CN116271251A - Tissue engineering small-caliber artificial blood vessel graft and preparation method and application thereof - Google Patents
Tissue engineering small-caliber artificial blood vessel graft and preparation method and application thereof Download PDFInfo
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- CN116271251A CN116271251A CN202310288913.7A CN202310288913A CN116271251A CN 116271251 A CN116271251 A CN 116271251A CN 202310288913 A CN202310288913 A CN 202310288913A CN 116271251 A CN116271251 A CN 116271251A
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- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/204—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with nitrogen-containing functional groups, e.g. aminoxides, nitriles, guanidines
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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Abstract
The invention discloses a tissue engineering small-caliber artificial blood vessel graft and a preparation method and application thereof, belonging to the technical field of biomedical materials. By modifying and related treating the inner layer of the small-caliber biological artificial vascular graft, thrombosis and intimal hyperplasia are prevented, and the patency rate after vascular grafting is improved. The endothelial cells with positive charges are planted on the outer surface of the blood vessel layer by layer, through an in vivo construction strategy, the scouring of hemodynamics is overcome, the adhesion and activation of immune cells and platelets are reduced, short-term acute thrombosis is prevented, meanwhile, the mesenchymal transition of the endothelium is inhibited, the endothelialization process of the inner surface of the blood vessel is realized through in vitro cell planting, the endothelialization process of the small-caliber artificial blood vessel is accelerated, and the capillary angiogenesis is promoted, so that the effect of the vascular implant on the weight plastic is effectively exerted, the unobstructed rate is improved, and the mature blood vessel is formed. Provides a small-caliber biological artificial blood vessel which can be used for clinical hemodialysis, peripheral blood vessel substitution and coronary bypass.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a tissue engineering small-caliber artificial blood vessel graft, and a preparation method and application thereof.
Background
According to the latest statistics, the incidence and mortality of cardiovascular diseases worldwide rise year by year. So that the clinical demands of vascular grafts are increasing worldwide. Small-caliber vascular grafts are required for peripheral vascular occlusion or lesion replacement, coronary artery bypass grafting, hemodialysis arteriovenous fistulization, and the like.
Currently, vascular remodeling is performed clinically with artificial blood vessels and autologous blood vessels as substitutes. Autologous blood vessels are ideal materials, but have limited sources and are difficult to replace as various blood vessels; heterogeneous blood vessels are widely available, but have strong immunological rejection reactions, leading to failure of the grafting procedure, and clinicians have to use artificial synthetic blood vessels as substitutes. Large-caliber artificial blood vessels replace human main arteries, but artificial blood vessels with the inner diameter smaller than 6mm do not always obtain satisfactory effects for replacing human arterioles or veins, and the main reasons are that the artificial blood vessels are blocked due to thrombosis and neointimal thickening, so that the development of arterioles, arteriovenous bypass and vein substitutes is a urgent problem to be solved at present.
To make the small-caliber biological artificial vascular graft have ideal long-term patency rate, the surface of the small-caliber biological artificial vascular graft needs to be modified. At present, two approaches for improving the blood compatibility of the surface of the artificial blood vessel material are mainly adopted: 1. endothelialisation of the surface of the artificial blood vessel. 2. Changing the chemical and physical characteristics of the material surface, such as increasing chemical groups, changing the material surface charge, increasing hydrophobicity, etc., to achieve the goal of reducing fibrin and platelet adhesion.
In recent years, tissue engineering technology shows great potential after implantation in the construction of small-caliber vascular grafts, and thrombosis and stenosis are main reasons for restricting the clinical application of the small-caliber biological vascular grafts. The realization of endothelialization is a key for guaranteeing the long-term patency of the small-caliber bioartificial blood vessel: the earlier the endothelialization of the lumen, the less the risk of thrombosis and can have an inhibitory effect on intimal hyperplasia.
Disclosure of Invention
The first object of the present invention is to provide a method for preparing a tissue engineering small-caliber artificial vascular graft, which relates to a method for preparing a vascular graft by layer-by-layer assembly, inhibiting thrombosis and endothelialization, realizing endothelialization of the inner surface of a blood vessel by planting endothelial cells, maintaining long-term patency, overcoming hemodynamic scouring in vivo by an in vivo construction strategy, reducing adhesion and activation of immune cells and platelets, realizing 'immune escape', preventing short-term acute thrombosis, simultaneously inhibiting endothelialization, realizing the endothelialization of the inner surface of the blood vessel by in vitro cell planting, accelerating the endothelialization process of the small-caliber artificial blood vessel, and promoting capillary angiogenesis, thereby realizing that the vascular graft effectively exerts antithrombotic and intimal hyperplasia effects in weight molding, improving the patency rate of the implantation of the small-caliber biological artificial blood vessel, and finally forming a mature blood vessel which is completely self-replaced. Solves the great demands of small-caliber biological artificial blood vessels and the technical problems of thrombus formation and vascular stenosis after transplantation in the prior art.
