Tissue-engineered heart valve with endothelial cell-responsive stiffness controllability and preparation method and application thereof

Abstract

The application discloses an endothelial cell responsive controllable-stiffness tissue engineering heart valve and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The application constructs a basic support close to the physiological stiffness of a natural valve through a low-concentration EDC / NHS, and simultaneously introduces a VEGF responsive DNA crosslinking network to moderately improve the initial stiffness, thereby providing excellent mechanical support on one hand and promoting early adhesion and proliferation of endothelial cells and accelerating endothelialization on the other hand. With the accumulation of VEGF secreted by endothelial cells, the DNA crosslinking network is triggered to be decrosslinked, the material stiffness is dynamically reduced to the physiological range, which is favorable to the migration and spreading of endothelial cells to form a complete endothelial layer, and simultaneously promotes the infiltration and functional differentiation of valve interstitial cells, thereby creating a favorable mechanical microenvironment for overall regeneration of the valve tissue. The tissue engineering heart valve prepared by the application has the functions of stiffness responsiveness, antithrombosis, endothelialization promotion and anti-calcification, and has an important clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an intelligent tissue-engineered valve capable of dynamically regulating the mechanical microenvironment to promote endothelialization and interstitial regeneration of heart valves, as well as its construction method and application. Background Technology

[0002] Tissue-engineered heart valves (TEHVs) aim to construct bioactive and growth-potential valve substitutes to overcome the limitations of traditional mechanical and bioprosthetic valves in terms of long-term durability, anticoagulation risk, and lack of growth capacity. One of the core challenges is constructing biomimetic scaffolds that mimic the extracellular matrix (ECM) of natural valves. These scaffolds must not only possess suitable mechanical properties to withstand the circulatory load of the heart but also provide dynamic biochemical and biophysical signals to guide cell differentiation, proliferation, and functional remodeling. In the scaffold construction strategy, cross-linking technology is crucial for regulating matrix stiffness, stabilizing the structure, and delaying degradation.

[0003] In current research and clinical applications, chemical cross-linking agents, especially glutaraldehyde (GA), have been widely used to fix bio-derived valve materials (such as porcine aortic valves or bovine pericardium). GA cross-linking can significantly enhance the mechanical strength and degradation resistance of materials, but this method has significant inherent drawbacks: First, GA cross-linking usually leads to a significant increase in matrix stiffness (elastic modulus can reach MPa), far exceeding the physiological stiffness range of natural cardiac valve interstitial cells (approximately 200-300 kPa). Studies have shown that this excessively high, non-physiological matrix stiffness can inhibit cell proliferation through mechanotransduction pathways, promote abnormal differentiation of fibroblasts into myofibroblasts, accelerate calcification, and hinder the normal spread and function of endothelial cells. Second, the GA cross-linking process produces cytotoxicity, and its residues may cause persistent inflammatory responses and calcification, which is detrimental to host cell infiltration and tissue remodeling. Third, the covalent network formed by GA cross-linking is usually irreversible and static, unable to respond to dynamic changes in the physiological environment or exogenous stimuli, thus limiting the possibility of constructing smart materials with dynamic adaptability and repair capabilities.

[0004] Deoxyribonucleic acid (DNA), as a natural biological macromolecule, has attracted increasing attention in the field of biomaterials due to its precise molecular recognition capabilities, programmable sequence design, good biocompatibility, and predictable response behavior. Of particular interest are DNA aptamers, a class of single-stranded DNA or RNA molecules obtained through in vitro screening, capable of binding to specific targets (such as proteins, cells, and small molecules) with high affinity and specificity. Integrating DNA aptamers into cross-linked networks can endow materials with specific responsiveness to aptamer targets, thereby translating biorecognition events into changes in the material's physicochemical properties (such as stiffness).

[0005] Although DNA hydrogels have been reported in fields such as drug controlled release and biosensing, their use as a primary crosslinking agent in constructing tissue-engineered heart valve matrix materials with adjustable stiffness in response to endothelial cells is currently undocumented in the literature or patents. Existing technologies lack a TEHV construction scheme that simultaneously meets the following requirements: 1) providing an initial mechanical microenvironment matching the physiological stiffness of the natural valve ECM; 2) possessing the ability to dynamically adjust stiffness in response to physiological or pathological signals (such as factors secreted by specific cells) to simulate the dynamic changes in stiffness during development and repair; and 3) the crosslinking agent itself possessing both biocompatibility and active biological functions (such as promoting specific cell recruitment or functional regulation).

