Tissue engineering heart valve for promoting endothelialization as well as preparation method and application of tissue engineering heart valve

Abstract

The invention discloses a tissue engineering heart valve for promoting endothelialization and a preparation method and application thereof, and belongs to the technical field of biomedical materials. Comprising the following steps: S1, preparing red cell membrane vesicles RBCM-Ab; s2, preparation of the 2dDR-PLGA-NPs (2, 2 '-(2, 2'-(2, 2 S3, preparation of RBCM-Ab / 2dDR (at) PLGA (poly (lactic-co-glycolic acid)) nanoparticles; s4, preparing the decellularized heart valve DHV; s5, preparation of the tissue engineering heart valve NP-CHS-DHV (tissue engineering heart valve); by means of the method, the tissue engineering heart valve NP-CHS-DHV capable of promoting endothelialization can be obtained. According to the invention, the blood compatibility is improved, the platelet adhesion and immune recognition are reduced, and the acceleration of endothelialization is also realized. The invention provides an innovative strategy, and the endothelialization efficiency and the clinical application prospect of the tissue engineering heart valve can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to an endothelialization-promoting tissue-engineered heart valve and its preparation method and application. Background Art

[0002] Current tissue-engineered heart valves (TEHVs) are a promising material to replace traditional artificial valves, but the delayed endothelialization of xenogeneic decellularized heart valves (xDHVs) severely limits their clinical application. Endothelialization depends on the migration of bone marrow-derived progenitor cells (EPCs) and endocardial cells, and existing strategies have not been able to effectively solve the problems of insufficient cell recruitment and adhesion. Summary of the Invention

[0003] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a preparation method of an endothelialization-promoting tissue-engineered heart valve, comprising the following steps: S1. Preparation of red blood cell membrane vesicles RBCM-Ab: Take whole blood and centrifuge it at 4 - 8°C and 1000 - 3000 rpm for 10 - 20 min, wash it with PBS solution to obtain purified red blood cells, resuspend the purified red blood cells in a PBS buffer containing 50 - 150 mM EDTA-K2, incubate at 4 - 8°C for 20 - 40 min, centrifuge at 11000 - 13000 rpm for 10 - 20 min at this temperature, and collect the precipitate to obtain RBCM; add 40 - 60 μL of CLS-PEG-SA with a concentration of 50 - 150 μM to 1 - 2 mL of RBCM suspension, oscillate and react at 15 - 20°C for 20 - 40 min, wash with PBS to obtain biotin-functionalized vesicles RBCM-Biotin; mix 40 - 60 μg of SA with 5 - 15 μg of biotinylated CD144 antibody, incubate at 20 - 30°C for 20 - 40 min to form an SA-antibody complex, take 50 - 100 μg of the complex and mix it with 1 - 5 mL of RBCM-Biotin, and react at 15 - 20°C for 20 - 40 min to obtain RBCM-Ab; S2. Preparation of 2dDR-PLGA-NPs nanoparticles: Dissolve 2dDR with 0.1 - 0.5% NaCl solution until the final concentration of 2dDR is 100 - 300 mg / mL to obtain the inner aqueous phase W1; add 1 - 10% ethanol to the ethyl acetate solution containing 1 - 10% PLGA to obtain the oil phase O; take 50 - 100 μL of the inner aqueous phase W1 and drop it into 350 - 400 μL of the oil phase O, and perform ice bath ultrasonic emulsification at a power of 40 - 60 W for 10 - 50 s to obtain the primary emulsion; use the 1 - 10% NaCl solution containing 1 - 5% poloxamer 188 as the outer aqueous phase W2; inject the primary emulsion into the outer aqueous phase W2, perform ice bath ultrasonic emulsification at a power of 40 - 60 W for 10 - 50 s, and perform magnetic stirring at 500 - 1500 rpm for 10 - 20 h to obtain the multiple emulsion; centrifuge at 5000 - 15000 rpm at 1 - 5 °C for 10 - 30 min, remove the supernatant, wash, and perform low-temperature freeze-drying for 12 - 36 h to obtain 2dDR-PLGA-NPs nanoparticles; S3. Preparation of RBCM-Ab / 2dDR@PLGA nanoparticles: Take 1 - 5 mL of RBCM-Ab and ultrasonically mix it with 1 - 10 mg of 2dDR-PLGA-NPs nanoparticles for 1 - 10 min, and extrude it through a 100 - 300 nm polycarbonate membrane using an extruder for 15 - 25 times to obtain RBCM-Ab / 2dDR@PLGA nanoparticles; S4. Preparation of decellularized heart valve DHV: Place the biological valve material in a 30 - 50 mM TRIS-HCl buffer containing 1 - 5% CHAPS and 1 - 5 mmol / l TnBP, shake it at room temperature for 12 - 36 h, rinse it with sterile water 5 - 10 times, 5 - 15 min each time, and then place it in a 30 - 50 mM TRIS-HCl buffer containing 1 - 5% CHAPS, 1 - 5 mmol / l TnBP, 0.5 - 1.5% ASB-14, and 1 - 5% SB 3 - 10, shake it at room temperature for 12 - 36 h to obtain the decellularized heart valve DHV; S5. Preparation of tissue-engineered heart valve NP-CHS-DHV: Prepare oxidized chondroitin sulfate OChS; mix DHV with a 10 - 20% OChS solution at a mass-to-volume ratio of 0.5:1 - 1:1, incubate it at 30 - 40 °C for 12 - 36 h to obtain Chs-DHV; mix 1 - 5 g of Chs-DHV with an EDC / NHS solution with a final concentration of 0.05 - 0.15 M, react it at 35 - 40 °C and 100 - 150 rpm for 1 - 5 h, add 1 - 5 mg of RBCM-Ab / 2dDR@PLGA nanoparticles, incubate it for 12 - 36 h, and wash it 1 - 3 times to obtain NP-CHS-DHV.

