; Figure 6 B. Figure 6 D and Figure 6 In F, the three columns from left to right in “PPs”, “media”, “adventitia”, “PPs site”, and “Vascular site” represent Ti-PVA@PDA-Se@TA, TA, and GLUT, respectively.

[0048] Figure 7 The figure shows the results of in vitro endothelialization function evaluation of bioprosthetic valves. Figure 7 A is a fluorescence image of the material surface adhesion after HUVECs were cultured for 24 h in the presence or absence of NO donor; Figure 7 B is the fluorescence image of the material surface adhesion after SMCs cultured for 24 h in the presence or absence of NO donor; Figure 7 C is the statistics of HUVECs adhesion with and without donors; Figure 7 D is the statistics of SMCs adhesion with and without donors; Figure 7 E is a confocal image of cell junction protein (ZO-1, red), cytoskeletal protein (Phalloidin, green) and DAPI (blue) after HUVECs were incubated on the bioprosthesis for 48 hours; Figure 7 F, HUVECs (green) and SMCs (red) were co-cultured to evaluate competitive adhesion on the bioprosthetic valve surface (n=5); Figure 7 G is the assessment of intracellular NO production by DAF-FM DA fluorescence (n=5); Figure 7 H is the wound healing assay to evaluate the migration ability of HUVECs (n=5); Figure 7 I is the wound healing assay to evaluate the migration ability of SMCs (n=5); Figure 7 J is the statistical analysis of competitive adhesion between HUVECs and SMCs; Figure 7 K is the migration distance of NO-positive cells in cells quantified by DAF-FM DA staining; Figure 7 K is the migration distance of HUVECs in the wound healing assay quantified by DAF-FM DA staining; Figure 7 M is the migration distance of SMCs in the wound healing assay using DAF-FM DA staining. Figure 7 E and Figure 7 In F, since it is impossible to distinguish different colors of fluorescence in the non-colored image, a color image was used.

[0049] Figure 8 The figure shows the evaluation results of in vivo endothelialization and hemodynamic performance. Figure 8 A is the immunofluorescence image of the bioprosthetic heart valves in each group 3 weeks after intravascular implantation (green is CD31, red is eNOS, and blue is DAPI); Figure 8 B is the comparison of the eNOS-positive area of ​​biological heart valves in each group; Figure 8 C, representative echocardiograms at day 0, week 1, and week 3 after endovascular transplantation (n=5); Figure 8 D is the statistical analysis of blood flow evaluated by echocardiography. Figure 8 In A, since it is impossible to distinguish different colors of fluorescence in the non-color image, a color image is used.

[0050] Figure 9 This is the result of the biological function evaluation of the bioprosthetic valve before and after near-infrared repair. Figure 9 A is a schematic diagram of the surface changes of the bioprosthesis before and after repair; Figure 9 B is the SEM image of platelet adhesion in damaged and repaired bioprosthetic valves; Figure 9 C is the SEM image of calcium deposition in damaged and repaired bioprosthetic valves; Figure 9D is the SEM image of HUVECs adhesion in damaged and repaired bioprosthetic valves; Figure 9 E is the evaluation of cell activity under different near-infrared irradiation times (living cells, Calcein-AM, green; dead cells, PI, red); Figure 9 F is the fluorescence image of HUVECs proliferation after long-term (2 min) NIR irradiation under the condition of supplementation of NO donor; Figure 9 G is the evaluation of NO production under conditions with and without near-infrared irradiation; Figure 9 H and Figure 9 I is the experimental setup for evaluating the self-healing performance in the fluid state; Figure 9 K is a representative image of the changes in the scratched area before and after the repair performance evaluation of the bioprosthetic valve under fluid conditions; Figure 9 J is a representative image of the local temperature changes before and after the repair of the bioprosthetic valve under fluid conditions. Figure 9 In E, since it is impossible to distinguish different colors of fluorescence in the non-color image, a color image is used.

[0051] Note: In the drawings of the specification, "Ti-PVA@PDA-Se@TA", "TP@PDA-Seca@TA", and "TP@PDA-SeCA@TA" all refer to the biological heart valve prepared in Example 1, "PVA@PDA-Se@TA", "P@PDA-Seca@TA", and "P@PDA-SeCA@TA" all refer to the biological heart valve prepared in Example 2, "PVA@TA" refers to the biological heart valve prepared in Comparative Example 1, "TA" refers to the biological heart valve prepared in Comparative Example 2, and "GLUT" and "Glut" all refer to the biological heart valve prepared in Comparative Example 3. DETAILED DESCRIPTION

[0052] The present invention will be further described below with reference to the embodiments.

[0053] Overall embodiment

[0054] A self-repairing biological heart valve comprises a polyhydroxy hydrogel protective layer loaded with polydopamine nanoparticles and modified with a tannic acid cross-linked pericardium; the polydopamine nanoparticles are distributed on the surface of the tannic acid cross-linked pericardium and / or in the polyhydroxy hydrogel protective layer.

[0055] As a specific embodiment, part or all of the polydopamine nanoparticles are modified on the surface of the tannic acid cross-linked pericardium.

[0056] As a specific embodiment, the polydopamine nanoparticles are compounded with an amino-containing NO catalytic releaser.

[0057] As a specific embodiment, antiplatelet drugs are dispersed in the polyhydroxy hydrogel protective layer.

[0058] As a specific embodiment, the amino-containing NO catalytic releaser is selenocystamine.

[0059] As a specific embodiment, the antiplatelet drug is tirofiban.

[0060] As a specific embodiment, the pericardium is porcine pericardium or bovine pericardium.

[0061] As a specific embodiment, the base material of the polyhydroxy hydrogel protective layer is polyvinyl alcohol.

[0062] A method for preparing the self-repairing biological heart valve comprises the following steps:

[0063] (1) Immersing the pericardium in a tannic acid solution for cross-linking to obtain a tannic acid-cross-linked pericardium;

[0064] (2) adding the tannic acid-crosslinked pericardium to a dopamine precursor solution to perform a polymerization reaction, removing the pericardium to obtain the tannic acid-crosslinked pericardium with polydopamine nanoparticles modified on its surface;

[0065] (3) Tannic acid-cross-linked pericardium modified with polydopamine nanoparticles on its surface was added to a polyhydroxy polymer solution and then transferred to a mold for freeze-thaw cycles to obtain a self-repairing biological heart valve.

[0066] As a specific embodiment, in step (1), the cross-linking time is not less than 48 hours.

[0067] As a specific embodiment, in step (1), the concentration of the tannic acid solution is 1 to 3 wt%.

[0068] As a specific embodiment, in step (2), the dopamine precursor solution contains an amino-containing NO catalytic releaser; in the dopamine precursor solution, the molar ratio between dopamine and the amino-containing NO catalytic releaser is 9 to 12:1.

