A tissue engineering small-caliber artificial blood vessel graft and its preparation method and application

Abstract

The present invention discloses a tissue engineering small-caliber artificial blood vessel graft and its preparation method and application, belonging to the field of biomedical material technology. By modifying the inner layer of the small-caliber biological artificial blood vessel graft and related treatment, thrombosis and intimal hyperplasia are prevented, and the patency rate after blood vessel transplantation is improved. Layers of self-assembled positively charged endothelial cells are planted on the outer surface of the blood vessel. Through the in vivo construction strategy, the hemodynamic scour is overcome, the adhesion and activation of immune cells and platelets are reduced, and short-term acute thrombosis is prevented. At the same time, endothelial mesenchymalization is inhibited, and the inner surface of the blood vessel is cellularized by in vitro cell planting, the endothelialization process of small-caliber artificial blood vessels is accelerated, and capillary formation is promoted, thereby realizing in vivo remodeling of vascular implants, effectively exerting anti-thrombosis and intimal hyperplasia effects, improving patency, and forming mature blood vessels. Provide a small-caliber biological artificial blood vessel that can be used clinically for hemodialysis, peripheral vascular replacement, and coronary artery bypass grafting.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a tissue engineering small-caliber artificial blood vessel graft and a preparation method and application thereof. Background Art

[0002] According to the latest statistics, the incidence and mortality of cardiovascular diseases are rising year by year around the world, which has led to an increasing demand for vascular grafts around the world. Small-caliber vascular grafts are needed for peripheral vascular occlusion or damage replacement, coronary artery bypass grafting, and hemodialysis arteriovenous fistulas.

[0003] At present, most clinical vascular reconstructions use artificial blood vessels and autologous blood vessels as substitutes. Autologous blood vessels are ideal materials, but their sources are limited and they are difficult to use as substitutes for various blood vessels; xenogeneic blood vessels are widely available, but they have strong immune rejection reactions, which leads to transplant failures. Clinicians have to use artificial synthetic blood vessels as substitutes. Large-caliber artificial blood vessels have been successfully used to replace human aortas, but artificial blood vessels with an inner diameter of less than 6 mm have not achieved satisfactory results in replacing human arterioles or veins. The main reason is that thrombosis and neointimal thickening cause artificial blood vessels to block. Therefore, the development of arterioles, arteriovenous bypass and venous substitutes has become an urgent problem to be solved.

[0004] In order to make small-caliber biological artificial blood vessel grafts have a relatively ideal long-term patency rate, their surfaces need to be modified. At present, there are two main ways to improve the blood compatibility of artificial blood vessel materials: 1. Endothelialization of artificial blood vessel surface. 2. Change the chemical and physical characteristics of the material surface, such as adding chemical groups, changing the surface charge of the material, increasing hydrophobicity, etc., to achieve the purpose of reducing fibrin and platelet adhesion.

[0005] In recent years, tissue engineering technology has shown great potential in the construction of small-caliber vascular grafts. After transplantation, the occurrence of thrombosis and stenosis is the main reason restricting the clinical application of small-caliber bioartificial vascular grafts. Achieving endothelialization is the key to ensuring the long-term patency of small-caliber bioartificial blood vessels: the earlier the lumen is endothelialized, the lower the risk of thrombosis, and it can inhibit abnormal proliferation of the intima. Summary of the invention

[0006] The first purpose of the present invention is to provide a method for preparing a tissue engineering small-caliber artificial vascular graft, which involves a layer-by-layer assembly to inhibit thrombosis and endothelial-mesenchymalization, and to achieve endothelialization of the inner surface of the blood vessel by planting endothelial cells, and to maintain a long-term patency of the vascular graft. Through an in vivo construction strategy, the hemodynamic flushing is overcome in vivo, the adhesion and activation of immune cells and platelets are reduced, and "immune escape" is achieved to prevent short-term acute thrombosis. At the same time, endothelial-mesenchymalization is inhibited, and the inner surface of the blood vessel is cellized by in vitro cell planting, the endothelialization process of small-caliber artificial blood vessels is accelerated, and capillary formation is promoted, thereby achieving in vivo remodeling of vascular implants, effectively exerting anti-thrombotic and intimal hyperplasia effects, improving the patency rate of small-caliber biological artificial blood vessel transplantation, and finally forming a fully self-replaced mature blood vessel. The huge demand for small-caliber biological artificial blood vessels in the prior art and the technical problems of post-transplant thrombosis and vascular stenosis are solved.