The second object of the present invention is to provide a method for preparing a tissue engineering small-caliber artificial blood vessel graft, wherein the third layer assembly of the artificial blood vessel realizes 'immune escape', prevents short-term acute thrombosis, simultaneously inhibits endothelial mesenchymal transition, realizes cell formation on the inner surface of the blood vessel by in vitro cell implantation, accelerates the endothelialization process of the small-caliber artificial blood vessel, promotes capillary angiogenesis, thereby realizing that the blood vessel graft can effectively play the effects of antithrombotic formation and intimal hyperplasia in weight molding, improving the implantation patency rate of the small-caliber biological artificial blood vessel, and finally forming a mature blood vessel which is completely self-replaced.
The invention is realized by the following technical scheme:
a process for preparing the small-caliber artificial vascular graft for tissue engineering includes such steps as treating the substrate of blood vessel to obtain the complete small-caliber artificial vascular scaffold, and using carbodiimide cross-linking agent to make PDA @ Covalent bonding of COF/LY nanoparticles to the surface of a small-caliber artificial vascular stent for the first timeLayer assembly, obtaining an artificial vascular graft i, characterized in that it further comprises:
and (3) second layer assembly: adopting a bifunctional group crosslinking agent to covalently crosslink the artificial blood vessel graft I, the anticoagulation high molecular weight hyaluronic acid with negative charges and the 4-25 disaccharide unit hyaluronic acid on the artificial blood vessel graft I to obtain an artificial blood vessel graft II;
and (3) third layer assembly: and adhering the endothelial cells with positive charges on the surface of the artificial vascular graft II by using electrostatic adsorption, and carrying out in-vitro cell culture to obtain the vascular surface-cellularized artificial vascular graft.
Preferably, in the second layer assembly, the bifunctional crosslinking reagent comprises MAL-PEG-NHS, MAL-PEG-NH 2 Any one of MAL-PEG-CHO or MAL-PEG-COOH;
one end of the difunctional group cross-linking agent contains a maleamide active group, and the other end contains an active group which reacts with amino or carboxyl.
Preferably, the crosslinking ratio of the negatively charged anticoagulated high molecular weight hyaluronic acid to the 4-25 disaccharide unit hyaluronic acid is 1:1-3;
the molecular weight of the anticoagulation high molecular weight hyaluronic acid is 100-150 ten thousand.
Preferably, the covalent crosslinking process is as follows: dissolving a bifunctional group cross-linking agent in PBS buffer solution with the concentration of 5-10mg/mL, placing at 2-8 ℃ to completely soak the artificial vascular graft I, and oscillating for 1-3 hours at 90-150r/min to obtain the artificial vascular graft I modified by the bifunctional group;
and (3) dissolving the anticoagulated high molecular weight hyaluronic acid with negative charges and 4-25 disaccharide units of hyaluronic acid into PBS solution with the concentration of 5-30mg/mL, placing the solution at the temperature of 2-8 ℃, completely soaking the artificial vascular graft I modified by the bifunctional groups, and oscillating for 1-3 hours at the speed of 100r/min to obtain the artificial vascular graft II.
Preferably, in the third layer assembly, the positively charged endothelial cells are obtained by layer-by-layer self-assembly of endothelial cells,
the specific method comprises the following steps: centrifuging endothelial cell suspension, and discarding supernatant; adding 2-10mL of 0.02-0.1% chitosan solution, re-suspending, oscillating for 4min, centrifuging, and discarding supernatant; adding D-Hank's for washing, centrifuging and discarding the supernatant; adding 2-10mL of 0.05% -0.1% sodium alginate solution for resuspension, centrifuging, and discarding the supernatant; adding 5mL of D-Hank's for washing, centrifuging and discarding the supernatant; the steps are repeated sequentially, and 5 layers of cells are assembled layer by layer.
Preferably, the 5-layer cells comprise a chitosan layer-sodium alginate layer-chitosan layer.
The purpose of the above 5-layer cell preparation is to make the assembly effect better and more stable.
Preferably, the third layer assembly process includes: selecting endothelial cells with positive charges, adding 700-100 mu L of EBM-2 culture medium, and resuspending; fixing the artificial blood vessel graft II in a blood vessel culture reactor, statically culturing for 1h in a 37 ℃ incubator, turning the reactor over for 180 ℃, continuously statically culturing for 1h in the 37 ℃ incubator, filling the reactor with an EBM-2 culture medium, statically culturing for 72h, and replacing the culture medium once a day to obtain the artificial blood vessel graft.
Preferably, the isolated vascular substrate comprises an isolated term gestation placental umbilical cord or a xenogeneic or allogenic blood vessel.
A tissue engineering small-caliber artificial blood vessel graft is obtained by a preparation method of the tissue engineering small-caliber artificial blood vessel graft.
The application of the tissue engineering small-caliber artificial blood vessel graft is clinically applied as a blood vessel substitute in hemodialysis, peripheral blood vessel substitution and coronary bypass.