[0069] As a specific embodiment, in step (2), the method for preparing the dopamine precursor solution includes the following steps: preparing dopamine into an alkaline solution, adding an amino-containing NO catalyst releaser solution thereto, wherein the volume ratio between the amino-containing NO catalyst releaser solution and the alkaline solution is 1:15-20, and the concentration of the amino-containing NO catalyst releaser solution is 0.8-1.3 mg / mL, and mixing to obtain a polydopamine nanoparticle dispersion.

[0070] As a specific implementation manner, in step (2), the polymerization reaction time is 20 to 30 hours.

[0071] As a specific embodiment, in step (3), the method for preparing the polyhydroxy polymer solution includes the following steps: taking the remaining liquid after removing the pericardium in step (2), and dissolving the polyhydroxy polymer therein.

[0072] As a specific embodiment, in step (3), the polyhydroxy polymer solution contains an antiplatelet drug.

[0073] As a specific embodiment, in step (3), the concentration of the polyhydroxy polymer in the polyhydroxy polymer solution is 25 to 30 wt%.

[0074] As a specific embodiment, in step (3), in the polyhydroxy polymer solution, the mass ratio between the polyhydroxy polymer and the antiplatelet drug is 1:0.015-0.025.

[0075] Example 1: Preparation of Biogenic Heart Valve (Ti-PVA@PDA-Se@TA PPs) The biogenic heart valve (Ti-PVA@PDA-Se@TA PPs) of this example was prepared by the following steps:

[0076] (1) Dissolve tannic acid powder in deionized water to form a tannic acid solution (1 wt%, pH=7.35); soak fresh porcine pericardium in the tannic acid solution for 48 hours, and then take it out to obtain tannic acid-crosslinked porcine pericardium.

[0077] (2) The tannic acid-cross-linked porcine pericardium prepared in step (1) is thoroughly washed with deionized water to remove unbound tannic acid on the surface.

[0078] (3) Dopamine powder was dissolved in an alkaline water-ethanol solution (0.25 mL ammonia water, 5 mL ethanol and 11.5 mL deionized water) and stirred for 30 minutes to prepare a 0.5 wt% dopamine solution; then 1 mL selenocystamine solution was added dropwise to the dopamine solution and mixed to obtain a dopamine precursor solution in which the concentration of selenocystamine was 1 mg / mL.

[0079] (4) adding the tannic acid-crosslinked porcine pericardium cleaned in step (2) to the dopamine precursor solution prepared in step (3), reacting for 24 hours under magnetic stirring, removing the pericardium, and obtaining tannic acid-crosslinked porcine pericardium with surface modified polydopamine-selenocystamine composite nanoparticles.

[0080] (5) Dispersing polyvinyl alcohol 1799 in the remaining liquid after removing the pericardium in step (4), heating to 90° C., and after the polyvinyl alcohol 1799 is completely dissolved, cooling to 50° C., adding tirofiban solution and stirring evenly to prepare a mixed solution of polyvinyl alcohol and tirofiban, wherein the concentration of polyvinyl alcohol is 26 wt % and the concentration of tirofiban is 5 mg / mL.

[0081] (6) Adding the tannic acid-cross-linked porcine pericardium surface-modified with polydopamine-selenocystamine composite nanoparticles prepared in step (4) to the mixed solution prepared in step (5), stirring for 30 minutes to prepare a hydrogel-porcine pericardium mixture.

[0082] (7) The hydrogel-porcine pericardium mixture prepared in step (6) was placed in a 0.2 mm deep silicone mold, tightly sealed, and subjected to freeze-thaw cycles. The freeze-thaw cycle was performed as follows: first, it was cooled to -20°C for 1 hour, then thawed at room temperature for 6 hours, and this process was repeated three times. After demolding, a bioprosthetic heart valve (Ti-PVA@PDA-Se@TA) was obtained.

[0083] Example 2: Preparation of biogenic heart valve (PVA@PDA-Se@TA PPs)

[0084] The biogenic heart valve (PVA@PDA-Se@TA PPs) of this embodiment was prepared by the following steps:

[0085] (1) Dissolve tannic acid powder in deionized water to form a tannic acid solution (1 wt%, pH=7.35); soak fresh porcine pericardium in the tannic acid solution for 48 hours, and then take it out to obtain tannic acid-crosslinked porcine pericardium.

[0086] (2) The tannic acid-cross-linked porcine pericardium prepared in step (1) is thoroughly washed with deionized water to remove unbound tannic acid on the surface.

[0087] (3) Dopamine powder was dissolved in an alkaline water-ethanol solution (0.25 mL ammonia water, 5 mL ethanol and 11.5 mL deionized water) and stirred for 30 minutes to prepare a 0.5 wt% dopamine solution; selenocystamine solution (1 mg / mL, 1 mL) was then added dropwise to the dopamine solution and mixed to obtain a dopamine precursor solution.

[0088] (4) adding the tannic acid-crosslinked porcine pericardium cleaned in step (2) to the dopamine precursor solution prepared in step (3), reacting for 24 hours under magnetic stirring, removing the pericardium, and obtaining tannic acid-crosslinked porcine pericardium with surface modified polydopamine-selenocystamine composite nanoparticles.

[0089] (5) Disperse polyvinyl alcohol 1799 in the remaining liquid after removing the pericardium in step (4), heat to 90°C, wait for the polyvinyl alcohol 1799 to be completely dissolved, and then cool to 50°C to prepare a 26 wt% polyvinyl alcohol solution.

[0090] (6) Adding the tannic acid-cross-linked porcine pericardium surface-modified with polydopamine-selenocystamine composite nanoparticles prepared in step (4) to the polyvinyl alcohol solution prepared in step (5), stirring for 30 minutes to prepare a hydrogel-porcine pericardium mixture.

[0091] (7) The hydrogel-porcine pericardium mixture prepared in step (6) was placed in a 0.2 mm deep silicone mold, tightly sealed, and subjected to freeze-thaw cycles. The freeze-thaw cycle was performed as follows: first, it was cooled to -20°C for approximately 1 hour, then thawed at room temperature for 6 hours, and this process was repeated three times. After demolding, a bioprosthetic heart valve (PVA@PDA-Se@TA) was obtained.

[0092] Comparative Example 1: Preparation of Bioheart Valve (PVA@TA PPs)

[0093] The biogenic heart valve (PVA@TA PPs) of this comparative example was prepared by the following steps:

[0094] (1) Dissolve tannic acid powder in deionized water to form a tannic acid solution (1 wt%, pH=7.35); soak fresh porcine pericardium in the tannic acid solution for 48 hours, and then take it out to obtain tannic acid-crosslinked porcine pericardium.

[0095] (2) The tannic acid-cross-linked porcine pericardium prepared in step (1) is thoroughly washed with deionized water to remove unbound tannic acid on the surface.

[0096] (3) A 26 wt% polyvinyl alcohol solution was prepared by dispersing polyvinyl alcohol 1799 powder in deionized water, heating the mixture to 90° C., and cooling the mixture to 50° C. after the polyvinyl alcohol 1799 was completely dissolved.