[0007] The second purpose of the present invention is to provide a method for preparing a tissue-engineered small-caliber artificial vascular graft, in which the third layer assembly of the artificial blood vessel realizes "immune escape", prevents short-term acute thrombosis, inhibits endothelial-mesenchymal transformation, and realizes vascular inner surface cellularization through in vitro cell planting, accelerates the endothelialization process of small-caliber artificial blood vessels, and promotes capillary formation, thereby realizing in vivo remodeling of vascular implants, effectively exerting anti-thrombotic and intimal hyperplasia effects, improving the patency rate of small-caliber biological artificial blood vessel transplantation, and ultimately forming a fully self-replacing mature blood vessel.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for preparing a tissue engineering small-caliber artificial vascular graft comprises: firstly treating an ex vivo vascular substrate to obtain a complete small-caliber artificial vascular stent; then using a carbodiimide crosslinking agent to crosslink PDA @ COF / LY nanoparticles are covalently bonded to the surface of a small-caliber artificial vascular stent for first-layer assembly to obtain an artificial vascular graft I, which is characterized by further comprising:

[0010] The second layer assembly: using a bifunctional group cross-linking agent, the artificial vascular graft I is covalently cross-linked with negatively charged anticoagulant high molecular weight hyaluronic acid and 4-25 disaccharide unit hyaluronic acid on the artificial vascular graft I to obtain artificial vascular graft II;

[0011] The third layer of assembly: using electrostatic adsorption to adhere positively charged endothelial cells to the surface of artificial vascular graft II, and conducting in vitro cell culture to obtain an artificial vascular graft with vascular surface cellularization.

[0012] Preferably, in the second layer assembly, the bifunctional group cross-linking agent includes any one of MAL-PEG-NHS, MAL-PEG-NH2, MAL-PEG-CHO or MAL-PEG-COOH;

[0013] One end of the bifunctional group cross-linking agent contains a maleamide active group, and the other end contains an active group that reacts with an amino group or a carboxyl group.

[0014] Preferably, the cross-linking ratio of the negatively charged anticoagulant high molecular weight hyaluronic acid and the 4-25 disaccharide unit hyaluronic acid is 1:1-3;

[0015] The molecular weight of the anticoagulant high molecular weight hyaluronic acid is 1-1.5 million.

[0016] Preferably, the covalent cross-linking process is as follows: a bifunctional group cross-linking agent is selected and dissolved in a PBS buffer solution at a concentration of 5-10 mg / mL, placed at 2-8°C, and completely immersed in the artificial vascular graft I, and after shaking at 90-150 r / min for 1-3 hours, an artificial vascular graft I modified with a bifunctional group is obtained;

[0017] Negatively charged anticoagulant high molecular weight hyaluronic acid and 4-25 disaccharide unit hyaluronic acid were selected and dissolved in PBS solution at a concentration of 5-30 mg / mL, placed at 2-8°C, and completely immersed in the artificial vascular graft I modified with the bifunctional groups. After oscillation at 100 r / min for 1-3 hours, artificial vascular graft II was obtained.

[0018] Preferably, in the third layer assembly, the positively charged endothelial cells are obtained by endothelial cell layer-by-layer self-assembly.

[0019] The specific method includes: centrifuging the endothelial cell suspension and discarding the supernatant; adding 2-10mL 0.02-0.1% chitosan solution to resuspend and mix, shaking for 4 minutes, centrifuging and discarding the supernatant; adding D-Hank's to wash, centrifuging and discarding the supernatant; then adding 2-10mL 0.05%-0.1% sodium alginate solution to resuspend, centrifuging and discarding the supernatant; adding 5mL D-Hank's to wash, centrifuging and discarding the supernatant; repeating the above steps in sequence to self-assemble into 5 layers of cells layer by layer.

[0020] Preferably, the five cell layers include chitosan layer-sodium alginate layer-chitosan layer-sodium alginate layer-chitosan layer.

[0021] The purpose of making the cells into five layers is to achieve a better and more stable assembly effect.

[0022] Preferably, the third layer assembly process includes: selecting positively charged endothelial cells and adding them to 700-100 μL EBM-2 culture medium to resuspend them; fixing the artificial vascular graft II in a vascular culture reactor, statically culturing in a 37°C incubator for 1 hour, flipping the reactor 180°, and continuing to statically culture in a 37°C incubator for 1 hour, filling the reactor with EBM-2 culture medium, statically culturing for 72 hours, changing the culture medium once a day, and obtaining an artificial vascular graft.

[0023] Preferably, the ex vivo vascular substrate includes an ex vivo full-term pregnancy placenta umbilical cord or a xenogeneic blood vessel or an allogeneic blood vessel.

[0024] A tissue engineering small-caliber artificial blood vessel graft is obtained by the preparation method of the tissue engineering small-caliber artificial blood vessel graft.

[0025] The invention discloses an application of a tissue-engineered small-caliber artificial vascular graft, wherein the artificial vascular graft is clinically used as a vascular substitute in hemodialysis, peripheral vascular replacement, and coronary artery bypass grafting.

[0026] Compared with the prior art, the present invention has at least the following technical effects:

[0027] The present invention provides a method for preparing a tissue-engineered small-caliber artificial vascular graft, which is aimed at the huge demand for small-caliber biological artificial blood vessels and the difficulties they face, such as thrombosis and vascular stenosis after transplantation, by modifying the surface of the material with self-assembled endothelial cells layer by layer to achieve cellularization of the inner surface of the blood vessel and promote the lateral migration of ECs. However, in the short term of vascular graft implantation, the vascular graft, as a foreign body, will activate platelets and cause acute thrombosis. In the medium and long term implantation period, in the complex pathological microenvironment, endothelial cells (ECs) can undergo endothelial-mesenchymal transformation (EndMT), thereby inducing pathological vascular remodeling and causing technical problems such as TEBV stenosis or blockage.