[0006] In summary, traditional chemical cross-linking methods, such as those using glutaraldehyde, result in excessively high matrix stiffness, cytotoxicity, and static unadjustability, which are detrimental to the construction of TEHVs that promote cell growth and functionalization. Therefore, there is an urgent need to develop a novel, biomolecular-based intelligent cross-linking strategy to construct tissue-engineered heart valve materials with biomimetic initial stiffness and dynamically adjustable stiffness under specific biological signal stimulation. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides an intelligent tissue-engineered valve capable of dynamically regulating the mechanical microenvironment to promote endothelialization and interstitial regeneration of heart valves, as well as its construction method and application.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a VEGF aptamer comprising the nucleotide sequence shown in SEQ ID NO.1.

[0009] Furthermore, the nucleotide sequence of the VEGF aptamer is shown in SEQ ID NO.2.

[0010] The second objective of this invention is to provide a VEGF-responsive DNA cross-linking agent, which contains the VEGF aptamer and has CHO capping at both ends.

[0011] Furthermore, the CHO end cap is provided by CHO-Primer 1 shown in SEQ ID NO.3 and CHO-Primer 2 shown in SEQ ID NO.4.

[0012] The third objective of this invention is to provide a tissue-engineered heart valve with controllable endothelial cell responsive stiffness, which is prepared by using the VEGF-responsive DNA crosslinking agent.

[0013] Furthermore, the tissue-engineered heart valve is a decellularized heart valve, and the VEGF-responsive DNA cross-linking agent is cross-linked onto it via EDC / NHS.

[0014] The fourth objective of this invention is to provide a method for preparing the VEGF-responsive DNA cross-linking agent, comprising the following steps: mixing the VEGF aptamer sequence, the CHO-Primer 1 sequence and the CHO-Primer 2 sequence in a molar ratio of (0.8-1.2):(0.8-1.2):(0.8-1.2), dissolving them in a DNA binding buffer, and reacting to obtain a DNA cross-linking agent with CHO ends capped at both ends.

[0015] The fifth objective of this invention is to provide a method for preparing the tissue-engineered heart valve, comprising the following steps: immersing the decellularized heart valve in a solution containing the VEGF-responsive DNA cross-linking agent, incubating it, and then incubating it in a solution containing EDC / NHS and pCBMA, so that the DNA cross-linking agent and pCBMA cross-link onto the decellularized heart valve.

[0016] Furthermore, the method for preparing the decellularized heart valve includes the following steps: placing a porcine aortic valve sequentially in a first decellularization buffer and a second decellularization buffer and shaking it, then rinsing to obtain a decellularized heart valve; the first decellularization buffer is a TRIS-HCl buffer containing CHAPS and TnBP; the second decellularization buffer is a TRIS-HCl buffer containing CHAPS, TnBP, ASB-14 and SB 3-10.

[0017] The sixth objective of this invention is to provide the application of the VEGF aptamer, the VEGF-responsive DNA crosslinking agent, or the tissue-engineered heart valve in the preparation of stiffness-responsive, antithrombotic, endothelialization-promoting, and anti-calcification functional materials.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a smart tissue-engineered valve and its construction method that can dynamically regulate the mechanical microenvironment to promote endothelialization and interstitial regeneration of heart valves. By using a low concentration of EDC / NHS to mildly crosslink the decellularized valve matrix, a basic scaffold with physiological stiffness close to that of a natural valve is first constructed. Based on this, a VEGF-responsive DNA aptamer crosslinking network is introduced, giving the material a moderately enhanced stiffness in the initial stage. This initial stiffness provides the valve with superior mechanical support performance and promotes early adhesion and proliferation of endothelial cells, thereby efficiently initiating and accelerating the endothelialization process.

[0019] As endothelial cells adhere and grow, the VEGF they secrete gradually accumulates, triggering specific decrosslinking of the DNA crosslinking network. This leads to a dynamic decrease in material stiffness to near the physiological range of a natural valve. This decrease in stiffness perfectly aligns with the next stage of tissue repair: a softer matrix facilitates the migration and spread of endothelial cells to form a complete endothelial layer, while also promoting the infiltration, growth, and functional differentiation of interstitial cells, creating a favorable mechanical microenvironment for the comprehensive regeneration (interstitialization) of valve tissue.