Compared with the prior art, the invention has at least the following technical effects:
the invention provides a preparation method of a tissue engineering small-caliber artificial blood vessel graft, which aims at the problems of great requirements and faced difficulties of a small-caliber biological artificial blood vessel, thrombus formation and vascular stenosis after transplantation, and realizes the cell formation of the inner surface of the blood vessel and promotes the transverse migration of ECs by modifying endothelial cells self-assembled layer by layer on the surface of a material. However, in the short term of vascular graft implantation, as a foreign vascular graft, platelets are activated, resulting in acute thrombosis, and in the middle-long term implantation period, in a complex pathological microenvironment, endothelial Cells (ECs) may undergo endothelial-mesenchymal transition (EndMT), thereby inducing pathological vascular remodeling, leading to technical problems of TEBV stenosis or blockage.
The technical proposal of the application is that PDA is modified in the inner layer of the small-caliber biological artificial blood vessel implant @ COF/LY utilizes COF nano-particles with high drug loading efficiency to load LY2157299, inhibits TGF-beta mediated EndMT by local slow release LY2157299, maintains endothelial integrity and ECs health, prevents thrombosis and intimal hyperplasia, and improves patency rate after vascular grafting. Meanwhile, in the middle layer of the surface of the blood vessel, the HA of a negative charge macromolecule and 4-25 disaccharide units is coupled to repel blood platelets, red blood cells and the like which are also negatively charged, thereby playing a key role in short-term acute thrombosis.
In addition, the endothelial cells with positive charges are planted on the outer surface of the blood vessel layer by layer, through an in vivo construction strategy, hemodynamic scouring is overcome in vivo, adhesion and activation of immune cells and platelets are reduced, immune escape is realized, short-term acute thrombosis is prevented, endothelial mesenchymal formation is inhibited, and in vitro cell planting is realized, the endothelialization process of small-caliber artificial blood vessels is accelerated, capillary angiogenesis is promoted, so that the vascular implant is molded in weight, the antithrombotic effect and intimal hyperplasia effect are effectively exerted, the implantation smoothness of the small-caliber biological artificial blood vessels is improved, and finally a mature blood vessel which is completely self-replaced is formed. Provides a small-caliber biological artificial blood vessel which can be used for clinical hemodialysis, peripheral blood vessel substitution and coronary bypass.
Drawings
FIG. 1 is a schematic view of a self-assembled Cheng Jiguang confocal microscope of Experimental example 1;
FIG. 2 is a graph showing changes in fluorescence intensity of FITC and Cy5 of cells during layer-by-layer self-assembly of endothelial cells detected by flow cytometry of Experimental example 2;
FIG. 3 is a graph showing the percentage of FITC+ cells after layer-by-layer self-assembly of endothelial cells of Experimental example 2;
FIG. 4 is a graph showing the percentage of Cy5+ cells after layer-by-layer self-assembly of endothelial cells of Experimental example 2;
FIG. 5 is a schematic diagram showing the change of cell membrane surface charge during the self-assembly of endothelial cells by Zeta potential detection according to Experimental example 3;
FIG. 6 is a schematic diagram of the layer-by-layer self-assembled endothelial cell transmission electron microscopy of Experimental example 4;
fig. 7 is a schematic diagram of scanning electron microscope observation of experimental example 5 after endothelial cell layer-by-layer self-assembly.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
The technical scheme of a specific implementation mode of the invention is as follows:
the preparation method of the tissue engineering small-caliber artificial blood vessel implant comprises the following steps:
1. preparation of a tissue engineering small-caliber artificial blood vessel graft stent material: the isolated fresh term gestation placenta umbilical cord or the heterogeneous blood vessel or the allogenic blood vessel is taken, and after pretreatment, decellularization and antigen removal, the non-immunogenicity vascular stent material is formed preliminarily, and the complete small-caliber artificial vascular stent material is formed through solidification, shaping, rehydration and sterilization.
1.1 the pretreatment steps comprise: 1% -5% heparin physiological saline solution is adopted, 100 mu L/mL penicillin and 0.1mg/mL streptomycin are added as transport preservation solution, the transport temperature is 2-8 ℃, the transport is carried out to a clean workshop within 12 hours, residual blood is washed, and the outer membrane is trimmed, so that the surface of the product is uniform.
1.2 decellularization: the CHAPS solution with the concentration of 4-10g/L is adopted for 8-16h, the SDS solution with the concentration of 0.4-1g/L is adopted for 8-16h, and the PBS buffer solution is used for washing 10 times.
1.3 antigen removal: the RNase and DNase are treated for 1-4h at 37 ℃ and 100r/min at a concentration of 10-30 mu L/mL.
1.4 curing and shaping: polytetrafluoroethylene or glass rod with different pore sizes is adopted to penetrate the implant, the implant is placed for 6-10h at the temperature of 30-50 ℃, and after complete solidification, the implant is rehydrated in normal saline for 5-10min and peeled.
1.5 sterilization method: sterilizing with chemical liquid, mixing 0.2-0.4% peracetic acid and 4-8% ethanol at a ratio of 1:1, soaking blood vessel at normal temperature, oscillating for 30-120 min, and washing with sterile water.
2. And (3) layer-by-layer assembly:
2.1 first layer assembly: synthesizing nano COF by combining TPB and DVA construction units through Schiff base reaction, loading LY2157299 on the COF to obtain COF/LY, and modifying dopamine/hexamethylenediamine on the surface of the COF/LY to obtain the surface aminated PDA @ COF/LY nanoparticles, the nanoparticles are covalently bound to the graft vessel stent surface by a carbodiimide crosslinking agent.
