An endothelialization-promoting tissue-engineered heart valve and its preparation method and application

Abstract

The present invention discloses an endothelialization-promoting tissue-engineered heart valve and its preparation method and application, belonging to the technical field of biomedical materials. It includes the following steps: S1, preparation of red blood cell membrane vesicles RBCM-Ab; S2, preparation of 2dDR-PLGA-NPs nanoparticles; S3, preparation of RBCM-Ab / 2dDR@PLGA nanoparticles; S4, preparation of decellularized heart valve DHV; S5, preparation of tissue-engineered heart valve NP-CHS-DHV; through this method, the endothelialization-promoting tissue-engineered heart valve NP-CHS-DHV can be obtained. The present invention not only improves blood compatibility, reduces platelet adhesion and immune recognition, but also realizes the acceleration of endothelialization. The present invention provides an innovative strategy, which can significantly improve the endothelialization efficiency and clinical application prospect of tissue-engineered heart valves.

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 for replacing traditional artificial valves, but the severe delay in 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 yet effectively solved 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:

[0004] In the first aspect, the present invention provides a preparation method of an endothelialization-promoting tissue-engineered heart valve, comprising the following steps:

[0005] S1. Preparation of red blood cell membrane vesicles RBCM-Ab:

[0006] Take whole blood and centrifuge it at 1000 - 3000 rpm for 10 - 20 min at 4 - 8°C, wash it with PBS solution to obtain purified red blood cells, resuspend the purified red blood cells in a PBS buffer solution 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, and 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;

[0007] S2. Preparation of 2dDR-PLGA-NPs nanoparticles:

[0008] 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 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;

[0009] S3. Preparation of RBCM-Ab / 2dDR@PLGA nanoparticles:

[0010] 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;

[0011] S4. Preparation of decellularized heart valve DHV:

[0012] 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;

[0013] S5. Preparation of tissue-engineered heart valve NP-CHS-DHV:

[0014] Preparation of oxidized chondroitin sulfate 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 RBCM - Ab / 2dDR@PLGA nanoparticles, incubate for 12 - 36 h, and wash 1 - 3 times to obtain NP - CHS - DHV.

[0015] Furthermore, 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.

[0016] Furthermore, add 50 μL of CLS - PEG - SA with a concentration of 100 μM to the 1 mL RBCM suspension, oscillate and react 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, and react at 18 °C for 30 min to obtain RBCM - Ab.

[0017] Furthermore, in step S2, use a 2dDR solution with a final concentration of 200 mg / mL dissolved in 0.5% NaCl as the inner aqueous phase W1; use an ethyl acetate solution containing 5% ethanol and 5% PLGA as the oil phase O.

[0018] Furthermore, 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 at a power of 50 W for 30 s; 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 W2 outer aqueous phase, ultrasonically treat in an ice bath again at a power of 50 W for 30 s, and then magnetically stir at 10,000 rpm for 12 h to obtain a multiple emulsion; Centrifuge the multiple emulsion at 10,000 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.

[0019] Further, in step S3, 1 mL of RBCM-Ab and 5 mg of 2dDR-PLGA-NPs nanoparticles were ultrasonically mixed for 5 min, and then extruded through a 200 nm polycarbonate membrane 20 times using an extruder to obtain RBCM-Ab / 2dDR@PLGA nanoparticles.

[0020] Further, in step S4, the biological valve material was 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.

[0021] Further, in step S5, DHV and a 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 3 times to obtain NP-CHS-DHV.

[0022] 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.

[0023] In a third aspect, the present invention further provides the application of the tissue-engineered heart valve in the preparation of materials with antithrombotic, endothelialization-promoting and anti-calcification functions.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 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 decellularized 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. Brief Description of the Drawings

[0026] Figure 1 Results of Coomassie brilliant blue imaging analysis of RBCM and RBCM-Ab synthesized in Example 1 of the present invention;

[0027] Figure 2 Results of the structure and morphology characterization of NP-Chs-DHV in Example 1 of the present invention;

[0028] Figure 3 Results of the component characterization of NP-Chs-DHV in Example 1 of the present invention;

[0029] Figure 4 Schematic diagram of the rabbit carotid artery implantation model experiment in Example 2 of the present invention;

[0030] Figure 5 Results of the in vivo blood compatibility characterization of NP-Chs-DHV in Example 2 of the present invention;

[0031] Figure 6 Results of histological staining after 28 days of abdominal aorta transplantation with NP-Chs-DHV in Example 2 of the present invention;

[0032] Figure 7 Results of staining analysis of macrophage phenotype, type I collagen, endothelial cells and interstitial cells after 28 days of abdominal aorta transplantation with NP-Chs-DHV in Example 2 of the present invention. Detailed implementation manners

[0033] For better illustration of the present invention, specific examples are listed as follows. Obviously, the described examples are only a part of the present invention, rather than all the examples. Based on the examples of the present invention, other examples obtained by those skilled in the art without creative efforts all fall within the protection scope of the present invention.