[0028] The technical solution of this application is to modify the PDA in the inner layer of a small-caliber biological artificial vascular graft. @ COF / LY uses COF nanoparticles with high drug loading efficiency to load LY2157299, and inhibits TGF-β-mediated EndMT by local sustained release of LY2157299, maintaining endothelial integrity and ECs health, preventing thrombosis and intimal hyperplasia, and improving the patency rate after vascular transplantation. At the same time, in the middle layer of the vascular surface, negatively charged macromolecules and HA with 4-25 disaccharide units are coupled to repel platelets and red blood cells that are also negatively charged, thus playing a key role in short-term acute thrombosis.

[0029] In addition, the outer surface of the blood vessel is planted with layers of self-assembled positively charged endothelial cells. Through the in vivo construction strategy, the hemodynamic scour is overcome in vivo, the adhesion and activation of immune cells and platelets are reduced, "immune escape" is achieved, and short-term acute thrombosis is prevented. At the same time, endothelial mesenchymalization is inhibited, and the inner surface of the blood vessel is cellularized through in vitro cell planting, which accelerates the endothelialization process of small-caliber artificial blood vessels and promotes capillary formation, thereby achieving in vivo remodeling of vascular implants, effectively exerting anti-thrombotic and intimal hyperplasia effects, improving the patency rate of small-caliber biological artificial blood vessels, and finally forming a fully self-replaced mature blood vessel. Provide a small-caliber biological artificial blood vessel that can be used clinically for hemodialysis, peripheral vascular replacement, and coronary artery bypass grafting. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the self-assembly process of Experimental Example 1 using a laser confocal microscope;

[0031] Figure 2 This is a schematic diagram of the changes in FITC and Cy5 fluorescence intensity of cells during the layer-by-layer self-assembly of endothelial cells detected by flow cytometry in Experimental Example 2;

[0032] Figure 3 Schematic diagram of the percentage of FITC+ cells after layer-by-layer self-assembly of endothelial cells in Experimental Example 2;

[0033] Figure 4 Schematic diagram of the percentage of Cy5+ cells after layer-by-layer self-assembly of endothelial cells in Experimental Example 2;

[0034] Figure 5 Schematic diagram of the change of cell membrane surface charge during endothelial cell self-assembly detection by Zeta potential in Experimental Example 3;

[0035] Figure 6 This is a schematic diagram of transmission electron microscopy observation of layer-by-layer self-assembled endothelial cells in Experimental Example 4;

[0036] Figure 7 This is a schematic diagram of scanning electron microscopy observation of layer-by-layer self-assembly of endothelial cells in Experimental Example 5. DETAILED DESCRIPTION

[0037] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0038] A technical solution of a specific implementation of the present invention is:

[0039] A method for preparing a tissue engineering small-caliber artificial blood vessel graft comprises the following steps:

[0040] 1. Preparation of tissue engineering small-caliber artificial vascular graft scaffold materials: Fresh full-term placenta umbilical cord or xenogeneic blood vessels or allogeneic blood vessels are taken out of the body, and after pretreatment, decellularization and antigen removal, non-immunogenic vascular scaffold materials are initially formed. After solidification, shaping, rehydration and sterilization, complete small-caliber artificial vascular scaffold materials are formed.

[0041] 1.1 The pretreatment steps include: using 1%-5% heparin saline solution, adding 100μL / mL penicillin and 0.1mg / mL streptomycin as the transport storage solution, the transport temperature is 2-8℃, transporting to the clean workshop within 12h, flushing the remaining blood, trimming the outer membrane to make its surface uniform.

[0042] 1.2 Decellularization: Treat with CHAPS solution at a concentration of 4-10 g / L for 8-16 h, or with SDS solution at a concentration of 0.4-1 g / L for 8-16 h, and wash with PBS buffer 10 times.

[0043] 1.3 Antigen removal: Use 10-30 μL / mL concentration of RNase and DNase for treatment at 37°C, 100 r / min for 1-4h.

[0044] 1.4 Solidification and shaping: Use specially made polytetrafluoroethylene or glass rods with different pore sizes to penetrate the graft, place it at 30-50℃ for 6-10 hours, and after it is completely solidified, rehydrate it in physiological saline for 5-10 minutes and peel it off.

[0045] 1.5 Sterilization method: Use chemical liquid sterilization, 0.2-0.4% peracetic acid and 4%-8% ethanol, mixed in a 1:1 ratio, soak the blood vessels at room temperature, shake on a shaker for 30min-120min, and wash with sterile water.

[0046] 2. Assemble layer by layer:

[0047] 2.1 First layer assembly: Nano-COF was synthesized by combining TPB and DVA building blocks through Schiff base reaction, and LY2157299 was loaded on COF to obtain COF / LY. After modifying the surface of COF / LY with dopamine / hexamethylenediamine, surface amino-modified PDA was obtained. @ COF / LY nanoparticles are covalently bonded to the surface of the graft vascular stent through a carbodiimide crosslinker.