PDA @ Preparation of COF/LY nanoparticles: dissolving 0.01-0.05mM TPB and 0.01-0.06mM MDVA in ethanol by ultrasonic treatment, mixing the two solutions, and adding 100-250 μl (12M) acetic acid. Mixing with high force, and standing at room temperature for 18-24h. Centrifuging at 5000-10000rpm to collect nanoparticles, washing with ethanol, and vacuum drying. The dried nanoparticles were sonicated in DMSO and then LY2157299 was dissolved in the suspension to prepare 0.5-1mg/mL of COF/LY. The mixture was sonicated for 2min and stored under high vacuum at room temperature for 24h. And (5) centrifugally collecting the precipitate, and washing with deionized water. 1-5mg of dopamine hydrochloride and 1-5mg of hexamethylenediamine are added into 5-10mL of Tris-Buffer to obtain a mixed solution. Dispersing the newly prepared COF/LY in the above solution, stirring at room temperature in the dark for 1-2h, centrifuging to collect particles, and washing with deionized water. Drying the granules in high vacuum to obtain PDA @ After COF/LY, the mixture was stored in a refrigerator at 4℃in the absence of light.
PDA @ COF/LY nanoparticles are assembled on the graft vessel surface: the small-caliber bioartificial vessel after decellularization was placed in 1-5mg/mL EDC solution (PBS, ph=7.4) and immersed overnight at 4 ℃.
2.2 second layer assembly: the cross-linking agent of the double functional groups is adopted, the small-caliber artificial vascular stent material assembled by the first layer is covalently cross-linked with high molecular weight hyaluronic acid with anticoagulation function and hyaluronic acid with 4-25 disaccharide units for promoting angiogenesis according to the proportion of 1:1, 1:2 or 1:3, and is connected with the outer layer of the vascular graft coating, so that the small-caliber artificial vascular graft assembled and modified layer by layer is obtained.
The cross-linking agent with double functional groups comprises MAL-PEG-NHS, MAL-PEG-NH2, MAL-PEG-CHO, MAL-PEG-COOH and the like, wherein one end of the cross-linking agent contains a maleamide active group, and the other end contains an active group which reacts with amino or carboxyl.
The crosslinking process is as follows: dissolving solid in PBS buffer solution at 5-10mg/mL, placing at 2-8deg.C, completely soaking small-caliber vein of umbilical vein, and oscillating at 100r/min for 1-3 hr. And then, dissolving high molecular weight (100-150 ten thousand) hyaluronic acid with negative charges and 4-25 disaccharide units of hyaluronic acid with anti-coagulation effect and angiogenesis promoting effect in PBS solution according to the proportion of 1:1, 1:2 and 1:3, wherein the concentration is 5-30mg/mL, placing the solution at 2-8 ℃, completely soaking the small-caliber biological artificial vascular graft modified by the bifunctional group, and oscillating for 1-3h at 90-150 r/min.
2.3 third layer assembly: the endothelial cells are self-assembled layer by layer to enable the endothelial cells to have positive charges, the endothelial cells and the hyaluronic acid with negative charges of the second layer are adhered to the surface of the blood vessel through electrostatic adsorption, and the surface of the blood vessel is cellular through in vitro cell culture, so that the small-caliber artificial blood vessel graft which is assembled and modified layer by layer is obtained.
Endothelial cell layer-by-layer self-assembly: the endothelial cell suspension was centrifuged at 1000rpm for 5min and the supernatant was discarded. Adding 2-10ml CH solution, suspending, and shaking with shaking table at 100rpm for 4min. Centrifuge at 1200rpm for 5min and discard supernatant. 5-10mL of D-Hank's was added for washing, centrifuged at 1200rpm for 5min, and the supernatant was discarded. Adding 2-10mLALG solution for resuspension, and shaking with shaking table at 100rpm for 4min. Centrifuge at 1000rpm for 5min and discard supernatant. 5-10mL of D-Hank's was added for washing, centrifuged at 1000rpm for 5min, and the supernatant was discarded. CH and ALG are sequentially and repeatedly added for layer-by-layer self-assembly, wherein the self-assembly layer 1 is C, the self-assembly layer 5 is CACACAC, the CAC is ((CA) 2C), the C is chitosan, and the A is sodium alginate.
The third layer assembly concrete flow: the layer-by-layer self-assembled endothelial cells were resuspended in 700-100. Mu.L of EBM-2 medium. The blood vessel is fixed in a blood vessel culture reactor, the reactor is statically cultured for 1h in a 37 ℃ incubator, the reactor is turned over for 180 DEG, the static culture is continued in the 37 ℃ incubator for 1h, the reactor is filled with EBM-2 culture medium, the static culture is carried out for 72h, and the culture medium is replaced once a day.
Example 1:
the allogenic umbilical vein is used as a vascular stent material, 1% -5% heparin physiological saline solution is adopted, 100 mu L/mL penicillin is added, 0.1mg/mL streptomycin is used as a transportation preservation solution, the transportation temperature is 2-8 ℃, the umbilical vein is transported to a clean workshop within 12 hours, residual blood is washed, and the umbilical length is trimmed to 20cm.