[0097] (4) adding the tannic acid-cross-linked porcine pericardium cleaned in step (2) to the polyvinyl alcohol solution prepared in step (3), stirring for about 30 minutes to obtain a hydrogel-porcine pericardium mixture.

[0098] (5) The hydrogel-porcine pericardium mixture prepared in step (4) was placed in a 0.2 mm deep silicone mold, tightly sealed, and subjected to freeze-thaw cycles. The freeze-thaw cycle was performed as follows: first, the mixture was cooled to -20°C for approximately 1 hour, then thawed at room temperature for 6 hours, and this process was repeated three times. The mixture was demolded to obtain a bioprosthetic heart valve (PVA@TA).

[0099] Comparative Example 2: Preparation of biological heart valves (TA PPs)

[0100] The bioprosthetic heart valve (TA PPs) of this comparative example was prepared by the following steps:

[0101] (1) Dissolve tannic acid powder in deionized water to form a tannic acid solution (1 wt%, pH=7.35); soak fresh porcine pericardium in the tannic acid solution for 48 hours, and then take it out to obtain tannic acid-crosslinked porcine pericardium.

[0102] (2) The tannic acid-cross-linked porcine pericardium prepared in step (1) is thoroughly washed with deionized water to remove unbound tannic acid on the surface, thereby obtaining a biological heart valve (TA).

[0103] Comparative Example 3: Preparation of biological heart valve (GLUT PPs)

[0104] The biological heart valve (GLUT PPs) of this comparative example was prepared by the following steps:

[0105] (1) Fresh porcine pericardium (PPs) was immersed in a GLUT solution (0.065% v / v, pH = 7.40) prepared in pure water for cross-linking and fixation;

[0106] (2) The mixture was shaken slowly at 100 rpm at room temperature for 7 days and then washed thoroughly with pure water to remove unbound glutaraldehyde on the surface to prepare GLUT PPs.

[0107] Test Case 1: Structural and functional characterization of biological heart valves

[0108] 1.1 Experimental methods

[0109] The biogenic heart valves prepared in each of the Examples and Comparative Examples were characterized for structure and function by the following methods:

[0110] (1) Each group of bio-heart valve samples was fixed with 2.5% v / v glutaraldehyde, and subjected to critical point drying and gradient dehydration for further observation.

[0111] (2) Scanning electron microscopy (SEM, ZEISS, GEMINI300, Germany) was used to observe the surface morphology and structure of the biological heart valve sample treated in step (1) and the "tannic acid cross-linked pig pericardium modified with polydopamine-selenocystamine composite nanoparticles" prepared in Example 1. Figure 1 A and Figure 1 B.

[0112] (3) Scanning electron microscope energy dispersive spectrometer was used to confirm the surface elements of the biological heart valve sample after step (1). The results are shown in Figure 1 C.

[0113] (4) The chemical composition of the biological heart valve samples treated in step (1) was characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR, Nicolet 6700, ThermoFisher). Figure 1 D.

[0114] (5) Water contact angle measurement: After the biological heart valve sample treated in step (1) is completely dried, it is attached to a glass slide using double-sided tape, and 3 μL of distilled water is dripped onto the pericardial surface. The contact angle is measured and photographed using a DSA 100 contact angle measurement system. The results are shown in Figure 1 E and Figure 1 G.

[0115] (6) Protein adhesion assay: The bio-heart valve samples treated in step (1) were placed in a 24-well plate and incubated with FITC-labeled bovine serum (1 mg / mL) at 37°C for 2 hours. The samples were rinsed with PBS three times and observed under a fluorescence microscope (Leica, DMI8, Germany). The fluorescence intensity of five matching microscope fields was analyzed using ImageJ software. The results are shown in Figure 2. Figure 1 F and Figure 1 H.

[0116] (7) Nitric oxide determination: The biological heart valve samples treated in step (1) were immersed in a mixed solution of S-nitrosoglutathione and glutathione at different concentrations. After the reaction was complete, the concentration of nitric oxide in each group was quantitatively determined using a Griess kit. The results are shown in Table 1. Figure 1 I.

[0117] 1.2 Experimental results analysis and conclusions

[0118] Scanning electron microscopy revealed porous polyethylene hydrogel encapsulation in the PVA@TA, PVA@PDA-Se@TA, and Ti-PVA@PDA-Se@TA groups, while exposed collagen fibers were visible in the GLUT and TA groups. Scanning energy spectrum analysis revealed a uniform distribution of selenium on the surfaces of the bioprosthetic valves in the PVA@PDA-Se@TA and Ti-PVA@PDA-Se@TA groups, whereas the surfaces of the GLUT, TA, and PVA@TA groups were primarily composed of carbon, nitrogen, and oxygen, with no selenium distribution. Contact angle measurements revealed significantly lower contact angles in the PVA@PDA-Se@TA and Ti-PVA@PDA-Se@TA groups compared to those in the GLUT, TA, and PVA@TA groups, indicating good hydrophilicity. Adhesion experiments with fluorescently labeled bovine serum revealed a high level of fluorescent protein adhered to the bioprosthetic valve surfaces in the GLUT and TA groups, while protein adhesion was significantly inhibited in the PVA@PDA-Se@TA and Ti-PVA@PDA-Se@TA groups, suggesting that the PVA@PDA-Se@TA and Ti-PVA@PDA-Se@TA materials exhibited superior anti-protein adhesion. Quantitative analysis of nitric oxide (NO) revealed a gradual increase in NO content with increasing donor concentration in the PVA@PDA-Se@TA and Ti-PVA@PDA-Se@TA groups, while NO production was absent in the absence of a donor. Significant NO production was observed in the GLUT, TA, and PVA@TA groups, regardless of the presence or absence of a NO donor.

[0119] Test Example 2: Damage Repair Function Evaluation

[0120] 2.1 Experimental Procedure

[0121] The biogenic heart valve prepared in Example 1 was evaluated for damage repair function using the following method:

[0122] (1) A crack of about 1 mm in length, less than 0.05 mm in width, and 0.1 mm in depth is made on the surface of the biological heart valve.

[0123] (2) The bio-heart valve treated in step (1) was placed in a 35 mm culture dish containing 1 mL of PBS buffer and illuminated with 808 nm near-infrared light (NIR) (output power 1 W / cm 2 ) Irradiate the damaged area on the surface for 0 to 30 seconds.

[0124] (3) An optical microscope, a scanning electron microscope, and a swept-source optical coherence tomography system were used to record the changes in the size of surface damage at NIR irradiation intervals of 0, 15, and 30 seconds. The results are shown in Figure 2 B.

[0125] (4) At the same time, a thermal infrared imager (China MobIR Air) was used to record the changes in surface temperature. The results are shown in Figure 2 C.

