Small diameter tissue engineered blood vessels and methods of making same

Abstract

The application discloses a small-bore tissue engineering blood vessel and a preparation method thereof. The preparation method of the small-bore tissue engineering blood vessel comprises the following steps: placing an electrostatic spinning polyethylene glycol tubular film in a columnar sandwich mold layer; mixing seed cells and a first solution to prepare a seed cell-protein mixed solution, and injecting the seed cell-protein mixed solution into the columnar sandwich mold layer containing the polyethylene glycol tubular film to form a composite gel; after the seed cell-protein-polymer composite gel is statically cultured to be stable, the seed cell-protein-polymer composite gel is continuously dynamically perfusion cultured under a simulated body arterial pulsatile flow pressure condition to obtain a tissue engineering blood vessel composed of seed cells and extracellular matrix; and the tissue engineering blood vessel is subjected to a decellularization method to remove cell components in a tube wall, so that a tissue engineering blood vessel composed of only extracellular matrix is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a small-caliber tissue-engineered blood vessel and a preparation method thereof. BACKGROUND

[0002] Cardiovascular disease (CVD) has always been a high-incidence and high-mortality disease, and is the leading cause of death worldwide. It is estimated that 4 million people die of CVD in China every year, accounting for more than 40% of the total deaths. Cardiovascular diseases such as coronary heart disease and peripheral arterial disease usually require surgical treatment, and the diseased blood vessels need to be repaired, replaced or bridged. Autologous arteries and veins are usually the first choice for blood vessel repair and replacement, but some patients cannot obtain autologous blood vessels due to various reasons.

[0003] Small-caliber (≤6mm) ePTFE and PET and other polymer material artificial blood vessels are prone to thrombosis, poor endothelialization, infection, low long-term patency rate and short service life after being implanted into the body. In order to improve the service life of small-caliber artificial blood vessels, researchers have been working to develop an artificial blood vessel with a structure and composition similar to autologous blood vessels. Tissue engineering technology provides a feasible solution for the manufacture of artificial blood vessels with properties similar to natural blood vessels. Biodegradable polymers or natural materials are used as scaffolds to provide a growth environment similar to the extracellular matrix for cells. During cell proliferation, the scaffold material degrades, and eventually an artificial blood vessel is constructed in vitro. Natural biomimetic tissue-engineered blood vessels have good biocompatibility, can quickly endothelialize after being implanted into the body, have a high long-term patency rate, and have advantages such as blood vessel regeneration and self-repair.

[0004] Natural protein hydrogels have properties similar to the extracellular matrix, good biocompatibility, and can be biodegradable in vitro, and can promote cell adhesion, migration, proliferation and differentiation. Combined with biodegradable polymer materials, the degradation rate of the gel / polymer scaffold system during in vitro culture can be more effectively regulated, the morphology of the gel components can be maintained stable, and the balance between cell proliferation / extracellular matrix formation and scaffold system degradation can be maintained. However, so far, there have been few reports on in vitro biomimetic culture of artificial blood vessels by combining natural protein hydrogels with polymers. SUMMARY

[0005] Therefore, the present application provides a small-caliber tissue-engineered blood vessel and a preparation method thereof, aiming to provide a new way of preparing a small-caliber tissue-engineered blood vessel.

[0006] In order to achieve the above-mentioned purpose, the present application also provides a preparation method of a small-caliber tissue-engineered blood vessel, which comprises the following steps:

[0007] The electrospun polyethylene glycol tubular film is placed in the middle layer of the tubular sandwich mold;

[0008] The seed cells are mixed with the first solution to form a seed cell-protein mixed solution, and are injected into the middle layer of the columnar sandwich mold containing the polyethylene glycol film to form a composite gel;

[0009] After the seed cell-protein-polymer composite gel is statically cultured to be stable, it is continuously dynamically perfusion cultured under the condition of simulating the pulsatile flow pressure of the body artery, and a tissue engineered blood vessel composed of seed cells and extracellular matrix is obtained.