[0034] The technical solutions of the present invention will be further described below with reference to the drawings and examples.

[0035] Example 1 Preparation of bifunctionalized decellularized nanoparticle crosslinked engineered valve group (NP-Chs-DHV)

[0036] 1. Preparation of CD144 antibody-engineered red blood cell membrane vesicles (RBCM-Ab)

[0037] 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.

[0038] The specific steps are as follows:

[0039] (1) Preparation of RBCM: Take the whole blood of SD rats, centrifuge at 2000 rpm for 10 min at 4°C to remove the serum, and wash 3 times with PBS solution with a pH of 7.4 to obtain purified red blood cells;

[0040] (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;

[0041] (3) Antibody conjugation:

[0042] Biotinylation modification: Add 50 μL of CLS-PEG-SA (MW 2000Da) with a concentration of 100 μM to every 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;

[0043] Antibody assembly: Pre-mix 50 μg of streptavidin (SA) and 10 μg of biotinylated CD144 antibody (manufacturer: Bio-techne; catalog number: BAF938) (molar ratio 1:1) at a molar ratio of 1:1, incubate at 25°C for 30 min to form an SA-antibody complex; then take 60 μg of the complex and mix it with 1 mL of RBCM-Biotin from whole blood, react at 18°C for 30 minutes, and complete the antibody-directed anchoring through the biotin-streptavidin cascade reaction to finally obtain RBCM-Ab.

[0044] 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.

[0045] 2.2 Preparation of 2dDR-PLGA-NPs nanoparticles

[0046] 2-Deoxy-D-ribose (2dDR) drug-loaded PLGA nanoparticles (2dDR-PLGA-NPs) were prepared by the double emulsion-solvent evaporation method.

[0047] [[ID=2X]](1) Inner aqueous phase (W1): Dissolve 2dDR in 0.5% NaCl solution to a final concentration of 200 mg / mL of 2dDR;

[0048] (2) Oil phase (O): An ethyl acetate solution containing 5% PLGA (LA:GA = 50:50, PLGA molecular weight MW: 3000 - 6000 Da, product number: P134566), with 5% ethanol added to enhance compatibility;

[0049] (3) Primary emulsion (W1 / O): 75 μL of W1 was dropped into 375 μL of O phase, and sonicated with a probe (50 W, ice bath, 30 s);

[0050] (4) External aqueous phase (W2): 5% NaCl solution containing 2% poloxamer 188;

[0051] (5) Double emulsion (W / O / W): 450 μL of the primary emulsion was injected into 4.5 mL of W2 phase and sonicated a second time (same parameters), and the solvent was evaporated by magnetic stirring (1000 rpm, 12 h);

[0052] (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.

[0053] 3. Preparation of nanoparticles encapsulated with red blood cell membrane vesicles RBCM-Ab [[ID=1?]]

[0054] Prepared by the extrusion method.

[0055] (1) Take 1 mL of the CD144 antibody-engineered red blood cell membrane vesicles RBCM-Ab prepared above;

[0056] (2) Mix 1 mL of RBCM-Ab vesicles with 5 mg of 2dDR-PLGA-NPs nanoparticles, and sonicate for 5 min at a power of 100 W under ice bath conditions;

[0057] (3) Use an Avanti mini extruder to extrude through a 200 nm polycarbonate membrane 20 times;

[0058] (4) Obtain RBCM-Ab / 2dDR@PLGA nanoparticles and store at 4°C.

[0059] 4. Preparation of decellularized heart valve DHV

[0060] It should be noted that there seems to be a small error in the tag "[[ID=1?]]" in the original text, which is likely a typo. I have translated it as "[[ID=1?]]" as it is, but it might need to be corrected in the original content.The porcine aortic valve was placed in a TRIS-HCl buffer solution (40 mM, pH 7.8) containing 2% 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) and 2 mmol / l tributylphosphine (TnBP), and continuously shaken at room temperature for 24 h for decellularization treatment. It was rinsed 6 times with sterile water, 10 min each time. Then it was placed in a TRIS-HCl buffer solution (40 mM, pH 7.8) containing 2% CHAPS, 2 mmol / l TnBP, 1% amidinosulfobetaine (ASB-14) and 2% sulfobetaine 10 (SB 3-10), and continuously shaken at room temperature for another 24 h for decellularization treatment to obtain the decellularized heart valve DHV.