[0004] Further, in step S1, whole blood is taken and centrifuged at 2000 rpm for 10 min at 4°C, washed 3 times with PBS solution to obtain purified red blood cells, which are resuspended in 0.25×PBS solution containing 100 mM EDTA-K2, incubated at 4°C for 30 min, and centrifuged at 12000 rpm for 10 min to obtain RBCM.

[0005] Further, 50 μL of CLS-PEG-SA with a concentration of 100 μM is added to the 1 mL RBCM suspension, and the mixture is oscillated and reacted at 18°C for 30 min, and then washed 3 times with PBS to obtain RBCM-Biotin; 50 μg of SA is mixed with 10 μg of biotinylated CD144 antibody, incubated at 25°C for 30 min to form an SA-antibody complex, 60 μg of the complex is taken and mixed with 1 mL of RBCM-Biotin, and reacted at 18°C for 30 min to obtain RBCM-Ab.

[0006] Further, in step S2, the 2dDR solution with a final concentration of 200 mg / mL dissolved in 0.5% NaCl is used as the inner aqueous phase W1; the ethyl acetate solution containing 5% ethanol and 5% PLGA is used as the oil phase O.

[0007] Further, the primary emulsion is obtained by dropping 75 μL of the inner aqueous phase W1 into 375 μL of the oil phase O and ultrasonically treating it in an ice bath at a power of 50 W for 30 s; the outer aqueous phase W2 is a 5% NaCl solution containing 2% poloxamer 188; 450 μL of the primary emulsion is injected into 4.5 mL of the outer aqueous phase W2 and ultrasonically treated in an ice bath at a power of 50 W for 30 s again, and then magnetically stirred at 1000 rpm for 12 h to obtain a multiple emulsion; the multiple emulsion is centrifuged at 10000 rpm for 20 min at 4°C, the supernatant is removed, washed 3 times with deionized water, and freeze-dried at -80°C for 24 h to obtain 2dDR-PLGA-NPs nanoparticles.

[0008] Further, in step S3, 1 mL of RBCM-Ab and 5 mg of 2dDR-PLGA-NPs nanoparticles are ultrasonically mixed for 5 min, and then extruded 20 times through a 200 nm polycarbonate membrane using an extruder to obtain RBCM-Ab / 2dDR@PLGA nanoparticles.