[0010] The tissue engineered blood vessel is decellularized to remove the cell components in the wall, and a tissue engineered blood vessel composed only of extracellular matrix is obtained.

[0011] Preferably, in the preparation method of the small-diameter tissue engineered blood vessel, when the first solution is a mixture of fibrin, collagen, hyaluronic acid, and gelatin solution, the proportion of each component is 1%-90%.

[0012] Preferably, in the preparation method of the small-diameter tissue engineered blood vessel, the step of mixing the seed cells with the first solution to form a seed cell-protein mixed solution, and injecting it into the middle layer of the columnar sandwich mold containing the polyethylene glycol film to form a gel, comprises:

[0013] The seed cells are mixed with the first solution to form a seed cell-protein mixed solution, and are injected into the middle layer of the columnar sandwich mold containing the polyethylene glycol film to form a composite gel.

[0014] Preferably, in the preparation method of the small-diameter tissue engineered blood vessel, the columnar sandwich mold comprises a first support wall, a second support wall, and a hollow shaft structure formed by a third support wall from the outside to the inside, the hollow shaft structure is located at the center, the diameter of the hollow shaft structure is 2-6mm, the inner diameter of the middle layer formed by the second support wall is 3-10mm, the inner diameter of the outer layer formed by the first support wall is 8-30mm, the length of the mold is 10-50cm, and a plurality of through-hole structures are uniformly distributed on the second support wall and the third support wall, the pore size of the through-hole structure is 10um-100um.

[0015] Preferably, in the preparation method of the small-diameter tissue engineered blood vessel, after the seed cell-protein-polymer composite gel is statically cultured to be stable, it is continuously dynamically perfusion cultured under the condition of simulating the pulsatile flow pressure of the body artery, and a tissue engineered blood vessel composed of seed cells and extracellular matrix is obtained.

[0016] After static culture of the seed cell-protein-polymer composite gel for 1-2 weeks, the tissue engineering blood vessel composed of seed cells and extracellular matrix is obtained by dynamic perfusion culture under the simulated body arterial pulsatile flow pressure condition for 2-12 weeks.

[0017] Preferably, in the preparation method of the small-diameter tissue engineering blood vessel, the simulated body arterial pulsatile flow pressure condition comprises:

[0018] After static in vitro culture for 1-2 weeks, the blood vessel is transferred to a pulsatile flow bioreactor, and then cultured under an arterial pulsatile flow pressure of 0-90 mmHg for 1-2 weeks, after which the arterial pulsatile flow pressure is increased to 90-130 mmHg for further culture, and in the last week of the culture process, the arterial pulsatile flow pressure is increased to 130-180 mmHg for culture.

[0019] Preferably, in the preparation method of the small-diameter tissue engineering blood vessel, the step of static culture of the seed cell-protein-polymer composite gel to stability and then dynamic perfusion culture under the simulated body arterial pulsatile flow pressure condition to obtain the tissue engineering blood vessel composed of seed cells and extracellular matrix, the culture condition of static culture comprises a culture medium, a culture medium supplement, and active cytokines, and the culture medium comprises one or more of low-sugar or high-sugar DMEM, DMEM / F-12, RPMI 1640, and MEM.

[0020] The culture medium supplement comprises one or two of fetal bovine serum FBS or FCS, bovine serum BS, calf serum CS, newborn calf serum NCS, goat serum, horse serum, human serum, pig serum, chicken serum, rabbit serum, and serum substitutes, and the proportion of the culture medium supplement in the total culture medium is 5%-50%.

[0021] The active cytokines comprise one or more combinations of fibroblast growth factor, angiogenin, angiotensin, endothelin, AcSDKP, angiostatin, vasculotensin, endothelial cell growth factor, endothelioma-derived motility factor, epidermal growth factor, endothelial cell viability maintenance factor, insulin-like growth factor, heparin-binding neurotrophic factor, human uterine angiogenic factor, platelet-derived endothelial cell growth factor, platelet-derived growth factor, placental growth factor / vascular permeability factor, transforming growth factor TGF, bone morphogenetic protein BMP, interleukin, chemokine ligand CCL and CXCL, and growth hormone, and the content of the active cytokines is 1 ng-100 ug / ml.