[0061] 5. Preparation of bifunctionalized decellularized nanoparticle crosslinked engineered valve NP-Chs-DHV

[0062] (1) Preparation of oxidized chondroitin sulfate OChS: Oxidized chondroitin sulfate (OChS) was prepared by the sodium periodate (NaIO4) oxidation method;

[0063] (2) Functionalization: The decellularized valve DHV was mixed with a 15% OChS solution at a mass-to-volume ratio of 0.8:1, and incubated 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;

[0064] (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.

[0065] Taking DHV and Chs-DHV as the control groups, and the bifunctionalized decellularized nanoparticle crosslinked engineered valve group (NP-Chs-DHV) prepared in Example 1 as the experimental group, the structure and morphology were characterized respectively.

[0066] The results are as Figure 2 shown: Figure 2 In, 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, and it can be observed that the crosslinking and modification on the surface morphology will not destroy the original structure of DHV, meeting the requirements for cell growth.

[0067] The component characterization results of NP-Chs-DHV are as Figure 3 shown. Figure 3In Figure A of , Fourier transform infrared spectroscopy was used to scan the surfaces of DHV, Chs-DHV, and NP-Chs-DHV, indicating that chondroitin sulfate (Chs) / nanoparticles were uniformly modified onto the surface of DHV. Figure B shows the results of sectioning 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, indicating that the RBCM-Ab-coated nanoparticles were successfully modified onto the surface of DHV.

[0068] Example 2 Verification of Heart Valve Endothelialization and Anti-Calcification Effect

[0069] Taking the acellular heart valve group (DHV group) and glutaraldehyde-crosslinked acellular heart valve group (GLU group) as control groups, and taking the bifunctionalized acellular nanoparticle-crosslinked engineered valve group (NP-Chs-DHV) prepared in Example 1 as the experimental group for subsequent experiments.

[0070] The DHV group was prepared by the method for preparing acellular heart valve DHV in step 4 of Example 1.

[0071] The preparation method of the GLU group was as follows: wipe the surface moisture of the above-prepared DHV, 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.

[0072] 1. Establish a rabbit carotid artery implantation model to evaluate the performance of the stent in a hemodynamic environment.

[0073] 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.

[0074] 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 responses.

[0075] Pre-adaptation treatment: Fast for 12 hours before surgery, with water available.

[0076] Experimental method: Establish a right carotid artery-left jugular vein shunt in rabbits, draw out the blood flow, prepare a 1 cm * 1 cm valve sheet into a valve roll and implant it into the arteriovenous shunt, run it under circulating blood for 3 h, take out the valve roll after 3 h, and detect the red blood cell adhesion on the valve surface. The schematic diagram of the model construction is shown in Figure 4 。

[0077] Two weeks later, the scaffolds were subjected to CD31 and DAPI immunofluorescence staining. Four weeks later, Doppler ultrasound was performed to examine the patency of the scaffolds, and then the scaffolds were stained with Masson and Von Kossa to show the ECM structure and calcification. Macrophage markers (CD68, iNOS, and CD206), endothelial cell markers (CD31 and vWF), stromal cell marker (vimentin), and type I collagen staining were used to evaluate the inflammation, cellularization, and remodeling of the implanted scaffolds, respectively.

[0078] 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 Figure 5 shown.

[0079] Figure 5 In it, Figure A is a 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 exhibited 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 exhibits excellent antithrombotic ability; Figure D shows that the in vitro platelet adhesion experiment also indicates that NP-Chs-DHV can effectively inhibit platelet adhesion and activation.

[0080] 2. Establish a rat abdominal aorta transplantation model

[0081] 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.

[0082] 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.

[0083] Preconditioning: 12 hours before surgery, fast but allow water intake.

[0084] 1) Anesthesia and skin preparation

[0085] Anesthesia: Intraperitoneal injection of sodium pentobarbital (40 mg / kg) or inhalation induction with isoflurane (4%) + maintenance anesthesia with inhalation of isoflurane (1.5 - 2%);

[0086] Skin preparation: Shave the abdomen, disinfect with iodophor + 75% alcohol, and drape with a sterile surgical drape;

[0087] 2) Exposure of the abdominal aorta

[0088] Incision: A midline abdominal incision (3-4 cm in length) is made, and the muscles are separated layer by layer to expose the abdominal cavity;

[0089] Positioning: Gently push the intestine to the right to expose the abdominal aorta (from the renal artery bifurcation to the iliac artery bifurcation);

[0090] Free blood vessels: Bluntly separate the connective tissue around the abdominal aorta, leaving a free segment of approximately 15 mm;

[0091] 3) Valve channel preparation

[0092] Valve shaping: A 5 × 5 mm NP-Chs-DHV valve sheet was rolled into a tube (inner diameter ≈ 1.5 mm) and fixed at the edges with continuous sutures of PDS 8-0 sutures.