Umbilical cord decellularization is carried out by adopting CHAPS solution with the concentration of 4.92g/L for 14h, SDS solution with the concentration of 0.52g/L for 14h, PBS buffer solution is washed for 10 times, 100r/min and 10 min/time.
The antigen was removed by treatment with RNase and DNase at a concentration of 10. Mu.L/mL at 37℃for 2h at 100 r/min.
The umbilical cord after cell removal and antigen removal is adopted, a special 3mm polytetrafluoroethylene rod is adopted to penetrate through umbilical veins, the umbilical cord is placed for 6 hours at the temperature of 40 ℃, after the umbilical cord is completely solidified, the umbilical cord is rehydrated in normal saline for 5 minutes, and the umbilical cord is peeled off.
The stripped umbilical cord is soaked in a mixed solution of 0.1% peracetic acid and 4% ethanol, and is oscillated for 2 hours by a shaking table at 25 ℃ for 100r/min, and is washed by sterile water until the peracetic acid test paper is unchanged.
The sterilized umbilical vein was placed in 400 μl of nanoparticle solution (1 mg/mL, PBS, ph=7.4), sonicated, and shaken at 300rpm for 2h at 4 ℃. Umbilical vein vessels were placed in 4mg/mL rat tail collagen and shaken at 300rpm for 2h at 4 ℃. The umbilical vein was again placed in the nanoparticle solution and shaken at 300rpm for 2h at 4 ℃. Umbilical vein vessels were placed in 5mg/mL EDC solution (PBS, ph=7.4) and soaked overnight at 4 ℃.
Taking out umbilical vein, dissolving bifunctional crosslinking agent MAL-PEG-NHS in PBS buffer solution at 5.5mg/mL, placing at 2-8deg.C, completely soaking umbilical vein small caliber blood vessel, and oscillating for 1 hr at 100 r/min.
Dissolving the hyaluronic acid with 150kda sulfhydryl active group and the micromolecular hyaluronic acid with 25 disaccharide units in PBS solution according to the proportion of 1:1, placing the mixture in 2-8 ℃ to completely soak the umbilical vein small caliber blood vessel modified by the bifunctional group, and oscillating for 1h at 100 r/min.
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 and resuspended, and the shaker was shaken at 100rpm for 4min. Centrifuge at 1200rpm for 5min and discard supernatant. 5mL of D-Hank's was added for washing, centrifuged at 1200rpm for 5min, and the supernatant was discarded. 2mL of 0.05% ALG solution was added and resuspended, and the shaker was shaken at 100rpm for 4min. Centrifuge at 1000rpm for 5min and discard supernatant. 5mL of D-Hank's was added for washing, centrifuged at 1000rpm for 5min, and the supernatant was discarded. CH and ALG are sequentially and repeatedly added for layer-by-layer self-assembly, wherein the self-assembly layer 1 is C, the self-assembly layer 5 is CACACAC, the CAC is ((CA) 2C), the C is chitosan, and the A is sodium alginate.
The layer-by-layer self-assembled endothelial cells were resuspended in 700-100. Mu.L of EBM-2 medium. The umbilical vein is fixed in a vascular culture reactor, the reactor is statically cultured for 1h in a 37 ℃ incubator, the reactor is turned over for 180 DEG, the static culture is continued in the 37 ℃ incubator for 1h, the reactor is filled with EBM-2 culture medium, the static culture is carried out for 72h, and the culture medium is replaced once a day. Thus obtaining the artificial vascular graft with small caliber assembled layer by layer.
The small-caliber artificial blood vessel of the same umbilical vein assembled layer by layer is obtained.
Example 2:
the allogenic blood vessel is taken as a small-caliber biological artificial blood vessel bracket, 1% -5% heparin physiological saline solution is adopted, 100 mu L/mL penicillin is added, 0.1mg/mL streptomycin is taken as a transportation preservation solution, the transportation temperature is 2-8 ℃, the blood vessel is transported to a clean workshop within 12 hours, residual blood is washed, and the adventitia of the blood vessel is trimmed to make the blood vessel uniform.
Allogeneic decellularization adopts CHAPS concentration of 4.92g/L solution to treat for 14h, PBS buffer solution washes 6 times, 100r/min,10 min/time.
The antigen was removed by treatment with RNase and DNase at a concentration of 10. Mu.L/mL at 37℃for 2h at 100r/min and washed 16 times with PBS.
Soaking the allogenic blood vessel after cell removal and antigen removal in a mixed solution of 0.1% peracetic acid and 4% ethanol, oscillating with a shaker at 25deg.C for 2h at 100r/min, and washing with sterile water until peracetic acid test paper is unchanged
Sterilized allogeneic blood vessels were placed in 400 μl of nanoparticle solution (1 mg/mL, PBS, ph=7.4), sonicated, and shaken at 300rpm for 2h at 4 ℃. The allogeneic blood vessels were placed in 4mg/mL rat tail collagen and shaken at 300rpm for 2h at 4 ℃. The vessel was again placed in the nanoparticle solution and shaken at 300rpm for 2h at 4 ℃. The vessel was placed in 5mg/mL EDC solution (PBS, ph=7.4) and soaked overnight at 4 ℃.