[0022] Preferably, in the preparation method of the small-diameter tissue-engineered blood vessel, the seed cells comprise one or more of vascular fibroblasts, subcutaneous connective tissue fibroblasts, skin fibroblasts, vascular smooth muscle cells, vascular endothelial cells, mesenchymal stem cells, embryonic stem cells and induced pluripotent stem cells differentiated into fibroblasts, smooth muscle cells and endothelial cells of adult humans or mammals.

[0023] To achieve the above-mentioned purpose, the present application further provides a small-diameter tissue-engineered blood vessel prepared by the preparation method of the small-diameter tissue-engineered blood vessel.

[0024] Preferably, in the small-diameter tissue-engineered blood vessel, the extracellular matrix proteins of the small-diameter tissue-engineered blood vessel comprise collagen I and collagen III.

[0025] To achieve the above-mentioned purpose, the present application further provides an application of the small-diameter tissue-engineered blood vessel, which is applied to chronic renal failure dialysis blood vessel access, coronary artery bypass, peripheral blood vessel replacement, and can also be applied to esophagus, trachea, ureter, urethra, biliary tract, fallopian tube or vas deferens replacement;

[0026] Or, applied to the preparation of allogeneic heart valve;

[0027] Or, applied to surgical operation patch.

[0028] The present application has the following beneficial effects:

[0029] The preparation method of the small-diameter tissue-engineered blood vessel provided by the present application places the electrospun polyethylene glycol tubular film in the middle layer of the columnar sandwich mold, mixes the seed cells with the first solution to prepare a seed cell-protein mixed solution, and injects the seed cell-protein mixed solution into the middle layer of the columnar sandwich mold containing the polyethylene glycol film scaffold to form a gel, then statically cultures the seed cell-protein-polymer composite gel until stable, continues dynamic perfusion culture under the condition of simulating the pulsatile flow pressure of the body artery, and obtains a tissue-engineered blood vessel composed of seed cells and extracellular matrix, finally removes the cell components in the wall of the tissue-engineered blood vessel by decellularization method to obtain a tissue-engineered blood vessel composed of only extracellular matrix, so that the small-diameter tissue-engineered blood vessel obtained in this way has the structure and composition of human autologous blood vessels, has good biocompatibility, antithrombosis, rapid endothelialization and promotion of tissue regeneration after being implanted into the body;

[0030] Further, the small-diameter tissue-engineered blood vessel can realize rapid endothelialization and promote vascular tissue regeneration after being implanted into the human body, effectively solves the problem of thrombosis and improves the long-term patency rate. That is, the small-diameter tissue-engineered blood vessel has good regeneration performance and can realize vascular regeneration in situ.

[0031] Further, the small-diameter tissue engineering blood vessel obtained by the application has a biomimetic extracellular matrix 3D structure composed of radially shaped protein fibers, and after replacing the pathological blood vessel or bypass surgery, the small-diameter blood vessel has high biocompatibility, is beneficial to cell adhesion and growth, realizes rapid endothelialization, prevents thrombosis, and has high long-term patency rate. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the specific embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0033] Figure 1 Flow chart of an embodiment of the small-diameter tissue engineering blood vessel of the present application;

[0034] Figure 2 Schematic diagram of the tubular sandwich mold of the present application;

[0035] 101-first support wall; 102-second support wall; 103-third support wall;

[0036] Figure 3 SEM morphology of the surface of the polyethylene glycol tubular film;

[0037] Figure 4 Ultrasonic image of the proximal end of the anastomotic stoma after the artificial blood vessel is implanted into the common carotid artery of a sheep for 24 weeks;

[0038] Figure 5 Ultrasonic image of the distal end of the anastomotic stoma after the artificial blood vessel is implanted into the common carotid artery of a sheep for 24 weeks.