[0126] (5) Mechanical property testing of self-healing hydrogel: A rectangular hydrogel of approximately 20 mm × 10 mm × 1 mm was prepared (polyvinyl alcohol hydrogel in a dispersion of polydopamine-selenocystamine composite nanoparticles prepared according to the method in Example 1). The original force, ultimate tensile strength before and after repair, Young's modulus, elongation, and tear strength of the hydrogel were measured and analyzed. The process is as follows: Figure 2 D, the results are shown in Figure 2 E~ Figure 2 G. The tensile test rate was set at 12.5 mm / min.

[0127] 2.2 Experimental results analysis and conclusions

[0128] Stereomicroscopy and scanning source optical coherence tomography revealed that the damaged area of ​​the Ti-PVA@PDA-Se@TA PPs was significantly smaller than its initial state after 15 seconds of near-infrared irradiation, and the damage disappeared significantly after 30 seconds of irradiation, with the surface of the bioprosthetic heart valve returning to its hydrogel-wrapped state. Thermal infrared imaging revealed that the local maximum temperature reached approximately 45 to 50 degrees Celsius after 30 seconds of near-infrared irradiation, a temperature range that is within the safe temperature range that human tissue can withstand. Mechanical property testing revealed that the mechanical properties of the hydrogel were significantly impaired after damage, but after self-repair, the Young's modulus returned to its initial level, and the ultimate tensile strength and elongation showed some improvement.

[0129] Test Example 3: Anti-thrombotic Ability Assessment

[0130] 3.1 In vitro thrombus adhesion experimental process

[0131] The biogenic heart valves prepared in Examples 1-2 and Comparative Examples 1-3 were subjected to an in vitro thrombus adhesion experiment by the following method: (1) fresh blood was collected from SD rats (8-12 weeks old) in a sodium citrate vacuum tube;

[0132] (2) Prepare the bioprosthetic heart valves (1×1 cm 2 ), after being fully washed with PBS buffer, each group of materials was placed in a 1.5 mL centrifuge tube, and 500 μL of whole blood was added and incubated at 37 °C for 1 h;

[0133] (3) After incubation is complete, slowly transfer the bio-heart valve sample to a new centrifuge tube and wash with PBS buffer three times, each time for 10 to 15 minutes;

[0134] (4) The biological heart valve sample treated in step (3) was immersed in glutaraldehyde solution (2.5% v / v) and fixed overnight, and the thrombus adhesion was observed using a scanning electron microscope. The results are shown in Figure 3 A and Figure 3 B.

[0135] 3.3 In vitro lactate dehydrogenase quantitative experimental process

[0136] The biological heart valves prepared in Examples 1-2 and Comparative Examples 1-3 were subjected to an in vitro lactate dehydrogenase quantitative test using the following method:

[0137] (1) Following step (1) in “3.2 Platelet Adhesion Experimental Procedure,” collect the experimental samples from each group after PRP incubation and place them in a 96-well plate for lactate dehydrogenase (LDH) quantification;

[0138] (2) According to the instructions, prepare the LDH detection working solution by mixing 1×INT solution, lactic acid solution and enzyme solution; (3) Add 60 μL of LDH detection working solution to each well, incubate at room temperature in the dark for 30 minutes, and measure the absorbance at a wavelength of 490 nm. The results are shown in the table. Figure 3 E and Figure 3 F.

[0139] 3.4 In vitro calcified blood adhesion experimental process

[0140] The biological heart valves prepared in Examples 1-2 and Comparative Examples 1-3 were subjected to an in vitro calcification blood adhesion experiment using the following method:

[0141] (1) Following step (1) in “3.2 Platelet Adhesion Experimental Procedure,” whole blood was collected from rats and pretreated with 100 mM CaCl2 solution to activate the coagulation process;

[0142] (2) Each group of bioprosthetic valve samples was placed in a 96-well plate, 200 μl of whole blood solution containing CaCl2 was added, and incubated at 37°C for 15 min;

[0143] (3) After incubation, transfer to a new 96-well plate and wash slowly with PBS buffer three times, each time for 10 to 15 minutes;

[0144] (4) Then, 300 μL of 0.5% Triton solution was added to each well and incubated at 37°C for 1 h;

[0145] (5) After the thrombus adhering to the surface of each group of samples is completely dissolved, the suspension is slowly collected and the absorbance of hemoglobin at 405 nm is measured on an enzyme-labeled instrument. The results are shown in Figure 3 C and Figure 3 D.

[0146] 3.5 Experimental process for evaluating antithrombotic ability in the intravascular implantation model

[0147] Male SD rats were fasted for 2 days, the abdominal aorta was exposed, and the surrounding tissues were stripped. The sterilized pericardium was cut into needle-like shapes (about 0.3 mm in diameter and about 10 cm in length). 1 / 23x8mm suture needle passed through the abdominal aorta. Blood vessels and surrounding tissues were collected 1 week and 3 weeks after surgery, fixed with PFA and subjected to scanning electron microscopy, histological analysis and immunofluorescence staining for anti-thrombotic ability evaluation. The results are shown in Figure 3 G and Figure 3 H.

[0148] 3.6 Experimental results analysis and conclusions

[0149] (1) In vitro thrombus adhesion experiments showed that a large number of thrombi adhered to the exposed collagen fibers in the GLUT and TA groups. Due to the strong hydrophilicity and anti-adhesive properties of PVA hydrogel, the surface thrombus adhesion in the PVA@TA group was significantly reduced. In contrast, Ti-PVA@PDA-Se@TA and PVA@PDA-Se@TA PPs showed more significant anti-thrombotic effects.

[0150] (2) To further evaluate the anticoagulant activity of each group of PPs, we activated rat whole blood with CaCl2 solution and conducted a recalcified thrombus adhesion experiment. The results showed that significant thrombus adhesion was observed in the GLUT and TA groups, while only a small amount of thrombus was observed in the PVA@TA group. In contrast, the Ti-PVA@PDA-Se@TA and PVA@PDA-Se@TA groups showed more pronounced effects than the other groups.

[0151] (3) The results of the platelet adhesion experiment showed that both the GLUT and TA groups showed a large number of platelets adhering to the surface-exposed collagen fibers. The LDH adhesion rate in the PVA@TA group was 75% (compared with GLUT), indicating that the PVA hydrogel coating had a more significant effect in reducing platelet adhesion. In addition, the anti-platelet adhesion effect of PVA@PDA-Se@TA PPs was almost similar to that of the PVA@TA group, with only 37% of the LDH adhesion (compared with GLUT, under the condition of providing NO donors). In contrast, the Ti-PVA@PDA-Se@TA group PPs significantly reduced platelet adhesion regardless of whether NO donors were provided, which was statistically significant compared with the other groups.

[0152] (4) We evaluated the feasibility of each group of bioprosthetic heart valves as blood-contact materials through hemolysis experiments. The results showed that no hemolysis occurred in any group of PPs, and the hemolysis rate was far lower than the national hemolysis requirement for blood-contact materials (less than 2%).