[0048] PDA @Preparation of COF / LY nanoparticles: 0.01-0.05mM TPB and 0.01-0.06mM DVA were dissolved in ethanol by ultrasonic treatment, the two solutions were mixed, and 100-250μL (12M) acetic acid was added. Mix vigorously and let stand at room temperature for 18-24h. The nanoparticles were collected by centrifugation at 5000-10000rpm, washed with ethanol, and dried in vacuum. The dried nanoparticles were ultrasonically dispersed in DMSO, and then LY2157299 was dissolved in the suspension to prepare 0.5-1mg / mL COF / LY. The mixture was ultrasonicated for 2min and stored under high vacuum at room temperature for 24h. The precipitate was collected by centrifugation and washed with deionized water. 1-5mg dopamine hydrochloride and 1-5mg hexamethylenediamine were added to 5-10mL Tris-Buffer to obtain a mixed solution. The newly prepared COF / LY was dispersed in the above solution, stirred in the dark for 1-2h at room temperature, and the particles were collected by centrifugation and washed with deionized water. The particles were dried under high vacuum to obtain PDA. @ After COF / LY, place in a 4°C refrigerator and protect from light.

[0049] PDA @ COF / LY nanoparticles were assembled on the surface of graft blood vessels: the decellularized small-caliber bioartificial blood vessels were placed in 1-5 mg / mL EDC solution (PBS, pH=7.4) and immersed at 4°C overnight.

[0050] 2.2 Second layer assembly: Using a cross-linking agent with bifunctional groups, the small-caliber artificial vascular scaffold material assembled in the first layer is covalently cross-linked with negatively charged high molecular weight hyaluronic acid with anticoagulant effect and hyaluronic acid with 4-25 disaccharide units that promotes angiogenesis in a ratio of 1:1 or 1:2 or 1:3, and connected to the outer layer of the vascular graft coating to obtain a small-caliber artificial vascular graft that is modified by layer-by-layer assembly.

[0051] The bifunctional cross-linking agents used include MAL-PEG-NHS, MAL-PEG-NH2, MAL-PEG-CHO, MAL-PEG-COOH, etc., which contain a maleamide active group at one end and an active group that reacts with an amino group or a carboxyl group at the other end.

[0052] The cross-linking process is as follows: take the solid and dissolve it in PBS buffer solution at a concentration of 5-10 mg / mL, place it at 2-8°C, completely immerse the small-caliber umbilical vein blood vessels, and oscillate at 100 r / min for 1-3 hours. Then, take the negatively charged high molecular weight (1 million to 1.5 million) hyaluronic acid with anticoagulant effect and the hyaluronic acid with 4-25 disaccharide units that promote angiogenesis, and dissolve the solid in PBS solution at a ratio of 1:1, 1:2, and 1:3 at a concentration of 5-30 mg / mL, place it at 2-8°C, completely immerse the small-caliber bioartificial vascular graft modified with the bifunctional group, and oscillate at 90-150 r / min for 1-3 hours.

[0053] 2.3 Third layer assembly: Endothelial cells are self-assembled layer by layer to make the endothelial cells positively charged, and adhere to the blood vessel surface through electrostatic adsorption with the second layer of negatively charged hyaluronic acid. The blood vessel surface is cellularized through in vitro cell culture to obtain a small-caliber artificial vascular graft modified by layer-by-layer assembly.

[0054] Endothelial cell layer-by-layer self-assembly: The endothelial cell suspension was centrifuged at 1000rpm for 5min, and the supernatant was discarded. 2-10mL CH solution was added to resuspend and mix, and shaken at 100rpm for 4min. Centrifuge at 1200rpm for 5min, and the supernatant was discarded. 5-10mL D-Hank's was added to wash, and the supernatant was discarded after centrifugation at 1200rpm for 5min. 2-10mL ALG solution was added to resuspend, and shaken at 100rpm for 4min. Centrifuge at 1000rpm for 5min, and the supernatant was discarded. 5-10mL D-Hank's was added to wash, and the supernatant was discarded after centrifugation at 1000rpm for 5min. CH and ALG were added repeatedly to perform layer-by-layer self-assembly. Self-assembly of 1 layer is C, and self-assembly of 5 layers is CACAC, which is denoted as ((CA)2C), where C represents chitosan and A represents sodium alginate.

[0055] The specific process of the third layer assembly: take the self-assembled endothelial cells layer by layer and add 700-100μL EBM-2 culture medium to resuspend. Fix the blood vessels in the vascular culture reactor, culture statically in a 37℃ incubator for 1h, flip the reactor 180°, continue to culture statically in a 37℃ incubator for 1h, fill the reactor with EBM-2 culture medium, culture statically for 72h, and change the culture medium once a day.

[0056] Embodiment 1:

[0057] Allogeneic umbilical vein is used as the vascular stent material, and 1%-5% heparin saline solution, 100μL / mL penicillin, and 0.1mg / mL streptomycin are used as the transportation storage solution. The transportation temperature is 2-8℃. It is transported to a clean workshop within 12 hours, residual blood is flushed, and the umbilical cord is trimmed to a length of 20cm.