Taking out the blood vessel, dissolving the bifunctional crosslinking agent MAL-PEG-NHS in PBS buffer solution with the concentration of 5.5mg/mL, placing the solution at 2-8 ℃, completely soaking the blood vessel, and oscillating for 1h at 100 r/min.
Dissolving the hyaluronic acid with 150kda sulfhydryl active group and the micromolecular hyaluronic acid with 25 disaccharide units in PBS solution according to the proportion of 1:1, placing the mixture in 2-8 ℃ to completely soak the blood vessel modified by the bifunctional group, and oscillating for 1h at 100 r/min.
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 and resuspended, and the shaker was shaken at 100rpm for 4min. Centrifuge at 1200rpm for 5min and discard supernatant. 5mL of D-Hank's was added for washing, centrifuged at 1200rpm for 5min, and the supernatant was discarded. 2ml of 0.05% ALG solution was added and resuspended, and the shaker was shaken at 100rpm for 4min. Centrifuge at 1000rpm for 5min and discard supernatant. 5mL of D-Hank's was added for washing, centrifuged at 1000rpm for 5min, and the supernatant was discarded. CH and ALG are sequentially and repeatedly added for layer-by-layer self-assembly, wherein the self-assembly layer 1 is C, the self-assembly layer 5 is CACACAC, the CAC is ((CA) 2C), the C is chitosan, and the A is sodium alginate.
The layer-by-layer self-assembled endothelial cells were resuspended in 700-100uL EBM-2 medium. The blood vessel is fixed in a blood vessel culture reactor, the reactor is statically cultured for 1h in a 37 ℃ incubator, the reactor is turned over for 180 DEG, the static culture is continued in the 37 ℃ incubator for 1h, the reactor is filled with EBM-2 culture medium, the static culture is carried out for 72h, and the culture medium is replaced once a day. Thus obtaining the artificial vascular graft with small caliber assembled layer by layer.
Correlation detection experiment:
1) Self-assembly process laser confocal microscopy (CLSM) observation
The whole experimental operation is carried out under the light-proof condition. During the layer-by-layer self-assembly of endothelial cells, after each layer of material was assembled and washed, 2mL of D-Hank's resuspended cells were added. 200. Mu.L of the cell suspension was added to a fresh 15mL off-line tube, and 800. Mu.L of 4% paraformaldehyde solution (PFA) was added to fix the cells, and after 15min, 5mL of DPBS solution was added and mixed well, and centrifuged at 1000rpm for 5min. After removing the supernatant, 200. Mu.L of DAPI solution (10. Mu.g/mL) was added, and after mixing, the mixture was left to stand for 10min, and 5mL of DPBS was added, and the mixture was centrifuged at 1000rpm for 5min, and the supernatant was discarded. 50 μl DPBS was added for resuspension. 25. Mu.L of the cell suspension was dropped on a slide glass, oven-dried at 37℃for 10 minutes in a constant temperature oven, and 10. Mu.L of an anti-fluorescence quenching capper was dropped, and a cover glass was slowly covered from one side (to prevent the generation of bubbles). The cover glass was fixed with nail polish, dried in the dark and ventilated, and then observed with a laser confocal microscope (LSM 900).
FIG. 1 is a schematic diagram showing the change of cell membrane surface materials in the process of observing cell layer-by-layer self-assembly by a laser confocal microscope. Wherein: red: sodium alginate; green: a chitosan; blue: and (3) cell nucleus. C represents chitosan, and A represents sodium alginate.
The self-assembled layer 1 is C, the self-assembled layer 5 is CACACAC, denoted (CA) 2C.
2) Flow cytometry detection for fluorescence intensity change after cell layer-by-layer self-assembly
The whole experimental operation is carried out under the light-proof condition. During the layer-by-layer self-assembly of endothelial cells, after each layer of material was assembled and washed, 2mL of D-Hank's resuspended cells were added. 200. Mu.L of the cell suspension was taken and filtered into a single cell suspension using a cell strainer. Cell surface FITC and Cy5 fluorescence intensity changes were detected using a flow cytometer (Accuri C6 Plus).
As shown in FIG. 2, a flow cytometer is shown to detect changes in fluorescence intensity of FITC and Cy5 of cells during layer-by-layer self-assembly of endothelial cells.
As shown in fig. 3, a schematic representation of fitc+ cell percentages after endothelial cell layer-by-layer self-assembly is shown.
As shown in fig. 4, a graph showing the percentage of Cy5+ cells after endothelial cell layer-by-layer self-assembly is shown.