[0153] Test Example 4: Anti-calcification Ability Assessment

[0154] 4.1 In vitro anti-calcification ability evaluation experimental process

[0155] Take the biological heart valves prepared in Example 1 and Comparative Examples 2 and 3, and use the following method (process as shown in FIG Figure 4 A), and in vitro anti-calcification ability was evaluated:

[0156] (1) Each group of bioprosthetic heart valves (1 cm × 1 cm) were incubated in 200 mM CaCl2 solution at 37°C for 2 h and washed three times with deionized water.

[0157] (2) The biological heart valve treated in step (1) was transferred to a 120 mM Na2HPO4 solution, incubated for another 2 h, and washed three times with deionized water.

[0158] (3) According to steps (1) and (2), the alternating immersion process in the CaCl2 and Na2HPO4 aqueous solutions was repeated 4 times to form hydroxyapatite (HAp) precipitation.

[0159] (4) After completing four alternating soaks, the bioprosthetic heart valve was immersed in a 200 mM CaCl2 aqueous solution at 37°C for 24 h to allow the mineralized amorphous calcium phosphate (ACP) to mature into HAp. The resulting PPs were fixed with paraformaldehyde and further subjected to scanning electron microscopy, energy dispersive spectrometer scanning, and alizarin red staining to evaluate their anti-calcification ability. The results are shown in Figure 4 B and Figure 4 C.

[0160] 4.2 In vivo anti-calcification ability evaluation experimental process

[0161] Take the biological heart valves prepared in Example 1 and Comparative Examples 2 and 3, and use the following method (process as shown in FIG Figure 4 D), and conduct in vivo anti-calcification ability assessment:

[0162] (1) Male SD rats (8-10 weeks, 220-250 g) were used as models for animal experiments and related operations.

[0163] (2) Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The pericardium (1×1 cm) of each group was disinfected with 70% ethanol and thoroughly rinsed with PBS. Five surgical incisions were made on the backs of rats in each group, and the bioprosthetic heart valves of each group were implanted in the subcutaneous bags. After 1 week and 4 weeks of implantation, the bioprosthetic heart valves and the surrounding fibrous capsule were removed, fixed, and the anti-calcification ability was evaluated. The results are shown in Figure 4 E~ Figure 4 G.

[0164] 4.3 Experimental results analysis and conclusions

[0165] (1) The results of the in vitro anti-calcification ability evaluation experiment showed that SEM, EDS calcium element scanning, and Alizarin red staining were used to evaluate the surface calcium salt deposition of each group after calcification induction. The results showed that GLUT and TA showed more obvious calcium salt deposition, while the Ti-PVA@PDA-Se@TA group showed significant improvement in comparison.

[0166] (2) The results of the in vivo anti-calcification ability evaluation experiment showed that the results of subcutaneous transplantation for 4 weeks further confirmed the anti-calcification performance of the Ti-PVA@PDA-Se@TA group under physiological conditions. The results of Alizarin Red staining showed that only a small amount of Alizarin Red-positive red areas were found in the PPs and fibrous capsule areas. To verify the effectiveness of the above anti-calcification effect in the hemodynamic environment, we collected the grafts 3 weeks after intravascular transplantation and performed Alizarin Red staining. We found that there was almost no calcification-positive area in the Ti-PVA@PDA-Se@TAPP and its surrounding proliferative tissue area, while the GLUT group showed a certain amount of calcium salt deposition in the area where the material contacted the blood vessel.

[0167] Test Example 5: Anti-inflammatory Ability Assessment

[0168] 5.1 In vitro anti-inflammatory ability evaluation experimental process

[0169] Take the biological heart valves prepared in Examples 1-2 and Comparative Examples 1-3, and use the following method (process as shown in FIG Figure 5 A), and in vitro anti-inflammatory ability evaluation:

[0170] (1) THP-1 cells were fluorescently labeled with the membrane dye DiD, incubated at 37°C for 30 minutes, and the precipitate was collected by centrifugation and the dye was discarded.

[0171] (2) Each bioprosthetic valve (1 cm × 1 cm) was sterilized by ultraviolet and placed at the bottom of a 24-well plate. Phorbol methyl ester (PMA) was added to the fluorescently labeled THP-1 cell suspension to induce the macrophage M0 phenotype. 5 The density of cells / mL was inoculated on the bioprostheses in each group.

[0172] (3) The cell culture well plates at the same cell density were used as the positive control group, in which the LPS and IL-4 intervention stimulation groups were used as the M1 macrophage and M2 macrophage control groups, respectively.

[0173] (4) Donors were added to the cell culture medium every 4 h, and the cell number and morphology were observed under a fluorescence microscope. At the same time, the number of iNOS-positive cells and CD206-positive cells was observed by immunofluorescence staining. The results are shown in Figure 5 B.

[0174] 5.2 Release of inflammatory factors

[0175] The bio-heart valves prepared in Examples 1-2 and Comparative Examples 1-3 were used to evaluate the release of inflammatory factors using the following method (see the results). Figure 5 C and 5D):

[0176] (1) Collect the cell culture supernatant from step 5.1 and measure the levels of inflammatory factors such as TNF-α and IL-10 according to the instructions;

[0177] (2) Set up standard wells, test sample wells, and blank wells respectively; first, add 300 μL of washing solution to each well and wash the plate 5 times. After the last wash, turn the plate upside down on absorbent paper and pat it dry appropriately, then discard the liquid in the well;

[0178] (3) Add detection antibody: dilute the detection antibody to the working concentration with diluent, add 100 μL of 1× detection antibody working solution to each well, seal the plate with sealing film and incubate at 100-300 rpm for 1 hour;

[0179] (4) After incubation is complete, wash the plate according to step (2), then add 100 μL of SA-HRP working solution and incubate at room temperature with shaking at 100-300 rpm for 30 minutes;

[0180] (5) Wash the plate according to step (2), and add 90 μL of TMB substrate solution to each well for color development, and incubate at room temperature in the dark (the reaction time should be controlled within 10 to 30 minutes, not more than 30 minutes. When the first 3 to 4 wells of the standard have a clear gradient of blue, and the gradient in the last 3 to 4 wells is not obvious, the reaction can be terminated);

[0181] (6) Add stop solution: Add 50 μL of stop solution to each well to stop the reaction. The blue color will immediately turn yellow (the order of adding the stop solution should be as similar as possible to the order of adding the substrate solution).

[0182] (7) After ensuring that there are no water droplets on the bottom of the plate and no bubbles in the wells, measure the absorbance at a wavelength of 450 nm using a microplate reader within 10 minutes.

[0183] 5.3 Assessment of oxidative stress (ROS) levels

[0184] (1) HUVEC without fluorescent labeling were inoculated into each group of samples and cultured for 24 hours, and then slowly washed with PBS buffer for 3 times;

[0185] (2) Reactive oxygen species fluorescent probe: Add the reactive oxygen species probe DCFH-DA (1:1000, 10 / μM / L) and Hoechst (10 μg / mL) to serum-free cell culture medium and incubate in the dark for 30 minutes;

[0186] (3) After incubation, each group of samples was gently rinsed with PBS buffer and observed and recorded under a fluorescence microscope. A positive control was established according to the manufacturer's instructions. A total of n = 3 samples were analyzed each time. The results are shown in Figure 5 E and 5F.