[0058] Umbilical cord decellularization was carried out by treating with CHAPS solution at a concentration of 4.92 g / L for 14 h, and with SDS solution at a concentration of 0.52 g / L for 14 h, followed by washing with PBS buffer 10 times at 100 r / min and 10 min / time.

[0059] Antigen removal was performed by treating with 10 μL / mL concentration of RNase and DNase at 37°C, 100 r / min for 2 h.

[0060] After decellularization and antigen removal, the umbilical cord is passed through the umbilical vein using a special 3mm polytetrafluoroethylene rod and placed at 40°C for 6 hours. After the umbilical cord is completely solidified, it is rehydrated in physiological saline for 5 minutes and the umbilical cord is peeled off.

[0061] The stripped umbilical cord was immersed in a mixed solution of 0.1% peracetic acid and 4% ethanol, shaken at 100 r / min at 25°C for 2 hours, and washed with sterile water until there was no change on the peracetic acid test paper.

[0062] Place the sterilized umbilical vein in 400 μL of nanoparticle solution (1 mg / mL, PBS, pH=7.4), ultrasonically disperse, and shake at 300 rpm for 2 hours at 4°C. Place the umbilical vein in 4 mg / mL rat tail collagen and shake at 300 rpm for 2 hours at 4°C. Place the umbilical vein in the nanoparticle solution again and shake at 300 rpm for 2 hours at 4°C. Place the umbilical vein in 5 mg / mL EDC solution (PBS, pH=7.4) and soak overnight at 4°C.

[0063] The umbilical vein was taken out, and the bifunctional group cross-linking agent MAL-PEG-NHS was dissolved in PBS buffer solution at a concentration of 5.5 mg / mL. The solution was placed at 2-8°C to completely immerse the small-caliber umbilical vein, and oscillated at 100 r / min for 1 hour.

[0064] Hyaluronic acid with 150kDa thiol-active groups and small-molecule hyaluronic acid with 25 disaccharide units were dissolved in PBS solution at a concentration of 5mg / mL in a 1:1 ratio. The solution was placed at 2-8°C to completely immerse the small-caliber umbilical vein blood vessels modified with the bifunctional groups, and oscillated at 100r / min for 1h.

[0065] Endothelial cell layer-by-layer self-assembly: The endothelial cell suspension was centrifuged at 1000rpm for 5min, and the supernatant was discarded. 2mL of 0.05% CH solution was added to resuspend and mix, and shaken at 100rpm for 4min. Centrifuged at 1200rpm for 5min, and the supernatant was discarded. 5mL of D-Hank's was added to wash, and the supernatant was discarded after centrifugation at 1200rpm for 5min. 2mL of 0.05% ALG solution was added to resuspend, and shaken at 100rpm for 4min. Centrifuged at 1000rpm for 5min, and the supernatant was discarded. 5mL of D-Hank's was added to wash, and the supernatant was discarded after centrifugation at 1000rpm for 5min. CH and ALG were added repeatedly to perform layer-by-layer self-assembly. Self-assembly of 1 layer is C, and self-assembly of 5 layers is CACAC, which is denoted as ((CA)2C), where C represents chitosan and A represents sodium alginate.

[0066] Take the layers of self-assembled endothelial cells and add 700-100 μL EBM-2 culture medium to resuspend. Fix the umbilical vein in the vascular culture reactor and culture it statically in a 37°C incubator for 1 hour. Flip the reactor 180° and continue to culture it statically in a 37°C incubator for 1 hour. Fill the reactor with EBM-2 culture medium and culture it statically for 72 hours. Change the culture medium once a day. Then you will get a small-caliber artificial vascular graft assembled layer by layer.

[0067] That is, a small-caliber artificial blood vessel of the same type of umbilical vein assembled layer by layer is obtained.

[0068] Embodiment 2:

[0069] Allogeneic blood vessels are used as small-caliber bioartificial blood vessel scaffolds, and 1%-5% heparin saline solution, 100μL / mL penicillin, and 0.1mg / ml streptomycin are used as transportation and preservation fluid. The transportation temperature is 2-8℃. The vessels are transported to a clean workshop within 12 hours, residual blood is flushed, and the vascular adventitia is trimmed to make it uniform.

[0070] Allogeneic decellularization was treated with CHAPS solution at a concentration of 4.92 g / L for 14 h and washed 6 times with PBS buffer at 100 r / min and 10 min / time.

[0071] Antigen removal was performed using 10 μL / mL concentration of RNase and DNase at 37°C, 100 r / min for 2 h, and then washed 16 times with PBS.

[0072] After decellularization and antigen removal, the allogeneic blood vessels were immersed in a mixed solution of 0.1% peracetic acid and 4% ethanol, shaken at 100 r / min at 25°C for 2 hours, and washed with sterile water until there was no change in the peracetic acid test paper.

[0073] The sterilized allogeneic blood vessels were placed in 400 μL of nanoparticle solution (1 mg / mL, PBS, pH=7.4), ultrasonically dispersed, and shaken at 300 rpm for 2 hours at 4°C. The allogeneic blood vessels were placed in 4 mg / mL rat tail collagen and shaken at 300 rpm for 2 hours at 4°C. The blood vessels were placed in the nanoparticle solution again and shaken at 300 rpm for 2 hours at 4°C. The blood vessels were placed in 5 mg / mL EDC solution (PBS, pH=7.4) and soaked overnight at 4°C.