[0020] This invention is the first to apply a VEGF-responsive DNA crosslinking system to tissue-engineered heart valves, enabling autonomous and dynamic regulation of material stiffness at different repair stages after implantation. This strategy not only improves the efficiency and quality of endothelialization but also actively creates mechanical conditions conducive to interstitial regeneration, enhancing the repair potential and long-term functionality of tissue-engineered valves, and has significant clinical translational potential. Attached Figure Description

[0021] Figure 1 The sequence of the VEGF-responsive DNA crosslinking agent synthesized in Example 1 of this invention and the results of EMSA electrophoresis analysis are shown. Figure 2 The results of the characterization of the tissue-engineered heart valve structure and morphology with controllable responsive stiffness in Embodiment 1 of the present invention; Figure 3 This is a characterization of the stiffness responsiveness of the tissue-engineered heart valve with controllable responsiveness stiffness in Embodiment 1 of the present invention; Figure 4 The results of staining analysis after endothelial cell culture of the tissue-engineered heart valve with controllable responsive stiffness in Embodiment 2 of the present invention; Figure 5 This is an experimental study of the rabbit carotid artery-venous bypass model and blood compatibility characterization in Example 2 of the present invention; Figure 6 This is the histological staining result of the tissue-engineered heart valve with controllable responsive stiffness subcutaneously embedded for 28 days in Example 2 of the present invention.

[0022] Figure 7 This is the result of transplanting tissue-engineered heart valves with controllable responsive stiffness into the rat abdominal aorta 28 days after the procedure in Example 2 of this invention. Detailed Implementation

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0024] The main contents of the technical solution of this invention are as follows: 1. Constructing VEGF aptamer cross-linking agents that are responsive to endothelial cells VEGF is an effective and specific growth factor that regulates the proliferation, migration, survival, and lumen formation of vascular endothelial cells. During the repair of heart valve injury or in vivo remodeling after tissue engineering implantation, the recruitment, attachment, and spreading of host endothelial cells and their precursor cells are highly dependent on the gradient and intensity of local VEGF signaling. Therefore, the intermediate functional domain of a known aptamer sequence with high affinity and specificity for VEGF (sequence SEQ ID NO.1: "5-CCGTCTTCCAGACAAGAGTGCAGGG-3") was selected, and "crosslinking arm" sequences were designed at both ends through direct extension. The "crosslinking arm" sequence is a segment that can form a stable double-stranded structure by base pairing with its complementary sequence. The final DNA crosslinking agent sequence is shown below:

[0025] Note: The underlined part is SEQ ID NO.1.

[0026] VEGF aptamers, CHO-Primer 1, and CHO-Primer 2 sequences were synthesized and mixed in a 1:1:1 molar ratio in DNA binding buffer (10×10⁻³ M HEPES, pH 8, containing 500×10⁻³ M NaCl and 50×10⁻³ M MgCl₂). The mixture was reacted at 37 °C for 1 h to obtain a responsive, degradable DNA cross-linking agent with CHO-terminated ends.

[0027] Through hybridization between the "crosslinking arm" and the "complementary crosslinking chain," the three components can form a DNA-based crosslinking agent. The aptamer backbone links two crosslinking chains, which carry aldehyde groups that can bind to amino groups on the valve material. The VEGF aptamer sequence, as a functional domain in the crosslinking agent, retains its active conformation for binding VEGF. When the backbone specifically binds to the VEGF protein, it dissociates from the two crosslinking chains, thus losing its crosslinking structure.

[0028] 2. Constructing tissue-engineered heart valves with controllable endothelial cell responsiveness stiffness. 2.1 Preparation of Decellularized Heart Valve DHV Porcine aortic valves were placed in TRIS-HCl buffer (40 mM, pH 7.8) containing 2% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and 2 mmol / L tributylphosphine (TnBP) and subjected to decellularization with continuous shaking at room temperature for 24 h. The valves were then rinsed 6 times with sterile water for 10 min each time. Finally, they were placed in TRIS-HCl buffer (40 mM, pH 7.8) containing 2% CHAPS, 2 mmol / L TnBP, 1% amidosulfobetaine (ASB-14), and 2% thiobetaine 10 (SB 3-10) and subjected to decellularization with continuous shaking for 24 h at room temperature to obtain decellularized heart valve DHV.

[0029] 2.2 Fabrication of tissue-engineered heart valves with controllable responsive stiffness A responsive, degradable DNA cross-linking agent with CHO-terminated ends was prepared (final concentration of cross-linking agent: 33.3 uM). Decellularized DHV valves were immersed in the solution and incubated at 37°C for 24 h. After rinsing with PBS, the membranes were immersed in EDC / NHS (2.5 × 10⁻³ MMES, containing 1.5 × 10⁻³ MMES) at pH 5.5. -3 MEDC and 3×10 -4 A decellularized valve material with responsive adjustment of matrix stiffness was obtained by incubating the solution of MNHS and pCBMA (MW: 5000, final concentration of 1 mg / ml) at room temperature for 8 h.

[0030] 2.3 Validation of the stiffness response of tissue-engineered valves The prepared valve material was directly embedded by OCT to create ice-cut specimens, and the stiffness was measured using an atomic force microscope (AFM) instrument (Multimode8, Bruker Corporation, USA).