[0009] Further, in step S4, the biological valve material is placed in a 40 mM TRIS-HCl buffer solution containing 2% CHAPS and 2 mmol / l TnBP, shaken at room temperature for 24 h, rinsed 6 times with sterile water for 10 min each time, and then placed in a 40 mM TRIS-HCl buffer solution containing 2% CHAPS, 2 mmol / l TnBP, 1.0% ASB-14 and 2% SB 3-10, shaken at room temperature for 24 h to obtain a decellularized heart valve DHV.

[0010] Furthermore, in step S5, DHV and 15% OChS solution were mixed at a mass-to-volume ratio of 0.8:1 and incubated at 37 °C for 24 h to obtain Chs-DHV; 2.5 g of Chs-DHV was mixed with an EDC / NHS solution with a final concentration of 0.1 M, reacted at 37 °C and 110 rpm for 3 h, 3 mg of RBCM-Ab / 2dDR@PLGA nanoparticles were added, incubated for 24 h, and washed three times to obtain NP-CHS-DHV.

[0011] In a second aspect, the present invention provides a tissue-engineered heart valve promoting endothelialization, which is prepared according to the above-mentioned preparation method.

[0012] In a third aspect, the present invention also provides the application of the tissue-engineered heart valve in the preparation of anti-thrombosis, endothelialization-promoting and anti-calcification functional materials.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a method for promoting endothelialization of heart valves and an artificial heart valve. The method accelerates the adhesion and migration of endothelial cells through nanoparticles modified with red blood cell membranes, and uses nanoparticles modified with red blood cell membranes functionalized with anti-VE-cadherin antibodies to bind to the surface of acellular valves (xDHV) to promote the endothelialization process. The core of the nanoparticles is loaded with 2-deoxy-D-ribose (2dDR), and the drug can be continuously released to stimulate the proliferation and directional migration of endothelial cells. In addition, through the functional design of the red blood cell membrane shell layer, not only the blood compatibility is improved, platelet adhesion and immune recognition are reduced, but also the acceleration of endothelialization is achieved. This method provides an innovative strategy that can significantly improve the endothelialization efficiency and clinical application prospects of tissue-engineered heart valves. Description of the Drawings

[0014] Figure 1 It is the Coomassie brilliant blue imaging analysis result of RBCM and RBCM-Ab synthesized in Example 1 of the present invention; Figure 2 It is the structural and morphological characterization result of NP-Chs-DHV in Example 1 of the present invention; Figure 3 It is the component characterization result of NP-Chs-DHV in Example 1 of the present invention; Figure 4 It is the experimental schematic diagram of the rabbit carotid artery implantation model in Example 2 of the present invention; Figure 5 It is the in vivo blood compatibility characterization result of NP-Chs-DHV in Example 2 of the present invention; Figure 6Results of histological staining 28 days after abdominal aorta transplantation with NP-Chs-DHV in Example 2 of the present invention; Figure 7 Results of staining analysis of macrophage phenotype, type I collagen, endothelial cells, and interstitial cells 28 days after abdominal aorta transplantation with NP-Chs-DHV in Example 2 of the present invention. Detailed implementation mode

[0015] To better illustrate the present invention, specific examples are listed below. Obviously, the described examples are only a part of the present invention, rather than all the examples. Based on the examples in the present invention, other examples obtained by those skilled in the art without creative work belong to the scope of protection of the present invention.

[0016] The technical solutions of the present invention will be further described below with reference to the drawings and examples.

[0017] Example 1 Preparation of bifunctional acellular nanoparticle cross-linked engineered heart valve group (NP-Chs-DHV) 1. Preparation of CD144 antibody-engineered red blood cell membrane vesicles (RBCM-Ab) To enhance endothelial cell adhesion, the red blood cell membrane vesicles (RBCM) were functionalized with CD144 antibody (RBCM-Ab) using a dual-terminal modified linker Chol-PEG-Biotin to achieve targeted conjugation.