[0074] The blood vessels were taken out, and the bifunctional crosslinker MAL-PEG-NHS was dissolved in PBS buffer solution at a concentration of 5.5 mg / mL. The solution was placed at 2-8°C to completely immerse the blood vessels and oscillated at 100 r / min for 1 hour.

[0075] Hyaluronic acid with 150 kDa thiol active groups and small molecule hyaluronic acid with 25 disaccharide units were dissolved in PBS solution at a concentration of 5 mg / mL in a 1:1 ratio. The solution was placed at 2-8°C to completely immerse the blood vessels modified with the bifunctional groups and oscillated at 100 r / min for 1 hour.

[0076] Endothelial cell layer-by-layer self-assembly: The endothelial cell suspension was centrifuged at 1000rpm for 5min, and the supernatant was discarded. 2mL of 0.05% CH solution was added to resuspend and mix, and shaken at 100rpm for 4min. Centrifuged at 1200rpm for 5min, and the supernatant was discarded. 5mL of D-Hank's was added to wash, and the supernatant was discarded after centrifugation at 1200rpm for 5min. 2mL of 0.05% ALG solution was added to resuspend, and shaken at 100rpm for 4min. Centrifuged at 1000rpm for 5min, and the supernatant was discarded. 5mL of D-Hank's was added to wash, and the supernatant was discarded after centrifugation at 1000rpm for 5min. CH and ALG were added repeatedly to perform layer-by-layer self-assembly. Self-assembly of 1 layer is C, and self-assembly of 5 layers is CACAC, which is denoted as ((CA)2C), where C represents chitosan and A represents sodium alginate.

[0077] Take the layers of self-assembled endothelial cells and add 700-100uL EBM-2 culture medium to resuspend. Fix the blood vessels in the vascular culture reactor, culture them statically in a 37℃ incubator for 1 hour, flip the reactor 180°, continue to culture them statically in a 37℃ incubator for 1 hour, fill the reactor with EBM-2 culture medium, culture them statically for 72 hours, and change the culture medium once a day. Then, a small-caliber artificial vascular graft assembled layer by layer is obtained.

[0078] Related testing experiments:

[0079] 1) Confocal laser scanning microscopy (CLSM) observation of the self-assembly process

[0080] The entire experimental operation was carried out under light-proof conditions. During the layer-by-layer self-assembly process of endothelial cells, after each layer of material was assembled and washed, 2mL D-Hank's was added to resuspend the cells. Take 200μL of cell suspension and add it to a new 15mL offline tube, add 800μL 4% paraformaldehyde solution (PFA) to fix the cells, add 5mL DPBS solution after 15min, mix well, and centrifuge at 1000rpm for 5min. After removing the supernatant, add 200μL DAPI solution (10μg / mL), mix well and let it stand for staining for 10min, add 5mL DPBS and mix well, centrifuge at 1000rpm for 5min, and discard the supernatant. Add 50μL DPBS to resuspend. Take 25μL of cell suspension and drop it on the slide, dry it in a constant temperature drying oven at 37℃ for 10min, add 10μL anti-fluorescence quenching sealing agent, and slowly cover the coverslip from one side (to prevent bubbles). The coverslip was fixed with nail polish, dried in a dark and ventilated environment, and then observed using a laser confocal microscope (LSM900).

[0081] like Figure 1 The figure shows the changes of cell membrane surface materials during the layer-by-layer self-assembly process observed by laser confocal microscopy. Red: sodium alginate; green: chitosan; blue: cell nucleus. C represents chitosan and A represents sodium alginate.

[0082] The self-assembly of 1 layer is C, and the self-assembly of 5 layers is CACAC, denoted as (CA)2C.

[0083] 2) Flow cytometric detection of fluorescence intensity changes after cell-layer self-assembly

[0084] The entire experimental operation was carried out under light-proof conditions. During the layer-by-layer self-assembly process of endothelial cells, after each layer of material was assembled and washed, 2 mL of D-Hank's was added to resuspend the cells. 200 μL of cell suspension was taken and filtered into a single cell suspension using a cell strainer. A flow cytometer (Accuri C6 Plus) was used to detect changes in FITC and Cy5 fluorescence intensity on the cell surface.

[0085] like Figure 2 Shown is a schematic diagram of the changes in FITC and Cy5 fluorescence intensity of cells during the layer-by-layer self-assembly of endothelial cells detected by flow cytometry.

[0086] like Figure 3 Shown is a schematic diagram of the percentage of FITC+ cells after layer-by-layer self-assembly of endothelial cells.

[0087] like Figure 4 Shown is a schematic diagram of the percentage of Cy5+ cells after layer-by-layer self-assembly of endothelial cells.