[0039] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0040] In the embodiments of the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B can mean that there are three cases of A alone, A and B together, and B alone. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0041] It should be noted that the terms "first", "second" and the like in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and do not necessarily have to describe a specific order or sequence.

[0042] The term "a plurality of" in the embodiments of the present application refers to two or more, and other quantifiers are similar.

[0043] To make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the embodiments of the present application will be described in detail below with reference to the drawings. However, those skilled in the art can understand that, in the embodiments of the present application, many technical details are presented in order to make the readers better understand the present application. However, the technical solutions claimed by the present application can be implemented even without these technical details and various changes and modifications based on the following embodiments. The division of the following embodiments is for the convenience of description, and should not constitute any limitation on the specific implementation modes of the present application, and the embodiments can be combined and referred to each other under the premise of no contradiction.

[0044] To achieve the above-mentioned purposes, the present application provides a preparation method of small-caliber tissue engineering blood vessels, please refer to Figure 1 The preparation method of the small-caliber tissue engineering blood vessels comprises:

[0045] Step S110, the electrospun polyethylene glycol tubular film is placed in the middle layer of the columnar sandwich mold;

[0046] It should be understood that the electrospun polyethylene glycol tubular film is placed in the middle layer of the sandwich columnar mold, wherein the middle layer is between the hollow shaft formed by the second support wall and the third support wall, and is attached to the third support wall, so that the electrospun polyethylene glycol tubular film stent is implanted in the middle layer of the columnar mold, wherein the polyethylene glycol tubular film is spun from a polyethylene glycol spinning solution with a concentration of 1-500 mg / ml, the fiber diameter is 5 nm-50 um, and the film thickness is 0.1-0.5 um.

[0047] Step S100, the seed cells are mixed with the first solution to form a seed cell-protein mixed solution, and are injected into the middle layer of the columnar sandwich mold containing the polyethylene glycol tubular film to form a composite gel;

[0048] The fibrin can also be a combination of fibrinogen and thrombin, which is not specifically limited here. The first solution can be a combination of one, two, three or four of fibrin (or a combination of fibrinogen and thrombin), collagen, hyaluronic acid and gelatin solution, and when the first solution is a mixture of multiple of them, the proportion of each component is 1%-90%. Preferably, the proportion of each component is 10%-80%, 20%-70%, 30%-60%, or 40%-50%.

[0049] In specific implementation, the seed cells are mixed with the first solution to form a seed cell-protein mixed solution, and are injected into the middle layer of the columnar sandwich mold containing the polyethylene glycol tubular film to form a composite gel.

[0050] wherein, referring to Figure 2 , the cylindrical sandwich mold comprises, from the outside to the inside, a first support wall 101 (located in the outer layer), a second support wall 102 (located in the middle layer), and a third support wall 103, the hollow shaft structure 103 formed by the third support wall has an inner diameter of 2-6 mm, the second support wall 102 forms a middle layer structure with an inner diameter of 3-10 mm, the first support wall 101 forms an outer layer structure with an inner diameter of 8-30 mm, the length of the cylindrical mold is 10-50 cm, a plurality of through-hole structures are uniformly distributed on the second support wall 102 and the third support wall 103, and the pore size of the through-hole structure is 10 um-100 um. The pore size of the through-hole structure can also be 40 um-70 um or 50 um-60 um. In other embodiments, the cylindrical sandwich mold can also have other structures, which are not specifically limited here.

[0051] The first support wall 101 and the second support wall 102 of the sandwich mold are made of non-degradable hard polymer biomaterials such as polytetrafluoroethylene (PTFE), polyethylene (PE), or polyether ether ketone (PEEK).

[0052] The third support wall 103 is made of non-degradable hard polymer biomaterials such as polytetrafluoroethylene (PTFE), polyethylene (PE), or polyether ether ketone (PEEK), or non-degradable soft polymer biomaterials such as medical silica gel and polyvinyl chloride (PVC).