[0031] 3. Provide the application of the tissue-engineered heart valve in the preparation of stiffness-responsive, antithrombotic, endothelialization-promoting, and anti-calcification functional materials.

[0032] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0033] Example 1: Construction of an endothelial cell-responsive VEGF aptamer crosslinking agent 1. The middle functional domain of a known aptamer sequence (SEQ ID NO.1: "5-CCGTCTTCCAGACAAGAGTGCAGGG-3") capable of high affinity and specific binding to VEGF was selected, and the "crosslinking arm" sequences at both ends were designed through direct extension. The "crosslinking arm" sequence is a segment that can form a stable double-stranded structure by base pairing with its complementary sequence. The final DNA crosslinking agent sequence is shown in Table 1 below.

[0034] Table 1. Details of DNA cross-linking agent-related sequences.

[0035] Note: The underlined part is SEQ ID NO.1.

[0036] VEGF aptamers, CHO-Primer 1, and CHO-Primer 2 sequences were synthesized and mixed in a 1:1:1 molar ratio in DNA binding buffer (10×10⁻³ M HEPES, pH 8, containing 500×10⁻³ M NaCl and 50×10⁻³ M MgCl₂). The mixture was reacted at 37 °C for 1 h to obtain a responsive, degradable DNA cross-linking agent with CHO-terminated ends.

[0037] Figure 1 Figure B shows a simulated binding pattern of the three strands. Figure C shows nucleic acid electrophoresis (lane 4 shows the DNA cross-linking agent after the three strands have bound). Figure D shows EMSA electrophoresis (lane 6 shows the DNA cross-linking agent unwinding and forming a complex with VEGF protein after co-incubation with VEGF). Figure 1 The binding of DNA cross-linking agents and the formation of DNA-protein complexes by unwinding under the action of VEGF were verified at the molecular level.

[0038] For nucleic acid electrophoresis, a 12% TBE-Page gel was used (using a 20 ml system as an example: 1 ml of 10xTBE; 10.2 ml of ddH2O; 8 ml of 30% Acr / Bis; 500 μL of glycerol; 20 μL of Page catalyst; and 120 μL of Page coagulant). The gel was incubated at 100V for 30 minutes (until the sample reached the lower part of the gel) before being photographed in a nucleic acid gel imaging system.

[0039] 2. Constructing tissue-engineered heart valves with controllable endothelial cell responsiveness stiffness. 2.1 Preparation of Decellularized Heart Valve DHV Porcine aortic valves were placed in TRIS-HCl buffer (40 mM, pH 7.8) containing 2% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and 2 mmol / L tributylphosphine (TnBP) and subjected to decellularization with continuous shaking at room temperature for 24 h. The valves were then rinsed 6 times with sterile water for 10 min each time. Finally, they were placed in TRIS-HCl buffer (40 mM, pH 7.8) containing 2% CHAPS, 2 mmol / L TnBP, 1% amidosulfobetaine (ASB-14), and 2% thiobetaine 10 (SB 3-10) and subjected to decellularization with continuous shaking for 24 h at room temperature to obtain decellularized heart valve DHV.

[0040] 2.2 Fabrication of tissue-engineered heart valves with controllable responsive stiffness A responsive degradable DNA cross-linking agent with CHO end capping (final concentration of cross-linking agent was 33.3 uM) was prepared. The decellularized valve DHV was immersed in the solution and incubated at 37 degrees Celsius for 24 h. After rinsing with PBS, it was immersed in a solution of EDC / NHS (2.5×10-3M MES, containing 1.5×10-3 M EDC and 3×10-4 M NHS) + pCBMA (MW: 5000, final concentration of 1 mg / ml) at pH 5.5 and incubated at room temperature for 8 h to obtain a decellularized valve material with responsive adjustment of matrix stiffness.

[0041] Figure 2 The image shows the macroscopic appearance of decellularized valves, DNA cross-linked valves, DNA-protected pCBMA-coated DNA cross-linked valves, and valves after incubation with VEGF165 protein (concentration of 1 ng / ul, incubation at 37°C for 4 h) (simulating the response of VEGF secreted by in vivo endothelial cells). The FAM-DNA fluorescence image confirms the successful loading of the DNA cross-linking agent, and the decrease in fluorescence after VEGF165 incubation demonstrates the VEGF protein responsiveness of tissue-engineered heart valves with controllable responsive stiffness.

[0042] Each group of valve materials was pre-cooled at -80℃ for 24 h, and then freeze-dried in a vacuum freeze dryer for 24 h. The freeze-dried valve materials were then fixed to the sample stage using conductive adhesive. For preparing the valve sample cross-section, the sample was subjected to liquid nitrogen fracturing and fixed to the side of the sample stage to avoid excessive sample height. After fixation, the samples were sputter-coated with gold for 120 s, placed in the sample chamber, and the instrument parameters were adjusted to observe the surface and cross-sectional morphology and collect data. Electron micrographs are shown below. Figure 2 As shown in the image.