[0093] Pretreatment: soak the tubing in heparinized saline (50 U / mL) for 10 min;

[0094] 4) Vascular transplantation (end-to-end anastomosis)

[0095] Blocking blood flow: Microvascular clips block the proximal and distal ends of the abdominal aorta;

[0096] Vascular transection: The abdominal aorta was transversely cut in the center of the occluded segment (length ≈ 5 mm);

[0097] Anastomosis operation (under microscope):

[0098] Proximal end: suture the rolled-up valve piece with 8 interrupted stitches using Prolene 10-0 suture (first fix the 3, 6, 9, and 12 o'clock directions);

[0099] Distal end: The rolled-up valve piece is anastomosed in the same way to ensure that the intima is aligned neatly without distortion or tension.

[0100] Open blood flow: first loosen the distal end clamp, then loosen the proximal end clamp, and observe the bleeding and pulsation.

[0101] 5) Intraoperative management

[0102] Anticoagulation: local spraying of heparin sodium (100 U / mL) to prevent thrombosis;

[0103] Anti-spasmodic: Papaverine (0.5 mg / mL) was instilled into the anastomotic site to relieve vasoconstriction;

[0104] 6) Experimental endpoint and sample collection

[0105] Time point: 14 days or 28 days according to the research purpose;

[0106] Euthanasia: Excessive inhalation of CO2 or intraperitoneal injection of sodium pentobarbital (150 mg / kg);

[0107] Specimen collection: Completely excise the transplanted vascular segment (including the anastomotic sites at both ends); Fix with 4% paraformaldehyde (for histology) or snap-freeze in liquid nitrogen (for molecular detection);

[0108] Histological detection: HE staining (for inflammation), Masson (for collagen), CD31 (for endothelialization).

[0109] 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 compared with DHV, it can also effectively inhibit valve degradation, while meeting the requirements of cell infiltration.

[0110] 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 macrophage polarization to the M2 phenotype, can effectively promote the regeneration of type I collagen, and partial endothelialization occurred 14 days after implantation, and complete endothelialization was achieved 28 days after implantation, showing excellent regenerative ability.

[0111] The above-described embodiments 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, It includes the following steps: S1. Preparation of RBCM-Ab: Take whole blood and centrifuge it at 1000 - 3000 rpm for 10 - 20 min, resuspend it in PBS buffer containing EDTA-K2, incubate it at 4 - 8 °C for 20 - 40 min, then centrifuge it at 11000 - 13000 rpm for 10 - 20 min to obtain an RBCM suspension; add 40 - 60 μL of 50 - 150 μM CLS-PEG-SA to 1 - 2 mL of the RBCM suspension, react at 15 - 20 °C for 20 - 40 min, and wash to obtain RBCM-Biotin; mix SA and 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, react at 15 - 20 °C for 20 - 40 min to obtain RBCM-Ab; S2. Preparation of 2dDR-PLGA-NPs: Use a 2dDR solution with a final concentration of 100 - 300 mg / mL dissolved in NaCl as the inner aqueous phase W1; use an ethyl acetate solution containing ethanol and 1 - 10% PLGA as the oil phase O; take 50 - 100 μL of W1 and drop it into 350 - 400 μL of the oil phase O, perform ice bath sonication at 40 - 60 W for 10 - 50 s to obtain primary emulsion; use an NaCl solution containing poloxamer 188 as the outer aqueous phase W2; inject the primary emulsion into the outer aqueous phase W2, perform ice bath sonication at 40 - 60 W for 10 - 50 s, and stir at 500 - 1500 rpm for 10 - 20 h to obtain multiple emulsion; centrifuge at 1 - 5 °C and 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: Take 1 - 5 mL of RBCM-Ab and 1 - 10 mg of 2dDR-PLGA-NPs, mix them for 1 - 10 min, and extrude with an extruder to obtain RBCM-Ab / 2dDR@PLGA; S4. Preparation of DHV: Place the bioprosthetic valve material in a 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, 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 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, 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, 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, wherein 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, wherein Add 50 μL of CLS - PEG - SA with a concentration of 100 μM to 1 mL of 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 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, 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.

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 at a power of 50 W for 30 s; 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 at a power of 50 W for 30 s again, and then stir magnetically at 1000 rpm for 12 h to obtain the 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, characterized in that, 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 by the preparation method according to 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, pro-endothelialization and anti-calcification functions.

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