[0018] The specific steps are as follows: (1) Preparation of RBCM: Take the whole blood of SD rats, centrifuge at 2000 rpm for 10 min at 4°C to remove serum, and wash 3 times with PBS solution with a pH of 7.4 to obtain purified red blood cells; (2) Hypotonic lysis: Resuspend the purified red blood cells in 0.25×PBS containing EDTA-K2 (100 mM), incubate at 4°C for 30 min, and then centrifuge at 12000 rpm for 10 min at this temperature to collect the pink precipitate, which is RBCM. Take 450 μL of PBS buffer to resuspend 1 g of RBCM from whole blood to obtain an RBCM suspension; (3) Antibody conjugation: Biotinylation modification: Add 50 μL of CLS-PEG-SA (MW 2000Da) with a concentration of 100 μM to each 1 mL of RBCM suspension from whole blood, react gently with shaking at 18°C for 30 min, and then wash 3 times with PBS to remove the unbound linker to obtain biotin-functionalized vesicles RBCM-Biotin; Antibody Assembly: 50 μg of streptavidin (SA) was premixed with 10 μg of biotinylated anti-CD144 antibody (manufacturer: Bio-techne; catalog number: BAF938) at a molar ratio of 1:1, incubated at 25 °C for 30 min to form an SA-antibody complex; subsequently, 60 μg of the complex was mixed with 1 mL of RBCM-Biotin from whole blood, reacted at 18 °C for 30 minutes, and the antibody was directionally anchored through the biotin-streptavidin cascade reaction to finally obtain RBCM-Ab.

[0019] The prepared RBCM-Ab was observed by gel electrophoresis, as Figure 1 shown, indicating that the anti-CD144 antibody was successfully modified onto the red blood cell membrane.

[0020] 2. Preparation of 2dDR-PLGA-NPs Nanoparticles The 2-deoxy-D-ribose (2dDR) drug-loaded PLGA nanoparticles (2dDR-PLGA-NPs) were prepared by the double emulsion-solvent evaporation method.

[0021] (1) Inner aqueous phase (W1): 2dDR was dissolved in 0.5% NaCl solution to a final concentration of 200 mg / mL of 2dDR; (2) Oil phase (O): An ethyl acetate solution containing 5% PLGA (LA:GA = 50:50, PLGA molecular weight MW: 3000 - 6000 Da, catalog number: P134566), with 5% ethanol added to enhance compatibility; (3) Primary emulsion (W1 / O): 75 μL of W1 was dropped into 375 μL of the O phase, and sonicated with a probe (50 W, ice bath, 30 s); (4) Outer aqueous phase (W2): 5% NaCl solution containing 2% poloxamer 188; (5) Double emulsion (W / O / W): 450 μL of the primary emulsion was injected into 4.5 mL of the W2 phase and sonicated again (same parameters), and the solvent was evaporated by magnetic stirring (1000 rpm, 12 h); (6) Purification: Centrifuged at 10000 rpm for 20 min at 4 °C, the supernatant was removed after centrifugation, washed 3 times with deionized water, and freeze-dried at -80 °C for 24 h to obtain 2dDR-PLGA-NPs nanoparticles.

[0022] 3. Preparation of Red Blood Cell Membrane Vesicles RBCM-Ab Encapsulating Nanoparticles Prepared by the extrusion method.

[0023] (1) Take 1 mL of the CD144 antibody-engineered red blood cell membrane vesicles RBCM-Ab prepared above; (2) Mix 1 mL of RBCM-Ab vesicles with 5 mg of 2dDR-PLGA-NPs nanoparticles, and ultrasonicate for 5 min under an ice bath condition with a power of 100 W. (3) Use an Avanti mini extruder to extrude through a 200 nm polycarbonate membrane 20 times. (4) Prepare RBCM-Ab / 2dDR@PLGA nanoparticles and store them at 4 °C.

[0024] 4. Preparation of decellularized heart valve DHV Place the porcine aortic valve in a TRIS-HCl buffer (40 mM, pH 7.8) containing 2% 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) and 2 mmol / l tributylphosphine (TnBP), and continuously shake at room temperature for 24 h for decellularization treatment. Rinse 6 times with sterile water, 10 min each time. Then place it in a TRIS-HCl buffer (40 mM, pH 7.8) containing 2% CHAPS, 2 mmol / l TnBP, 1% amidinosulfobetaine (ASB-14), and 2% sulfobetaine 10 (SB 3-10), and continue to shake at room temperature for 24 h for decellularization treatment to obtain the decellularized heart valve DHV.