[0043] 2.3 Validation of the stiffness response of tissue-engineered valves The prepared valve materials (decellularized valves, DNA cross-linked valves, DNA-protected pCBMA-coated DNA cross-linked valves, and valves incubated with VEGF165 protein) were directly OCT-embedded to prepare ice-cut specimens. Stiffness was measured using an atomic force microscope (AFM) (Multimode8, Bruker Corporation, USA). The stiffness measurement results are as follows: Figure 3 As shown in the first image. Figure 3 The second image shows pCBMA-DNA-DAV immersed in ECM endothelial cell culture medium after sterilization. ECM-iVECs are those with only ECM culture medium added. ECM+iVECs are those with valve endothelial cells cultured in ECM culture medium for 3 days, then embedded by OCT and ice-cut for AFM measurement of matrix stiffness. The stiffness measurement results show the responsiveness of pCBMA-DNA-DAV valves to endothelial cells.

[0044] Example 2: Verification of the effects of heart valves on promoting endothelialization, preventing thrombosis, and preventing calcification. 1. The ability of tissue-engineered heart valves with controllable responsive stiffness to promote endothelial cell adhesion, proliferation, migration, and mesenchymal formation. (1) Adhesion experiment of iVEC Sample processing: Using a punch, each group of valve materials was prepared into discs with a diameter of 6.35 mm, placed in a 96-well plate, with 3 replicates for each group, and sterilized with 75% alcohol for 1.5 h, and then rinsed 3 times with sterile PBS solution for later use. Cell seeding: When the cultured iVECs reach 80%-90% confluence, digest with 1 ml of 0.25% trypsin for 1 min, then add an equal volume of culture medium to stop digestion. Centrifuge at 1000 rpm for 5 min, remove the supernatant, resuspend in culture medium, count the cells, and dilute the cell suspension to a cell density of 5 × 10⁻⁶ cells / mL. 4 / mL. Place the prepared valve material with the central chamber facing up in a 48-well plate, and add 500 μL of diluted cell suspension to complete the seeding; Cell live / dead staining: 2 h and 4 h after cell seeding, the culture medium was aspirated and the cells were rinsed once with PBS solution. The staining working solution was prepared by mixing Calcein-AM and PI dye in a 1:1 ratio. 100 μL of the working solution was added to valve samples seeded with HUVECs 3 d and 5 d, and the samples were incubated in a 37℃ incubator in the dark for 30 min before fluorescence photography.

[0045] (2) Proliferation experiment of iVEC The preparation, cell culture, and seeding methods for each group of valve materials are the same as above.

[0046] Cell live / dead staining: 1 day, 3 days and 5 days after cell seeding, Calcein-AM and PI dye were mixed in a 1:1 ratio to prepare staining working solution. 100 μL of the working solution was added to the valve samples seeded with iVECs 3 days and 5 days after seeding, and the samples were incubated in a 37℃ incubator in the dark for 30 min before fluorescence photography.

[0047] (3) Cell migration experiment The preparation, cell culture, and seeding methods for each group of valve materials are the same as above.

[0048] Cell scratching: Two days after cell seeding, the valve sample was pressed at the valve edge with sterile forceps, and a cell scratch of uniform width was made at the valve midline with a 1 ml sterile pipette tip. After changing the medium, the cells were cultured for another day. Calcein-AM staining working solution was prepared, and 100 μL of the working solution was added to the valve sample. The sample was incubated in a 37°C incubator in the dark for 30 min. The valve midline position was fixed with a fluorescence microscope, and the distance the cells migrated towards the center was observed.

[0049] Immunofluorescence staining: Two days after cell seeding, the valve samples were aspirated from the culture medium and washed three times with PBS solution. The cells were then fixed with 4% paraformaldehyde fixative for 30 min and ruptured with 0.2% Triton X-100 at room temperature for 15 min. Primary antibodies MMP2 / MMP9 of different species were added at a 1:100 ratio and incubated overnight at 4°C. The cells were washed three times with PBS, and the corresponding secondary antibodies were added and incubated at 37°C in the dark for 30 min. The cells were then incubated with DAPI staining solution at room temperature for 2 min and washed with PBS. The images were observed and acquired using a laser confocal microscope.