[0187] 5.4 In vivo anti-inflammatory ability evaluation experimental process

[0188] The biogenic heart valves prepared in Examples 1-2 and Comparative Examples 1-3 were evaluated for their in vivo anti-inflammatory abilities using the following method:

[0189] (1) Male SD rats (8-10 weeks, 220-250 g) were used as models for animal experiments and related operations.

[0190] (2) Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The pericardium (1×1 cm) of each group was disinfected with 70% ethanol and thoroughly rinsed with PBS. Five surgical incisions were made on the backs of rats in each group, and the bioprosthetic heart valves of each group were implanted in the subcutaneous pockets. After 1 week and 4 weeks of implantation, the bioprosthetic heart valves and the surrounding fibrous capsule were removed, fixed, and the anti-inflammatory ability was evaluated. The results are shown in Figure 5 G~ Figure 5 J.

[0191] 5.5 Experimental process for evaluating anti-inflammatory ability in the intravascular implantation model

[0192] The biogenic heart valves prepared in Example 1 and Comparative Examples 2 and 3 were evaluated for their anti-inflammatory ability in an intravascular implantation model using the following method:

[0193] Male SD rats were fasted for 2 days, the abdominal aorta was exposed, and the surrounding tissues were stripped. The sterilized pericardium was cut into needle-like shapes (about 0.3 mm in diameter and about 10 cm in length). 1 / 23x8mm suture needle passed through the abdominal aorta. Blood vessels and surrounding tissues were collected 1 week and 3 weeks after surgery, fixed with PFA and subjected to scanning electron microscopy, histological analysis and immunofluorescence staining for anti-inflammatory ability evaluation. The results are shown in Figure 6 .

[0194] 5.6 Experimental Results Analysis and Conclusions

[0195] (1) The results of the in vitro anti-inflammatory ability evaluation experiment showed that the GLUT and TA groups showed more obvious DiD fluorescently labeled THP-1 cells and iNOS positive cells on the surface. In contrast, the PVA@TA group showed a certain degree of improvement in the adhesion and differentiation of inflammatory cells. The inhibitory effect of the Ti-PVA@PDA-Se@TA and PVA@PDA-Se@TA groups was more significant; the adhesion of THP-1 cells treated with PMA and the expression of pro-inflammatory cytokines (TNF-α) were significantly reduced, suggesting that the anti-adhesive properties of PVA hydrogel combined with NO exerted a significant inhibitory effect. The expression level of the anti-inflammatory cytokine IL-10 was not significant among the groups. The main reason was that the number of macrophages adhering to the surface of the Ti-PVA@PDA-Se@TA and PVA@PDA-Se@TA groups was limited, resulting in no significant difference.

[0196] (2) In vivo anti-inflammatory ability evaluation results showed that the GLUT and TA groups were surrounded by thick fibrous capsules and had obvious CD68-positive cell infiltration. In contrast, the thickness of the fibrous capsule and macrophage infiltration of the Ti-PVA@PDA-Se@TA and PVA@PDA-Se@TA groups were significantly reduced at 1 and 4 weeks after implantation, suggesting that these two groups of PPs achieved better biocompatibility by effectively inhibiting the inflammatory response.

[0197] (3) We further evaluated the infiltration of circulating monocytes / macrophages and PP remodeling in each group of PPs in the hemodynamic environment through an intravascular transplantation model. The results showed that compared with GLUT and TA, the thickness of the fibrous capsule around the PPs in the Ti-PVA@PDA-Se@TA group was reduced by 27% and 40%, respectively. In addition, immunofluorescence staining and quantitative analysis of macrophage-specific markers CD86 (M1 macrophages) and CD206 (M2 macrophages) showed that after 3 weeks of implantation, the infiltration of CD86+ macrophages in the peripheral area of ​​the Ti-PVA@PDA-Se@TA group material was less than that in the peripheral area and vascular area of ​​the TA and GLUT groups, indicating that our modification strategy did not aggravate macrophage infiltration in the in vivo hemodynamic environment.

[0198] Test Example 6: Endothelialization Function Assessment

[0199] 6.1 In vitro endothelial cell adhesion assay

[0200] The biological heart valves prepared in Example 1 and Comparative Examples 1 to 3 were subjected to an in vitro endothelial cell adhesion test using the following method:

[0201] (1) Cut each group of biological heart valves into 1 cm × 1 cm pieces, sterilize with ultraviolet light, and place them at the bottom of a 24-well plate.

[0202] (2) Human umbilical vein endothelial cells (HUVECs) labeled with GFP were cultured at a rate of 1×10 5 Cells were seeded at a density of 100 μM / mL on the bio-heart valves of each group. 10 μM S-nitrosoglutathione and 10 μM glutathione were added to the cell culture medium every 4 hours as donors. After 24 hours of cell culture, the bio-heart valves of each group were slowly washed with PBS and the number and morphology of endothelial cells attached to their surfaces were observed under a fluorescence microscope. The results are shown in Figure 2. Figure 7 A and Figure 7 C.

[0203] 6.2 In vitro smooth muscle cell adhesion assay

[0204] The biological heart valves prepared in Example 1 and Comparative Examples 1 to 3 were subjected to an in vitro smooth muscle cell adhesion experiment using the following method:

[0205] Smooth muscle cells (SMCs) fluorescently labeled with membrane dye DiI were cultured at a rate of 1×10 5 Cells were seeded at a density of 100 μM S-nitrosoglutathione and 10 μM glutathione as donors into the cell culture medium every 4 hours. After 24 hours of cell culture, the bio-heart valves of each group were slowly washed with PBS and the number and morphology of cells adhering to the surface smoothness agent were observed by fluorescence microscopy. Figure 7 B and Figure 7 D.

[0206] 6.3 In vitro competitive adhesion assay between endothelial cells and smooth muscle cells

[0207] The biological heart valves prepared in Example 1 and Comparative Examples 1 to 3 were subjected to an in vitro competitive adhesion experiment between endothelial cells and smooth muscle cells using the following method:

[0208] Human umbilical vein endothelial cells and smooth muscle cells fluorescently labeled with membrane dye were placed at 2×10 5 Cells were seeded at a density of 100 μM / mL on the bio-heart valves of each group. 10 μM S-nitrosoglutathione and 10 μM glutathione were added to the cell culture medium every 4 hours as a donor. After 24 hours of cell culture, the bio-heart valves of each group were slowly washed with PBS and the number and morphology of endothelial cells and smooth muscle cells adhered to their surfaces were observed under a fluorescence microscope. The results are shown in Figure 2. Figure 7 F and Figure 7 J.