3) Cell membrane Zeta potential change detection in self-assembly process
Self-assembling solutions were formulated using D-Hank's solution: 0.02% CH and 0.05% ALG were reserved. One flask (T25 flask) was taken to grow about 80% of endothelial cells, the medium was aspirated off, and 2mL of DPBS was added to wash 3 times for 30s each. 1.5mL of 0.25% pancreatin digest was added, placed in culture for digestion for 2min, and 4.5mL of complete medium was added to terminate digestion. The cell suspension was added to a 15mL centrifuge tube, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Adding 1mL of D-Hank's for resuspension, adding 100 mu L of cell suspension into 1.4mL of D-Hank's, uniformly mixing, and then carrying out Zeta potential detection; the remaining cells were centrifuged at 1000rpm for 5min, and the supernatant was discarded. 2mL of CH solution was added to the mixture and the mixture was resuspended and shaken for 4min at 100rpm on a shaker. Centrifuge at 1000rpm for 5min and discard supernatant. 5mL of the Hank's was added and mixed well, centrifuged at 1200rpm for 5min, and the supernatant was discarded. 1mL of the D-Hank's was added to the suspension, 100. Mu.L of the cell suspension was added to 1.4mL of the D-Hank's, and the mixture was subjected to Zeta potential detection. After each self-assembly and washing, 1mL of D-Hank's are added for re-suspension, 100 mu L of cell suspension is added into 1.4mL of D-Hank's, and Zeta potential detection is carried out after uniform mixing, and five layers are self-assembled in total.
As shown in FIG. 5, the Zeta potential is used to detect changes in cell membrane surface charge during endothelial cell self-assembly. The results show that: zeta potential is the average of 20 repeated measurements of the instrument.
The data shown in FIGS. 2-5 above were all obtained from 3 independent replicates and are expressed as mean.+ -. Variance.
* : p <0.05,: p <0.01,: p <0.001,: p <0.0001, indicating significant statistical differences.
4) Layer-by-layer self-assembled endothelial cell transmission electron microscope observation
Drawing materials: taking 5 layers of self-assembled endothelial cells, centrifuging at 1000rpm for 5min, and discarding the supernatant;
fixing: 1mL of 2.5% glutaraldehyde solution was added and the mixture was fixed at 4℃for 12 hours in a refrigerator. The PBS was rinsed twice for 20min each. 200. Mu.L of 2% osmium acid fixative was added to fix for 2h. Rinsing with PBS twice for 10min each;
dehydrating: dehydration was performed using a gradient of 50%, 70%, 80% and 90% acetone for 10min each. Dehydration was performed 2 times with 100% acetone for 10min each. Dehydrating with anhydrous acetone treated with anhydrous calcium chloride for 2 times each for 10min;
penetration: treating the anhydrous acetone-resin mixed solution (1:1) at 40 ℃ for 2 hours;
embedding: embedding the sample in an embedding groove by using a pure embedding agent; oven at 40 ℃ overnight; oven at 60 ℃ for 48 hours;
slicing: positioning a semi-thin slice, slicing by an ultrathin slicing machine, wherein the slice thickness is 50-100nm; double-dyeing of lead citrate and uranium acetate; transmission electron microscopy (JEM-1400 Plus) was used.
As shown in FIG. 6, the cell membrane surface material was observed five times after endothelial cell self-assembly by transmission electron microscopy. Arrows indicate self-assembled material of cell membrane surface.
5) Scanning electron microscope observation after endothelial cell layer-by-layer self-assembly
Spreading a cell climbing sheet: placing a cell climbing sheet (polylysine coating) with the diameter of 9mm in a 48-hole cell culture plate, soaking and cleaning the cell climbing sheet in sterile DPBS for 10min, and sucking and discarding the DPBS;
inoculating cells: endothelial cells were lysed into a cell suspension, counted in a cell counting plate and the cell density was adjusted to 5X 10 using MEM complete medium 4 Adding 250 mu L of cell suspension into each hole, shaking uniformly, and then standing in a cell culture box for culturing for 24 hours;
cell layer-by-layer self-assembly: a solution of 0.02% CH and 0.05% ALG was prepared using D-Hank's solution for use. Cells were washed 3 times with 0.5mL of D-Hank' per well, 30s each. 500. Mu.LCH was added and the mixture was shaken at 60rpm for 4min. Add 500. Mu. L D-Hank' to wash 3 times for 30s each. 500. Mu.LALG was added and shaken at 60rpm for 4min. Mu. L D-Hank's were added and washed 3 times for 30s each. CH and ALG are alternately self-assembled layer by layer, and 5 layers ((CA) 2C) are assembled on the surface of a cell membrane;
fixing: after the cell layer self-assembly was completed, 0.5mL of DPBS was added to wash three times for 1min each time, 0.5mL of 2.5% glutaraldehyde solution was added and the mixture was fixed at 4℃for 12h in a refrigerator. Adding 0.5mL of DPBS to wash twice for 10min each time;
dehydrating: gradient dehydration is carried out by using 70 percent ethanol, 80 percent ethanol, 90 percent ethanol and 95 percent ethanol for one time, and dehydration is carried out by using absolute ethanol for two times, and each time is carried out for 5 minutes;
replacement: respectively replacing with 70%, 80%, 90% and 95% tertiary butanol once, and replacing with anhydrous tertiary butanol twice for 5min each time;
and (5) metal spraying: adhering a sample to a sample holder by using a conductive double-sided adhesive tape, and placing under a metal spraying instrument special for a scanning electron microscope to spray metal for 30s;
and (3) observation: the observation was performed using a scanning electron microscope (ZEISS Cross Beam 340).