[0025] 5. Preparation of bifunctionalized decellularized nanoparticle crosslinked engineered valve NP-Chs-DHV (1) Preparation of oxidized chondroitin sulfate OChS: Prepare oxidized chondroitin sulfate (OChS) by the sodium periodate (NaIO4) oxidation method. (2) Functionalization: Mix the decellularized valve DHV with a 15% OChS solution at a mass-volume ratio of 0.8:1, and incubate at 37 °C and 110 rpm for 24 h to allow the reaction of OChS with the amino groups of DHV to obtain Chs-DHV. (3) Nanoparticle crosslinking: Take 2.5 g of valve Chs-DHV and react with a final concentration of 0.1 M EDC / NHS solution at 37 °C and 110 rpm for 3 h; add 3 mg of RBCM-Ab / 2dDR@PLGA nanoparticles and incubate for 24 h; wash 3 times with deionized water to obtain the bifunctionalized decellularized nanoparticle crosslinked engineered valve NP-CHS-DHV.

[0026] Use DHV and Chs-DHV as control groups, and use the bifunctionalized decellularized nanoparticle crosslinked engineered valve group (NP-Chs-DHV) prepared in Example 1 as the experimental group, and perform structural and morphological characterizations respectively.

[0027] The results are as Figure 2 shown: Figure 2In it, Figure A is a gross photograph, and the original appearance of DHV will not be significantly changed after modification; Figure B is the SEM image of the surface and cross-section of the decellularized heart valve. The surface morphology shows that crosslinking and modification will not destroy the original structure of DHV and meet the requirements for cell growth.

[0028] The characterization results of the NP-Chs-DHV components are as Figure 3 shown. Figure 3 In it, in Figure A, Fourier transform infrared spectroscopy scans the surfaces of DHV, Chs-DHV, and NP-Chs-DHV, indicating that chondroitin sulfate (Chs) / nanoparticles are uniformly modified onto the surface of DHV. In Figure B, the results of slicing NP-Chs-DHV and labeling the red blood cell membrane and anti-CD144 antibody on the surface of NP-Chs-DHV with anti-goat antibody and CD47 antibody show that the RBCM-Ab-coated nanoparticles are successfully modified onto the surface of DHV.

[0029] Example 2 Verification of heart valve endothelialization and anti-calcification effect Taking the decellularized heart valve group (DHV group) and the glutaraldehyde-crosslinked decellularized heart valve group (GLU group) as the control groups, and taking the bifunctionalized decellularized nanoparticle-crosslinked engineered valve group (NP-Chs-DHV) prepared in Example 1 as the experimental group for subsequent experiments.

[0030] The DHV group was prepared by the method for preparing the decellularized heart valve DHV in step 4 of Example 1.

[0031] The preparation method of the GLU group is as follows: Wipe the surface moisture of the prepared DHV above, take 2 g of DHV and put it into 1.5 mL of 0.625% glutaraldehyde solution, crosslink at 37 °C in an incubator at 110 rpm for 24 h to obtain GLU.

[0032] 1. Establish a rabbit carotid artery implantation model to evaluate the performance of the stent in the hemodynamic environment.

[0033] Animal source: Japanese white rabbits (weight 2.5 - 3.5 kg, male), purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd.; the experimental protocol was approved by the institutional animal ethics committee and complied with the ARRIVE guidelines.

[0034] Feeding conditions: Temperature 22 ± 2 °C, humidity 50 ± 10%, 12-hour light / dark cycle; single-cage feeding, cage size ≥ 60 × 40 × 40 cm, equipped with sterile bedding (changed daily); fed with standard pellet feed (crude protein ≥ 16%), free access to water (sterilized drinking water); adapt to the environment for at least 7 days before the experiment to reduce stress response.

[0035] Pre-adaptation treatment: Fast for 12 hours before surgery without restricting water intake.

[0036] Experimental method: A right carotid artery-left jugular vein shunt was established in rabbits to draw out blood flow. A 1 cm × 1 cm valve sheet was prepared into a valve roll and implanted into the arteriovenous shunt. It was run for 3 h under circulating blood. After 3 h, the valve roll was taken out and the erythrocyte adhesion on the valve surface was detected. The schematic diagram of model construction is shown in Figure 4 .