[0209] 6.4 Characterization of Endothelial Cell Tight Junction Protein (ZO-1) Experimental Procedure

[0210] After culturing for 24 hours on the surface of each group of bioprosthetic heart valves, non-fluorescently labeled HUVEC cells were plated and fixed with 4% PFA at room temperature for 30 minutes, washed three times with PBS, each time for 5-10 minutes; then the membrane was permeabilized with 0.25% Triton-100 for about 5-10 minutes and washed three times with PBS; after blocking with 5% BSA solution for about 1 hour, anti-ZO-1 antibody (1:400, Proteintech, China) was added and incubated at 4°C overnight; the above samples were slowly washed three times with PBS buffer for 10-15 minutes each time, and fluorescent secondary antibody was added and incubated for about 45 minutes; after sufficient PBS washing, 488 fluorescently labeled phalloidin staining solution was added for cytoskeleton staining, incubated for 30 minutes, washed three times with PBS, and mounted with a mounting medium containing DAPI; intercellular tight junction protein (ZO-1) and cytoskeleton protein were observed and photographed under a confocal microscope. The results are shown in Figure 2. Figure 7 E.

[0211] 6.5 Experimental process for determination of NO level in cells HUVEC without fluorescent labeling was seeded at a density of 1×105 / mL in the biological heart valve prepared in Example 1 and Comparative Examples 1 to 3 and cultured at 37°C for 24h. After the incubation was completed, the culture supernatant was discarded, and the NO quantitative fluorescent probe DAF-FM DA (1:1000 dilution, final concentration of 5μM / L) was added to the well plate and incubated at 37°C for 30min. Then the wells were fully washed 3 times with PBS buffer for 10 to 15 minutes each time to remove the unreacted fluorescent reagent, and the number of fluorescent cells on the surface of each group of PPs samples in the FITC channel was photographed under a fluorescence inverted microscope. The results are shown in Figure 2. Figure 7 G and 7K.

[0212] 6.6 Endothelial cell migration assay process

[0213] PPs samples were prepared in accordance with Example 1 and Comparative Examples 1 to 3. GFP-HUVECs were seeded into the PPs samples at a cell density of 5×10 5 cells / mL and cultured for 24 h in the presence of 10 μM GSNO and 10 μM GSH. After the GFP-HUVECs were uniformly attached to the surface of the PPs samples, the sample surfaces were scratched with a sterile coverslip to create a neat scratch boundary. The samples were washed with PBS buffer and then transferred to a culture medium containing 2% fetal bovine serum. The cell migration process was recorded using a fluorescence microscope at 0, 12, and 24 h, respectively. The results are shown in FIG. Figure 7 H and 7L.

[0214] 6.7 Smooth Muscle Cell Migration Experimental Procedure

[0215] Prepare each group of PPs samples according to Example 1 and Comparative Examples 1-3; DiI-labeled SMCs were seeded into each group of PPs samples at a cell density of 5×105 cells / mL and cultured for 24 hours under the conditions of 10μM GSNO and 10μM GSH donors. After the SMCs were evenly attached to the sample surface, the cell layer was slowly scratched with a coverslip, washed with PBS buffer, and then transferred to a culture medium containing 2% serum. The cell migration process was recorded using a fluorescence microscope at 0, 12, and 24 hours. The results are shown in Figure 2. Figure 7 I and 7M.

[0216] 6.8 Experimental Procedure for Evaluating Endothelial Function in an Intravascular Implantation Model

[0217] The biogenic heart valves prepared in Example 1 and Comparative Examples 2 and 3 were evaluated for endothelialization function in an intravascular implantation model using the following method:

[0218] Male SD rats were fasted for 2 days, the abdominal aorta was exposed, and the surrounding tissues were stripped. The sterilized pericardium was cut into needle-like shapes (about 0.3 mm in diameter and about 10 cm in length). 1 A 23×8mm suture needle was inserted through the abdominal aorta. Blood vessels and surrounding tissues were collected 1 week and 3 weeks after surgery, fixed with PFA, and subjected to scanning electron microscopy, histological analysis, and immunofluorescence staining for endothelial function assessment. Figure 8 .

[0219] 6.9 Experimental Results Analysis and Conclusions

[0220] (1) The results of in vitro endothelial and smooth muscle cell adhesion experiments showed that: under the condition of providing NO donor, Ti-PVA@PDA-Se@TA PPs can effectively promote HUVEC adhesion and proliferation. After 24 hours of culture, a significant endothelial cell layer can be seen on the surface of PPs. Under the condition of no donor, HUVEC showed no obvious adhesion and proliferation. Compared with HUVEC, the Ti-PVA@PDA-Se@TA group can effectively inhibit SMCs adhesion under the condition of providing NO donor. However, the proliferation of HUVEC and the inhibition of SMCs in the GLUT, TA and PVA@TA PPs groups were significantly hindered regardless of whether NO donor was added. The above results are mainly due to the fact that PVA and xenografts failed to effectively establish a microenvironment that promotes endothelial cell growth and proliferation. SeCA modification can achieve in situ NO production, thereby constructing an endothelial cell-friendly immune microenvironment to promote its adhesion and proliferation.

[0221] (2) In the intravascular implantation model, we collected grafts transplanted 3 weeks after intravascular implantation for immunofluorescence analysis of CD31 and eNOS. The results showed that a complete endothelial cell layer with both CD31 and eNOS positive expression was observed in the Ti-PVA@PDA-Se@TA group, extending along the lumen. Although CD31-positive 'endothelial-like' cells were also observed in the TA and GLUT groups, their morphology was inconsistent with that of normal endothelial cells, and there was no expression or co-localization of eNOS.

[0222] (3) In the intravascular implantation model, we evaluated the hemodynamics of the abdominal aorta material transplantation area and the surrounding area by echocardiography at 1 and 3 weeks after transplantation. The results showed that the vascular patency rates of the GLUT, TA, and Ti-PVA@PDA-Se@TA groups were 56%, 40%, and 62%, respectively. The blood flow velocity in the Ti-PVA@PDA-Se@TA group was 1.56 times and 1.16 times higher than that in the TA and GLUT groups, respectively, indicating that the Ti-PVA@PDA-Se@TA group did not cause significant vascular obstruction or proliferative remodeling.

[0223] Test Example 7: Evaluation of biological functions before and after self-repair

[0224] 7.1 Sample preparation

[0225] The bioprosthetic valve sample (1×1 cm 2 ) and were divided into a damaged group and a repair group (damaged group: a crack of about 1 mm in length, less than 0.05 mm in width, and 0.1 mm in depth was made on the surface of the sample; repair group: the damaged part of the sample was repaired by irradiating it with NIR for 30 seconds). Figure 9 As shown in A.

[0226] 7.2 Experimental process of evaluating antithrombotic ability before and after self-repair

[0227] (1) A rat platelet adhesion experiment was performed according to "3.2 In vitro platelet adhesion experiment procedure" in Test Example 3.