[0050] (4) EndoMT fluorescence characterization of cells Immunofluorescence staining: Five days after cell seeding, the valve samples were aspirated from the culture medium and washed three times with PBS solution. The cells were then fixed with 4% paraformaldehyde fixative for 30 min and ruptured with 0.2% Triton X-100 at room temperature for 15 min. Primary antibodies CD144 / Vimentin of different species were added at a 1:100 ratio and incubated overnight at 4°C. The cells were washed three times with PBS, and the corresponding secondary antibodies were added and incubated at 37°C in the dark for 30 min. The cells were then incubated with DAPI staining solution at room temperature for 2 min and washed with PBS. The images were then observed and acquired using a laser confocal microscope.

[0051] 2. Rabbit arteriovenous shunt experiment to evaluate the blood compatibility of tissue-engineered heart valves with controllable responsive stiffness. (1) Cut each group of valve materials into strips of 1 cm × 1.5 cm, sterilize with 75% alcohol for 1.5 h, and then rinse 3 times with sterile PBS solution for later use. (2) Take 2.5-3 kg of Japanese rabbits and anesthetize them by injecting 1% sodium pentobarbital at a dose of 4 mL / kg through the marginal ear vein. At the same time, inject physiological saline containing heparin (100 U / kg) for systemic anticoagulation. (3) Remove hair from the neck of the Japanese big-eared rabbit, cut the skin along the midline, and separate the left carotid artery and the right jugular vein; (4) Roll up the prepared valve material and place it into the inner wall of the PVC tube, and connect it to the left carotid artery and the right jugular vein at random to establish circulation; (5) After 2 hours of circulation, the valve material was removed from the PVC tube and gently rinsed 3 times with sterile PBS solution, and then photographed and recorded. (6) SEM observation: The valve material was fixed overnight with 2.5% glutaraldehyde solution; 30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100% ethanol solutions were prepared, and the samples were placed in the ethanol solutions from low concentration to high concentration for gradient dehydration, 15 min each time; after the samples were freeze-dried, SEM sample preparation was performed, and after gold sputtering, the samples were placed in the sample chamber to observe the number and morphology of red blood cells adhering to the sample surface.

[0052] 3. Evaluation of the anti-calcification performance of valve materials using a subcutaneous embedding experimental model. (1) Sample preparation: Cut the valve material of each group into a square of 1 cm × 1 cm and sterilize it. Store it in sterile PBS solution at 4°C for later use. Set up 5 replicates for each group.

[0053] (2) Subcutaneous embedding surgical method: 1) Take male SD rats weighing 80-100 g, anesthetize them by inhalation with isoflurane, and prepare the skin on the back of the rats with a skin preparation knife; 2) Make a small incision along the midline of the back and use forceps to bluntly separate the skin and muscle tissue; 3) Place each group of valve materials subcutaneously and as deep as possible, at least 1 cm away from the skin incision, ensuring that the sample is laid flat within it; 4) Fix the sample to the back muscle with 8-0 surgical sutures, then suture the skin incision with 5-0 surgical sutures and disinfect it; 5) The embedded valve material was removed 28 days after the operation for subsequent experiments.

[0054] (3) Histological staining: The valve materials described above were fixed in 4% paraformaldehyde and then embedded in paraffin.

[0055] HE staining After sectioning the specimen, dewax it with xylene (20 min, twice), then place it in anhydrous ethanol for 2 min, 95% ethanol for 1 min, 80% ethanol for 1 min, and 75% ethanol for 1 min in sequence. Repeat the above steps again. Finally, wash with distilled water for 2 min, blot dry, and then stain according to the following steps: a) Stain with hematoxylin for 3-5 min, rinse with tap water and blot dry, then differentiate with hydrochloric acid ethanol for 30 s, rinse with tap water again and blot dry. b) Stain with eosin for 3-5 minutes; c) Dehydrated sequentially with 85%, 95% and anhydrous ethanol (5 min, 2 times), permeated in xylene for 15 min, and sealed with neutral resin. d) Observe and photograph under a microscope.

[0056] Masson staining The steps for specimen acquisition, fixation, sectioning, and dewaxing are the same as before. Masson staining is performed as follows: a) Stain with hematoxylin for 3-5 min, rinse with tap water and blot dry, then differentiate with hydrochloric acid ethanol for 30 s, rinse with tap water again and blot dry. b) Bluing solution returns to blue for 3-5 minutes, followed by a 1-minute rinse with water; c) Stain with Ponceau S and Flavescent Stain for 5-10 min, then treat with 1% phosphomolybdic acid solution for 1-2 min; d) Stain in aniline blue staining solution for 1-2 min, then differentiate using 1% glacial acetic acid; e) The dehydration and mounting steps are the same as above. Observe and photograph under a microscope.

[0057] Von kossa staining: 1) Incubate with 1% silver nitrate solution under ultraviolet light for 45 minutes, then rinse with distilled water; 2) Incubate with 3% sodium thiosulfate for 5 minutes, then rinse with tap water for 30 seconds; 3) Counterstain for 5 min, then rinse with ethanol for 1 min; 4) After mounting, observe and photograph the slide under a microscope.