As shown in FIG. 7, the coverage of the self-assembled material on the surface of endothelial cell membrane was observed by scanning electron microscopy.
Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A process for preparing the small-caliber artificial vascular graft for tissue engineering includes such steps as treating the substrate of blood vessel to obtain the complete small-caliber artificial vascular scaffold, and using carbodiimide cross-linking agent to make PDA @ The COF/LY nano-particles are covalently bonded to the surface of the small-caliber artificial blood vessel stent for first layer assembly to obtain the artificial blood vessel graft I, which is characterized by further comprising:
and (3) second layer assembly: adopting a bifunctional group crosslinking agent to covalently crosslink the artificial blood vessel graft I, the anticoagulation high molecular weight hyaluronic acid with negative charges and the 4-25 disaccharide unit hyaluronic acid on the artificial blood vessel graft I to obtain an artificial blood vessel graft II;
and (3) third layer assembly: and adhering the endothelial cells with positive charges on the surface of the artificial vascular graft II by using electrostatic adsorption, and carrying out in-vitro cell culture to obtain the vascular surface-cellularized artificial vascular graft.
2. According to claimThe method for preparing tissue engineering small caliber artificial blood vessel graft as described in 1, wherein in the second layer assembly, the bifunctional group crosslinking agent comprises MAL-PEG-NHS, MAL-PEG-NH 2 Any one of MAL-PEG-CHO or MAL-PEG-COOH;
one end of the difunctional group cross-linking agent contains a maleamide active group, and the other end contains an active group which reacts with amino or carboxyl.
3. The method for preparing a tissue engineering small caliber artificial blood vessel graft according to claim 2, wherein the crosslinking ratio of the negatively charged anticoagulated high molecular weight hyaluronic acid and the 4-25 disaccharide unit hyaluronic acid is 1:1-3;
the molecular weight of the anticoagulation high molecular weight hyaluronic acid is 100-150 ten thousand.
4. The method for preparing a tissue engineering small caliber artificial blood vessel graft according to claim 2, wherein the covalent crosslinking process is as follows: dissolving a bifunctional group crosslinking agent in PBS buffer solution with the concentration of 5-10mg/mL, placing the solution at the temperature of 2-8 ℃ to completely soak the artificial vascular graft I, and oscillating for 1-3 hours at the speed of 90-150r/min to obtain the artificial vascular graft I modified by the bifunctional group;
and (3) dissolving the anticoagulated high molecular weight hyaluronic acid with negative charges and 4-25 disaccharide units of hyaluronic acid into PBS solution with the concentration of 5-30mg/mL, placing the solution at the temperature of 2-8 ℃, completely soaking the artificial vascular graft I modified by the bifunctional groups, and oscillating for 1-3 hours at the speed of 100r/min to obtain the artificial vascular graft II.
5. The method for preparing a tissue engineering small caliber artificial blood vessel graft according to claim 1, wherein in the third layer assembly, the positively charged endothelial cells are obtained by adopting endothelial cell layer-by-layer self-assembly,
the specific method comprises the following steps: centrifuging endothelial cell suspension, and discarding supernatant; adding 2-10mL of 0.02-0.1% chitosan solution, re-suspending, oscillating for 4min, centrifuging, and discarding supernatant; adding D-Hank's for washing, centrifuging and discarding the supernatant; adding 2-10mL of 0.05% -0.1% sodium alginate solution for resuspension, centrifuging, and discarding the supernatant; adding 5mL of D-Hank's for washing, centrifuging and discarding the supernatant; the steps are repeated sequentially, and 5 layers of cells are assembled layer by layer.
6. The method for preparing a tissue engineering small caliber artificial blood vessel graft according to claim 5, wherein the 5 layers of cells comprise a chitosan layer-sodium alginate layer-chitosan layer.
7. The method of claim 5, wherein the third layer assembly process comprises: selecting endothelial cells with positive charges, adding 700-100 mu L of EBM-2 culture medium, and resuspending; fixing the artificial blood vessel graft II in a blood vessel culture reactor, statically culturing for 1h in a 37 ℃ incubator, turning the reactor over for 180 ℃, continuously statically culturing for 1h in the 37 ℃ incubator, filling the reactor with an EBM-2 culture medium, statically culturing for 72h, and replacing the culture medium once a day to obtain the artificial blood vessel graft.
8. The method for preparing a tissue engineering small caliber artificial blood vessel graft according to claim 1, wherein the isolated blood vessel base material comprises isolated term gestation placental umbilical cord or xenogeneic blood vessel or allogenic blood vessel.
9. A tissue engineering small-caliber artificial blood vessel graft, characterized in that the tissue engineering small-caliber artificial blood vessel graft is obtained by the preparation method of the tissue engineering small-caliber artificial blood vessel graft as set forth in any one of claims 1 to 7.
10. Use of the tissue engineering small caliber artificial blood vessel graft according to claim 8, wherein the artificial blood vessel graft is clinically used as a blood vessel substitute in hemodialysis, peripheral blood vessel substitution and coronary bypass.
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