[0037] After 2 weeks, the stent was subjected to CD31 and DAPI immunofluorescence staining. After 4 weeks, Doppler ultrasound was used to examine the patency of the stent, and then the stent was subjected to Masson and Von Kossa staining to show the ECM structure and calcification. Macrophage markers (CD68, iNOS, and CD206), endothelial cell markers (CD31 and vWF), interstitial cell marker (vimentin), and type I collagen staining were used to evaluate the inflammation, cellularization, and remodeling of the implanted stent, respectively.

[0038] The blood compatibility characterization of acellular heart valves (DHV group), glutaraldehyde-crosslinked acellular heart valves (GV group), and bifunctionalized acellular nanoparticle-crosslinked engineered valve group (NP-Chs-DHV group) was as follows Figure 5 shown.

[0039] Figure 5 In it, Figure A is the schematic diagram of the rabbit carotid artery model. Figure B shows that after 2 h of rabbit carotid artery circulation, a large amount of thrombus appeared on the surface of DHV, while NP-Chs-DHV showed excellent antithrombotic ability; Figure C is the SEM analysis result of the surface of the sample after 2 h of carotid artery circulation, and the result shows that NP-Chs-DHV shows excellent antithrombotic ability; Figure D shows that the in vitro platelet adhesion experiment also shows that NP-Chs-DHV can effectively inhibit platelet adhesion and activation.

[0040] 2. Establish a rat abdominal aorta transplantation model Animal source: SD rats (body weight 150 - 180 g, male), purchased from Wuhan Beite Biotechnology Co., Ltd.; the experimental protocol was approved by the institutional animal ethics committee and complied with the ARRIVE guidelines.

[0041] Feeding conditions: Temperature 22 ± 2 °C, humidity 50 ± 10%, 12-hour light / dark cycle; single-cage feeding, cage size 465 × 285 × 230 mm, equipped with sterile bedding (changed daily); fed with standard pellet feed (crude protein ≥ 16%), free access to water (sterilized drinking water); adapted to the environment for at least 7 days before the experiment to reduce stress response.

[0042] Preconditioning: 12 hours before surgery, fast but do not restrict water intake.

[0043] 1) Anesthesia and skin preparation Anesthesia: Intraperitoneal injection of sodium pentobarbital (40 mg / kg) or induction by inhaled isoflurane (4%) + maintenance of anesthesia with inhaled isoflurane (1.5 - 2%); Skin preparation: Shave the abdomen, disinfect with povidone-iodine + 75% alcohol, and drape with a sterile fenestrated sheet; 2) Exposure of the abdominal aorta Incision: Midline abdominal incision (length 3 - 4 cm), gradually separate the muscles layer by layer to expose the abdominal cavity; Localization: Gently push the intestines to the right side to expose the abdominal aorta (from the renal artery bifurcation to the iliac artery bifurcation); Vessel dissection: Bluntly dissect the connective tissue around the abdominal aorta, leaving a free segment of about 15 mm; 3) Preparation of the valve conduit Valve shaping: Roll the 5×5 mm valve sheet NP-Chs-DHV into a tube (inner diameter ≈ 1.5 mm), and continuously suture the edges with PDS 8-0 thread for fixation; Pretreatment: Immerse the conduit in heparinized saline (50 U / mL) for 10 min; 4) Vascular transplantation (end-to-end anastomosis) Blood flow occlusion: Occlude the proximal and distal ends of the abdominal aorta with a microvascular clamp; Vessel truncation: Horizontally cut the abdominal aorta in the center of the occluded segment (length ≈ 5 mm); Anastomosis operation (under the microscope): Proximal end: Perform 8 interrupted sutures on the rolled valve sheet with Prolene 10-0 thread (first fix the 3, 6, 9, and 12 o'clock directions); Distal end: Complete the anastomosis of the rolled valve sheet in the same way, ensuring that the intima is neatly aligned without distortion or tension; Release of blood flow: First release the clamp at the distal end, then release the clamp at the proximal end, and observe bleeding and pulsation.