[0058] (4) Immunofluorescence staining: The steps for specimen acquisition, fixation, sectioning, and dewaxing to water are the same as before. Fluorescent staining is then performed as follows: 1) Antigen retrieval: Immerse the slides in sodium citrate antigen retrieval solution and boil for 15 min. After removing them, rinse them three times with PBS solution for 5 min each time. 2) Blocking and perforation: Add 5% goat serum to the slide and block at room temperature for 30 min. Then rinse 3 times with PBS solution and perforate the slide with 0.2% Triton X-100 for 15 min. 3) Incubate with primary antibody: Add primary antibody (iNOS / Arg1) diluted 1:100 and incubate overnight at 4°C; 4) Incubation with secondary antibody: Rinse three times with PBS solution, add the corresponding secondary antibody, and incubate at 37°C in the dark for 30 min; 5) Incubation with DAPI: Rinse three times with PBS solution, add 1:1000 DAPI solution and incubate at room temperature for 10 min, then rinse and mount. 6) Observe and photograph the slides under a fluorescence microscope.

[0059] 4. Evaluation of the in vivo regeneration capacity of valve materials using an abdominal aortic transplantation model. Animal source: Male SD rats (weighing 150-180 g), purchased from Wuhan Beinte Biotechnology Co., Ltd.; The testing protocol was approved by the agency's animal ethics committee and complies with the ARRIVE guidelines.

[0060] Rearing conditions: Temperature 22±2℃, humidity 50±10%, 12-hour light / dark cycle; single-cage rearing, cage size 465×285×230 ​​mm, equipped with sterile bedding (changed daily); fed standard pelleted feed (crude protein ≥16%), with free access to drinking water (sterile drinking water); allow at least 7 days for acclimatization before the experiment to reduce stress response.

[0061] Preconditioning: Fasting is allowed for 12 hours before surgery, but water is allowed.

[0062] 1) Anesthesia and skin preparation Anesthesia: Intraperitoneal injection of sodium pentobarbital (40 mg / kg) or inhalation of isoflurane (4%) for induction and maintenance anesthesia for two days. Alkane inhalation (1.5-2%); Preparation: Shave the abdomen, disinfect with iodine solution and 75% alcohol, and drape with a sterile drape; 2) Exposure of the abdominal aorta Incision: Midline abdominal incision (3-4 cm in length), muscle layers are separated to expose the abdominal cavity; Positioning: Gently push the intestines to the right to expose the abdominal aorta (from the bifurcation of the renal artery to the bifurcation of the iliac artery). Free vessel: Bluntly dissect the connective tissue around the abdominal aorta, leaving a free segment of about 15 mm; 3) Valve conduit preparation Valve reshaping: Roll a 5×5 mm NP-Chs-DHV valve piece into a tube (inner diameter ≈ 1.5 mm), and connect it with a PDS 8-0 suture. Continue suturing and fixing the edges; Pretreatment: Immerse the tubing in heparinized saline (50 U / mL) for 10 min; 4) Vascular grafting (end-to-end anastomosis) Blocking blood flow: Microvascular clamps block the proximal and distal ends of the abdominal aorta; Transecting the blood vessel: Transversely cut the abdominal aorta in the center of the blocked segment (length ≈ 5 mm). Anastomosis procedure (under a microscope): Proximal end: The valve piece, rolled into a tubular shape, is sutured with 8 interrupted stitches using Prolene 10-0 sutures (first fix stitches 3, 6, 9, and 12). point direction); Distal end: The valve piece rolled into a tube is anastomosed in the same way to ensure that the endometrium is aligned neatly without twisting or tension; To restore blood flow: first loosen the distal clamp, then loosen the proximal clamp, and observe for bleeding and pulsation.

[0063] 5) Intraoperative management Anticoagulation: Topical application of heparin sodium (100 U / mL) to prevent thrombosis; Antispasmodic treatment: Infusion of papaverine (0.5 mg / mL) into the anastomosis site to relieve vasoconstriction; 6) Experimental endpoint and sample collection Time point: 28 days post-surgery, as determined by the study objectives; Euthanasia: CO2 inhalation overdose or intraperitoneal injection of sodium pentobarbital (150 mg / kg); Material harvesting: The entire transplanted blood vessel segment (including both anastomoses) is harvested; fixation is performed using 4% paraformaldehyde (histological) or liquid fixation. Nitrogen-freezing (molecular detection); Histological examination: HE staining (inflammation), Masson (collagen), CD31 (endothelialization), Vimentin (mesenchymal remodeling), Col 1 (stromal remodeling).