[0228] (2) Scanning electron microscopy was used to observe the platelet adhesion on the surface of the damaged and repaired groups, and the results were analyzed and evaluated. Figure 9 B.

[0229] 7.3 Experimental process of evaluating anti-calcification ability before and after self-repair

[0230] (1) An anti-calcification test was performed according to "4.1 In vitro anti-calcification ability evaluation test process" in Test Example 4.

[0231] (2) Scanning electron microscopy was used to observe the platelet adhesion on the surface of the damaged and repaired groups, and the results were analyzed and evaluated. Figure 9C.

[0232] 7.4 Experimental process for evaluating endothelialization ability before and after self-repair

[0233] (1) An endothelial cell adhesion test was performed according to "6.1 In vitro endothelial cell adhesion test procedure" in Test Example 6.

[0234] (2) Scanning electron microscopy was used to observe the adhesion and proliferation of endothelial cells in the damaged and repaired groups, and the results were analyzed and evaluated. Figure 9 D.

[0235] 7.5 Experimental process of evaluating cell activity before and after self-repair

[0236] (1) An endothelial cell adhesion test was performed according to "6.1 In vitro endothelial cell adhesion test procedure" in Test Example 6.

[0237] (2) Calcein-AM / PI staining was used to evaluate cell viability at different near-infrared (1 W) irradiation times, such as 0 s, 30 s, and 60 s. The results are shown in Figure 9 E.

[0238] 7.6 Experimental Process of Cell Proliferation Evaluation Before and After Self-Repair

[0239] (1) An endothelial cell adhesion test was performed according to "6.1 In vitro endothelial cell adhesion test procedure" in Test Example 6.

[0240] (2) Evaluation of cell proliferation and growth after intentional damage of some cells by near-infrared irradiation (power 1W) for 120s under the condition of NO donor. The results are shown in Figure 9 F.

[0241] 7.7 Experimental process for evaluating NO production levels before and after self-repair

[0242] (1) An endothelial cell adhesion test was performed according to "6.1 In vitro endothelial cell adhesion test procedure" in Test Example 6.

[0243] (2) Giress experiment evaluated the NO production level of the pericardium in the Ti-PVA@PDA-Se@TA group before and after NIR irradiation under the condition of providing NO donor. The results are as follows Figure 9 G.

[0244] 7.8 Experimental process for evaluating self-repair function under fluid state

[0245] Experimental process and equipment Figure 9 H and Figure 9 As shown in Figure 1, the specific process is as follows:

[0246] (1) Sample preparation: Prepare tubular (1×2cm2 ) and simulated heart valve (4×4cm 2 , sutured in a latex tube with a radius of 2 cm to make a Y-shaped incision) experimental sample, and scratched cracks with a length of 1 mm, a width less than 0.05 mm, and a depth of 0.1 mm at multiple locations on the surface.

[0247] (2) Establishment of fluid channel: The tubular sample from step (1) was slowly rolled and placed on the inner wall of the silicone tube, and the assembled silicone tube was connected to a syringe pump (LONGER, China) to construct a fluid environment; the heart valve-like experimental sample from step (1) was installed in the tubular fluid channel, and the upper and lower ends were connected to the syringe pump through latex tubes.

[0248] (3) In-fluid repair step: prepare a near-infrared optical fiber (MDL-808nm, China) and slowly insert it from the end of the silicone tube in the fluid channel and extend it to the sample in the center of the tube; with the assistance of the near-infrared optical fiber (6W), and under the monitoring of the flow state (2mL / min) and temperature detector, perform in-situ repair for about 30 seconds; after the fluid repair process is completed, turn off the laser power and slowly pull out the optical fiber, then photograph and observe the repaired sample under an optical microscope, and record and analyze the scratch area. The results are shown in Figure 9 J and Figure 9 K.

[0249] 7.9 Experimental Results Analysis and Conclusions

[0250] The results showed that PPs exhibited good damage repair capabilities at a flow rate of 2 mL / min with the assistance of approximately 6 W NIR optical fibers, while monitoring the local temperature, which rose from close to body temperature to 45-50+°C. In addition, in order to simulate the human body environment to a greater extent, we also prepared PPs that resembled heart valves and placed them in the fluid channel to observe the repair ability. The results showed that after 30 seconds of near-infrared irradiation, the repair effect of surface damage on PPs could be clearly observed. The above results indicate that the self-repairing hydrogel modification strategy not only enhances the mechanical properties of cardiac biovalve, but also intervenes and repairs under near-infrared conditions even in the event of damage, thereby reducing the mechanical and functional fragility of damaged PPs, achieving not only structural repair but also functional recovery.

[0251] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0252] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A self-repairing biological heart valve, characterized in that: The self-repairing biological heart valve comprises a polyhydroxy hydrogel protective layer loaded with polydopamine nanoparticles and modified with a tannic acid cross-linked pericardium; the polydopamine nanoparticles are distributed on the surface of the tannic acid cross-linked pericardium and in the polyhydroxy hydrogel protective layer; the polydopamine nanoparticles are compounded with selenocystamine; and the polyhydroxy hydrogel is a polyvinyl alcohol hydrogel.

2. The self-repairing biological heart valve according to claim 1, characterized in that: Antiplatelet drugs are dispersed in the polyhydroxy hydrogel protective layer.

3. The self-repairing biological heart valve according to claim 2, characterized in that: The antiplatelet drug is tirofiban.

4. The self-repairing biological heart valve according to claim 1, characterized in that: Part or all of the polydopamine nanoparticles are modified on the surface of the tannic acid cross-linked pericardium.

5. A method for preparing the self-repairable biological heart valve according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Soaking the pericardium in a tannic acid solution for cross-linking to obtain tannic acid-cross-linked pericardium; (2) adding the tannic acid-crosslinked pericardium to a dopamine precursor solution containing selenocystamine to carry out a polymerization reaction, removing the pericardium to obtain the tannic acid-crosslinked pericardium with polydopamine nanoparticles on its surface; (3) Adding tannic acid cross-linked pericardium with surface modified polydopamine nanoparticles into a polyhydroxy polymer solution, and then transferring it into a mold for freeze-thaw cycles to obtain a self-repairing biological heart valve; the polyhydroxy polymer is polyvinyl alcohol.

6. The preparation method according to claim 5, characterized in that In step (2), the molar ratio of dopamine to selenocystamine in the dopamine precursor solution is 9-12:

1.

7. The preparation method according to claim 5, characterized in that In step (3), the method for preparing the polyhydroxy polymer solution comprises the following steps: taking the remaining liquid after removing the pericardium in step (2), and dissolving the polyhydroxy polymer therein.

8. The preparation method according to claim 5, characterized in that In step (3), the polyhydroxy polymer solution contains an antiplatelet drug.

9. The preparation method according to claim 7, characterized in that In step (3), in the polyhydroxy polymer solution, the mass ratio between the polyhydroxy polymer and the antiplatelet drug is 1:0.015-0.025.

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