[0044] 5) Intraoperative management Anticoagulation: Locally spray heparin sodium (100 U / mL) to prevent thrombosis; Antispasm: Infuse papaverine (0.5 mg / mL) at the anastomosis site to relieve vasoconstriction; 6) Experimental endpoint and sample collection Time points: Set at 14 days and 28 days according to the research purpose; Euthanasia: Overdose inhalation of CO2 or intraperitoneal injection of sodium pentobarbital (150 mg / kg); Specimen collection: Completely excise the transplanted vascular segment (including the anastomosis sites at both ends); Fix with 4% paraformaldehyde (for histological examination) or quick-freeze in liquid nitrogen (for molecular detection); Histological examination: HE staining (for inflammation), Masson (for collagen), CD31 (for endothelialization).

[0045] Figure 6 The histological staining results 28 days after abdominal aorta transplantation. From Figure 6 the results, it can be seen that NP-Chs-DHV can effectively inhibit valve calcification and can also effectively inhibit valve degradation compared with DHV, while meeting the requirements of cell infiltration.

[0046] Figure 7 The results of staining analysis of macrophage phenotype, type I collagen, endothelial cells and interstitial cells 28 days after abdominal aorta transplantation. From Figure 7 the results, it can be seen that the NP-Chs-DHV group can significantly induce the polarization of macrophages to the M2 phenotype, can effectively promote the regeneration of type I collagen, and at the same time partial endothelialization occurred 14 days after implantation and complete endothelialization was achieved 28 days after implantation, showing excellent regenerative ability.

[0047] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of an endothelialization-promoting tissue-engineered heart valve, characterized in that, Including the following steps: S1. Preparation of RBCM-Ab: Centrifuge 1000 - 3000 rpm for 10 - 20 min with whole blood, resuspend in PBS buffer containing EDTA-K2, incubate at 4 - 8 °C for 20 - 40 min, then centrifuge 11000 - 13000 rpm for 10 - 20 min to obtain RBCM suspension; add 40 - 60 μL of 50 - 150 μM CLS-PEG-SA to 1 - 2 mL of RBCM suspension, react at 15 - 20 °C for 20 - 40 min, wash to obtain RBCM-Biotin; mix SA and biotinylated CD144 antibody, incubate at 20 - 30 °C for 20 - 40 min to form SA-antibody complex, mix 50 - 100 μg of the complex with 1 - 5 mL of RBCM-Biotin, react at 15 - 20 °C for 20 - 40 min to obtain RBCM-Ab; S2. Preparation of 2dDR-PLGA-NPs: Use 2dDR solution with a final concentration of 100 - 300 mg / mL dissolved in NaCl as the inner aqueous phase W1; use ethyl acetate solution containing ethanol and 1 - 10% PLGA as the oil phase O; add 50 - 100 μL of W1 dropwise into 350 - 400 μL of oil phase O, sonicate in an ice bath at 40 - 60 W for 10 - 50 s to obtain primary emulsion; use NaCl solution containing poloxamer 188 as the outer aqueous phase W2; inject the primary emulsion into the outer aqueous phase W2, sonicate in an ice bath at 40 - 60 W for 10 - 50 s, stir at 500 - 1500 rpm for 10 - 20 h to obtain multiple emulsion; centrifuge at 1 - 5 °C, 5000 - 15000 rpm for 10 - 30 min, remove the supernatant, wash and freeze-dry at low temperature to obtain 2dDR-PLGA-NPs; S3. Preparation of RBCM-Ab / 2dDR@PLGA: Mix 1 - 5 mL of RBCM-Ab and 1 - 10 mg of 2dDR-PLGA-NPs for 1 - 10 min, extrude with an extruder to obtain RBCM-Ab / 2dDR@PLGA; S4. Preparation of DHV: Place the bioprosthetic valve material in 30 - 50 mM TRIS-HCl buffer containing 1 - 5% CHAPS and 1 - 5 mmol / l TnBP, shake at room temperature for 12 - 36 h, rinse 5 - 10 times, 5 - 15 min each time, then place in 30 - 50 mM TRIS-HCl buffer containing 1 - 5% CHAPS, 1 - 5 mmol / l TnBP, 0.5 - 1.5% ASB-14 and 1 - 5% SB 3 - 10, shake at room temperature for 12 - 36 h to obtain DHV; S5. Preparation of NP-CHS-DHV: Prepare OChS; Mix DHV with a 10 - 20% OChS solution at a mass - to - volume ratio of 0.5:1 - 1:1, and incubate at 30 - 40 °C for 12 - 36 h to obtain Chs - DHV; Mix 1 - 5 g of Chs - DHV with an EDC / NHS solution with a final concentration of 0.05 - 0.15 M, react at 35 - 40 °C and 100 - 150 rpm for 1 - 5 h, add 1 - 5 mg of the product obtained in step S3, incubate for 12 - 36 h, and wash to obtain NP - CHS - DHV.