[0064] The results of staining analysis of endothelial cells after culturing tissue-engineered heart valves with controllable responsive stiffness are as follows: Figure 4 As shown, the results indicate that the tissue-engineered heart valve prepared by this invention has good cell compatibility, can support the adhesion and proliferation of endothelial cells in the early stage, promotes the rapid formation of the endothelial layer, and promotes the migration of valve endothelial cells and the acquisition of some interstitial cell phenotypes after VEGF response, thus promoting the completion of interstitial cellization of heart valve materials. This is of great significance for preventing thrombosis, valve degeneration and promoting in situ regeneration of valves.

[0065] Rabbit carotid arteriovenous bypass model experiment and blood compatibility characterization, such as Figure 5 As shown, the results indicate that the tissue-engineered heart valve prepared by this invention has excellent blood compatibility, can effectively inhibit erythrocyte adhesion and coagulation activation, and has good anti-thrombotic ability.

[0066] Histological staining results of tissue-engineered heart valves with controllable responsive stiffness after subcutaneous embedding for 28 days are as follows: Figure 6 As shown, the results indicate that the tissue-engineered heart valve prepared in this invention can: inhibit inflammatory responses and reduce calcification inducing factors; regulate macrophage polarization towards the M2 type, and block the cellular pathway of calcification. Therefore, the tissue-engineered heart valve prepared in this invention possesses long-term anti-calcification potential.

[0067] Gross appearance, ultrasound examination, histological staining, and immunofluorescence staining results of a tissue-engineered heart valve abdominal aortic transplantation model with controllable responsive stiffness after 28 days are as follows: Figure 7 As shown, the results indicate that, compared to the obvious tumor-like dilatation of the conduit created by simple decellularized valves and the obvious calcification of glutaraldehyde valves, the tissue-engineered heart valve with controllable responsive stiffness maintained structural and hemodynamic stability. Further immunofluorescence staining showed that the tissue-engineered valve achieved complete endothelialization of the inner surface of the conduit (CD31 positive) and matrix remodeling (positive interstitial cell markers and mouse collagen 1 positive) 28 days after the operation, demonstrating excellent in vivo regeneration capacity.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A VEGF aptamer, characterized in that, It contains the nucleotide sequence shown in SEQ ID NO.

1.

2. The VEGF aptamer according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A VEGF-responsive DNA cross-linking agent, characterized in that, It includes the VEGF aptamer as described in any one of claims 1-2, and has CHO end caps at both ends.

4. The VEGF-responsive DNA cross-linking agent according to claim 3, characterized in that, The CHO end caps are provided by CHO-Primer 1 shown in SEQ ID NO. 3 and CHO-Primer 2 shown in SEQ ID NO.

4.

5. A tissue-engineered heart valve with controllable endothelial cell responsive stiffness, characterized in that, It is prepared by using the VEGF-responsive DNA cross-linking agent as described in any one of claims 3-4.

6. The tissue-engineered heart valve according to claim 5, characterized in that, The tissue-engineered heart valve is a decellularized heart valve, and the VEGF-responsive DNA cross-linking agent is cross-linked onto it via EDC / NHS.

7. The method for preparing the VEGF-responsive DNA cross-linking agent according to any one of claims 3-4, characterized in that, Includes the following steps: The VEGF aptamer sequence, CHO-Primer 1 sequence, and CHO-Primer 2 sequence were mixed in a molar ratio of (0.8-1.2):(0.8-1.2):(0.8-1.2) and dissolved in DNA binding buffer to obtain a DNA cross-linking agent with CHO ends.

8. The method for preparing the tissue-engineered heart valve according to any one of claims 5-6, characterized in that, Includes the following steps: The decellularized heart valve was immersed in a solution containing the VEGF-responsive DNA cross-linking agent according to any one of claims 3-4, and after incubation, it was placed in a solution containing EDC / NHS and pCBMA for incubation, so that the DNA cross-linking agent and pCBMA cross-linked onto the decellularized heart valve.

9. The preparation method according to claim 8, characterized in that, The method for preparing the decellularized heart valve includes the following steps: placing a porcine aortic valve in a first decellularization buffer and a second decellularization buffer in sequence and shaking it, then rinsing to obtain a decellularized heart valve; the first decellularization buffer is a TRIS-HCl buffer containing CHAPS and TnBP; the second decellularization buffer is a TRIS-HCl buffer containing CHAPS, TnBP, ASB-14 and SB 3-10.

10. The use of the VEGF aptamer according to any one of claims 1-2, the VEGF-responsive DNA crosslinker according to any one of claims 3-4, or the tissue-engineered heart valve according to any one of claims 5-6 in the preparation of stiffness-responsive, antithrombotic, endothelialization-promoting, and anti-calcification functional materials.

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