[0088] 3) Detection of changes in cell membrane Zeta potential during self-assembly

[0089] Use D-Hank's solution to prepare the self-assembly solution: 0.02% CH and 0.05% ALG for later use. Take a bottle (T25 culture flask) with about 80% endothelial cells, discard the culture medium, add 2mL DPBS to wash 3 times, 30s each time. Add 1.5mL 0.25% trypsin digestion solution, place in culture for digestion for 2min, and add 4.5mL complete culture medium to terminate digestion. Add the cell suspension to a 15mL centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant. Add 1mL D-Hank's to resuspend, take 100μL of cell suspension and add it to 1.4mL D-Hank's, mix well and perform Zeta potential detection; the remaining cells are centrifuged at 1000rpm for 5min, and the supernatant is discarded. Add 2mL CH solution to resuspend and mix well, and shake on a shaker at 100rpm for 4min. Centrifuge at 1000rpm for 5min and discard the supernatant. Add 5mL D-Hank's and mix well. Centrifuge at 1200rpm for 5min and discard the supernatant. Add 1mL D-Hank's to resuspend. Take 100μL of cell suspension and add it to 1.4mL D-Hank's. Mix well and perform Zeta potential detection. After each self-assembly and washing, add 1mL D-Hank's to resuspend. Take 100μL of cell suspension and add it to 1.4mL D-Hank's. Mix well and perform Zeta potential detection. A total of five layers are self-assembled.

[0090] like Figure 5 The figure shows the change of cell membrane surface charge during endothelial cell self-assembly by Zeta potential. The results show that the Zeta potential is the average value of 20 repeated measurements by the instrument.

[0091] Above Figure 2-5 The data presented in the figure were obtained from 3 independent repeated experiments and are expressed as mean ± variance.

[0092] *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, indicating significant statistical differences.

[0093] 4) Transmission electron microscopy observation of layer-by-layer self-assembled endothelial cells

[0094] Sampling: Take 5 layers of self-assembled endothelial cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0095] Fixation: Add 1 mL of 2.5% glutaraldehyde solution and fix in a 4°C refrigerator for 12 h. Rinse twice with PBS, 20 min each time. Add 200 μL of 2% osmium acid fixative and fix for 2 h. Rinse twice with PBS, 10 min each time.

[0096] Dehydration: Dehydrate with 50%, 70%, 80% and 90% acetone in a gradient manner for 10 min each time. Dehydrate with 100% acetone twice for 10 min each time. Dehydrate with anhydrous acetone treated with anhydrous calcium chloride twice for 10 min each time.

[0097] Infiltration: Anhydrous acetone-resin mixture (1:1) at 40°C for 2h;

[0098] Embedding: embed the sample in the embedding tank with pure embedding agent; bake in 40℃ oven overnight; bake in 60℃ oven for 48h;

[0099] Sectioning: Semi-thin section positioning, ultra-thin sectioning, section thickness 50-100nm; lead citrate and uranyl acetate double staining; transmission electron microscope (JEM-1400Plus) observation.

[0100] like Figure 6 As shown, transmission electron microscopy was used to observe the cell membrane surface material after endothelial cells self-assembled five times. The arrow indicates the self-assembled material on the cell membrane surface.

[0101] 5) Scanning electron microscopy observation of endothelial cell layer-by-layer self-assembly

[0102] Spread cell slides: Place a 9 mm diameter cell slide (coated with poly-lysine) in a 48-well cell culture plate, soak and wash with sterile DPBS for 10 min, and discard the DPBS;

[0103] Cell inoculation: Endothelial cells were digested into a cell suspension, counted with a cell counting plate, and the cell density was adjusted to 5 × 10 cells / mL using MEM complete medium. 4 250 μL of cell suspension was added to each well, and the cells were cultured in a cell culture incubator for 24 h after shaking evenly.

[0104] Layer-by-layer self-assembly of cells: Use D-Hank's solution to prepare 0.02% CH solution and 0.05% ALG solution for later use. Add 0.5mL D-Hank' to each well and wash the cells 3 times, 30s each time. Add 500μL CH and shake at 60rpm for 4min. Add 500μL D-Hank' and wash 3 times, 30s each time. Add 500μL ALG and shake at 60rpm for 4min. Add 500μL D-Hank's and wash 3 times, 30s each time. CH and ALG are self-assembled layer by layer alternately, and 5 layers ((CA)2C) are assembled on the cell membrane surface;

[0105] Fixation: After the cells are self-assembled, add 0.5 mL DPBS to wash three times, 1 min each time. Add 0.5 mL 2.5% glutaraldehyde solution and fix in a 4°C refrigerator for 12 h. Add 0.5 mL DPBS to wash twice, 10 min each time.

[0106] Dehydration: Dehydrate with 70%, 80%, 90% and 95% ethanol gradient once, dehydrate with anhydrous ethanol twice, each time for 5 minutes;

[0107] Replacement: Use 70%, 80%, 90%, 95% tert-butanol to replace once, and anhydrous tert-butanol to replace twice, each replacement lasts 5 minutes;

[0108] Gold spraying: stick the sample on the sample holder with conductive double-sided tape, place it under the gold spraying instrument for scanning electron microscope, and spray gold for 30 seconds;

[0109] Observation: Observation was performed using a scanning electron microscope (ZEISS Cross Beam 340).