2. The preparation method according to claim 1, characterized in that, In step S1, take whole blood, centrifuge at 2000 rpm for 10 min at 4 °C, wash 3 times with PBS solution to obtain purified red blood cells, resuspend them in a 0.25×PBS solution containing 100 mM EDTA - K2, incubate at 4 °C for 30 min, and centrifuge at 12000 rpm for 10 min to obtain RBCM.

3. The preparation method according to claim 2, characterized in that, Add 50 μL of CLS - PEG - SA with a concentration of 100 μM to the 1 mL RBCM suspension, react with shaking at 18 °C for 30 min, wash 3 times with PBS to obtain RBCM - Biotin; Mix 50 μg of SA with 10 μg of biotinylated CD144 antibody, incubate at 25 °C for 30 min to form an SA - antibody complex, take 60 μg of the complex and mix it with 1 mL of RBCM - Biotin, react at 18 °C for 30 min to obtain RBCM - Ab.

4. The preparation method according to claim 1, characterized in that, In step S2, a 2dDR solution with a final concentration of 200 mg / mL dissolved in 0.5% NaCl is used as the inner aqueous phase W1; An ethyl acetate solution containing 5% ethanol and 5% PLGA is used as the oil phase O.

5. The preparation method according to claim 4, characterized in that, The primary emulsion is obtained by dropping 75 μL of the inner aqueous phase W1 into 375 μL of the oil phase O and ultrasonically treating in an ice bath for 30 s at a power of 50 W; The outer aqueous phase W2 is a 5% NaCl solution containing 2% poloxamer 188; Inject 450 μL of the primary emulsion into 4.5 mL of the outer aqueous phase W2, ultrasonically treat in an ice bath again for 30 s at a power of 50 W, and then magnetically stir at 1000 rpm for 12 h to obtain a multiple emulsion; Centrifuge the multiple emulsion at 10000 rpm for 20 min at 4 °C, remove the supernatant, wash 3 times with deionized water, and freeze - dry at - 80 °C for 24 h to obtain 2dDR - PLGA - NPs nanoparticles.

6. The preparation method according to claim 1, wherein In step S3, take 1 mL of RBCM - Ab and ultrasonically mix it with 5 mg of 2dDR - PLGA - NPs nanoparticles for 5 min, and extrude through a 200 - nm polycarbonate membrane 20 times using an extruder to obtain RBCM - Ab / 2dDR@PLGA nanoparticles.

7. The preparation method according to claim 1, wherein In step S4, the biological valve material is placed in a 40 mM TRIS-HCl buffer containing 2% CHAPS and 2 mmol / l TnBP, shaken at room temperature for 24 h, rinsed 6 times with sterile water for 10 min each time, and then placed in a 40 mM TRIS-HCl buffer containing 2% CHAPS, 2 mmol / l TnBP, 1.0% ASB-14 and 2% SB 3-10, shaken at room temperature for 24 h to obtain a decellularized heart valve DHV.

8. The preparation method according to claim 1, wherein In step S5, DHV is mixed with a 15% OChS solution at a mass-to-volume ratio of 0.8:1 and incubated at 37 °C for 24 h to obtain Chs-DHV; 2.5 g of Chs-DHV is mixed with an EDC / NHS solution with a final concentration of 0.1 M, reacted at 37 °C and 110 rpm for 3 h, 3 mg of RBCM-Ab / 2dDR@PLGA nanoparticles are added, incubated for 24 h, and washed 3 times to obtain NP-CHS-DHV.

9. A tissue-engineered heart valve promoting endothelialization, characterized in that, Prepared according to the preparation method described in any one of claims 1-8.

10. Use of the tissue-engineered heart valve according to claim 9 in the preparation of materials with antithrombotic, endothelialization-promoting and anti-calcification functions.

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