[0110] like Figure 7 As shown, the coverage of self-assembled materials on the surface of endothelial cell membrane was observed by scanning electron microscopy.

[0111] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a tissue engineering small-caliber artificial blood vessel graft, firstly treating an ex vivo blood vessel substrate to obtain a complete small-caliber artificial blood vessel stent, It is characterized in that Also includes: First layer assembly: Nano-COF (covalent organic framework compound) was synthesized by combining TPB (1,3,5-tris(4-aminophenyl)benzene) and DVA (2,5-divinyl-1,4-benzenedicarbaldehyde) building blocks through Schiff base reaction, and LY2157299 was loaded on COF to obtain COF / LY. After modifying the surface of COF / LY with dopamine / hexamethylenediamine, surface amino-modified PDA was obtained. @ COF / LY nanoparticles, the nanoparticles are covalently bonded to the surface of the graft vascular stent through a carbodiimide crosslinker to obtain an artificial vascular graft I; The second layer assembly: using a bifunctional group cross-linking agent, the artificial vascular graft I is covalently cross-linked with negatively charged anticoagulant high molecular weight hyaluronic acid and 4-25 disaccharide unit hyaluronic acid on the artificial vascular graft I to obtain artificial vascular graft II; The bifunctional group cross-linking agent includes MAL-PEG-NHS, MAL-PEG-NH 2 , any one of MAL-PEG-CHO or MAL-PEG-COOH; One end of the bifunctional cross-linking agent contains a maleamide active group, and the other end contains an active group that reacts with an amino group or a carboxyl group; The cross-linking ratio of the negatively charged anticoagulant high molecular weight hyaluronic acid and the 4-25 disaccharide unit hyaluronic acid is 1:1-3; The molecular weight of the anticoagulant high molecular weight hyaluronic acid is 1-1.5 million; The third layer of assembly: using electrostatic adsorption to adhere positively charged endothelial cells to the surface of artificial vascular graft II, and conducting in vitro cell culture to obtain an artificial vascular graft with vascular surface cellularization.

2. The method for preparing the tissue engineering small-caliber artificial vascular graft according to claim 1, It is characterized in that The covalent cross-linking process is as follows: a bifunctional group cross-linking agent is selected and dissolved in a PBS buffer solution with a concentration of 5-10 mg / mL, placed at 2-8°C, and completely immersed in the artificial vascular graft I, and after shaking at 90-150 r / min for 1-3 hours, the artificial vascular graft I modified with the bifunctional group is obtained; Negatively charged anticoagulant high molecular weight hyaluronic acid and 4-25 disaccharide unit hyaluronic acid were selected and dissolved in a PBS solution with a concentration of 5-30 mg / mL, placed at 2-8°C, and the artificial vascular graft I modified with the bifunctional groups was completely immersed. After oscillation at 100 r / min for 1-3 hours, the artificial vascular graft II was obtained.

3. The method for preparing the tissue engineering small-caliber artificial vascular graft according to claim 1, It is characterized in that In the third layer assembly, the positively charged endothelial cells are obtained by layer-by-layer self-assembly of endothelial cells. The specific method includes: centrifuging the endothelial cell suspension and discarding the supernatant; adding 2-10 mL of 0.02-0.1% chitosan solution to resuspend and mix, shaking for 4 minutes, centrifuging and discarding the supernatant; adding D-Hank's to wash, centrifuging and discarding the supernatant; then adding 2-10 mL of 0.05% -0.1% sodium alginate solution to resuspend, centrifuging and discarding the supernatant; adding 5 mL of D-Hank's to wash, centrifuging and discarding the supernatant; repeating the above steps in sequence to self-assemble into 5 layers of cells layer by layer.

4. The method for preparing the tissue engineering small-caliber artificial vascular graft according to claim 3, It is characterized in that The five layers of cells include a chitosan layer-a sodium alginate layer-a chitosan layer-a sodium alginate layer-a chitosan layer.

5. The method for preparing the tissue engineering small-caliber artificial vascular graft according to claim 4, It is characterized in that The third-layer assembly process includes: selecting positively charged endothelial cells and adding 700-100 μL EBM-2 culture medium to resuspend them; fixing the artificial vascular graft II in a vascular culture reactor, statically culturing in a 37°C incubator for 1 hour, flipping the reactor 180°, and continuing to statically culture in a 37°C incubator for 1 hour, filling the reactor with EBM-2 culture medium, statically culturing for 72 hours, changing the culture medium once a day, and obtaining an artificial vascular graft.

6. The method for preparing the tissue engineering small-caliber artificial vascular graft according to claim 1, It is characterized in that The ex vivo vascular substrate includes an ex vivo full-term pregnancy placenta umbilical cord or a xenogeneic blood vessel or an allogeneic blood vessel.

7. A tissue engineering small-caliber artificial vascular graft, It is characterized in that The invention is obtained by the preparation method of the tissue engineering small-caliber artificial vascular graft according to any one of claims 1 to 6.

8. Use of the tissue engineering small-caliber artificial vascular graft as claimed in claim 7 in preparing a vascular substitute for clinical hemodialysis, peripheral vascular replacement, and coronary artery bypass grafting.

Images