[0053] In addition, the seed cells include one or more of vascular fibroblasts, subcutaneous connective tissue fibroblasts, skin fibroblasts, vascular smooth muscle cells, vascular endothelial cells, mesenchymal stem cells, embryonic stem cells, and fibroblasts, smooth muscle cells, and endothelial cells differentiated from induced pluripotent stem cells of adult humans or mammals. Preferably, the seed cells are derived from a single donor or cell bank, and are primary-15th generation cells, more preferably 3rd-10th generation cells.

[0054] Preferably, the density of the seed cells is 10 2 / ml-10 10 / ml, more preferably 10 4 / ml-10 7 / ml.

[0055] Step S200, after the seed cell-protein-polymer composite gel is statically cultured to be stable, continue dynamic perfusion culture under the condition of simulating the pulsatile flow pressure of the body artery, and obtain a tissue engineered blood vessel composed of seed cells and extracellular matrix;

[0056] In a specific implementation, the seed cell-protein-polymer composite gel is statically cultured for 1-2 weeks, and then is dynamically perfusion cultured for 2-12 weeks under simulated arterial pulsatile flow pressure conditions to obtain a tissue engineered blood vessel composed of seed cells and extracellular matrix.

[0057] In the present embodiment, the simulated arterial pulsatile flow pressure conditions include:

[0058] After static in vitro culture for 1-2 weeks, the blood vessel is transferred to a pulsatile flow bioreactor and cultured under arterial pulsatile flow at a pressure of 0-90 mmHg (in the present embodiment, the arterial pulsatile flow pressure is 10 mmHg, 20 mmHg, 30 mmHg, 40 mmHg, 50 mmHg, 60 mmHg, or 80 mmHg) for 1-2 weeks, after which the arterial pulsatile flow is increased to 90-130 mmHg (in the present embodiment, the arterial pulsatile flow is 95 mmHg, 100 mmHg, 110 mmHg, or 120 mmHg) for continued culture, and in the last week of the culture process, the arterial pulsatile flow pressure is increased to 130-180 mmHg (in the present embodiment, the arterial pulsatile flow is 140 mmHg, 150 mmHg, 160 mmHg, or 170 mmHg) for culture.

[0059] In addition, the culture conditions for static and dynamic culture include a culture medium, a culture medium supplement, and active cytokines, and the culture medium includes one or more of low-sugar or high-sugar DMEM, DMEM / F-12, RPMI 1640, and MEM.

[0060] Preferably, the culture medium supplement includes one or two of fetal bovine serum (FBS or FCS), bovine serum (BS), calf serum (CS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), human serum, pig serum, chicken serum, rabbit serum, and a serum substitute, and the culture medium supplement accounts for 5%-50% of the total culture medium; in addition, the culture medium supplement that can be added can include, but is not limited to, one or more of amino acids such as L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0061] The active cytokines include one or more combinations of fibroblast growth factor, angiogenin, angiotensin, endothelin, AcSDKP, angiostatin, vasculotensin, endothelial cell growth factor, endothelioma-derived motility factor, epidermal growth factor, endothelial cell viability maintenance factor, insulin-like growth factor, heparin-binding neurotrophic factor, human uterine angiogenic factor, platelet-derived endothelial cell growth factor, platelet-derived growth factor, placental growth factor / vascular permeability factor, transforming growth factor TGF, bone morphogenetic protein BMP, interleukin, chemokine ligand (CCL and CXCL), and growth hormone, and the content of the active cytokines is 1 ng-100 ug / ml.

[0062] In step S300, the tissue engineering blood vessel is subjected to a decellularization process to remove the cell components in the vessel wall, and a tissue engineering blood vessel composed only of extracellular matrix is obtained.

[0063] The decellularization process can be one of a chemical reagent decellularization process, a biological enzymatic decellularization process, and a physical decellularization process, or a combination of two of them, and is not specifically limited herein.

[0064] It should be noted that the decellularization technology can be combined with chemical reagents and enzymes. Preferably, sodium dodecyl sulfate (SDS), EDTA, Triton X-100, DNase, and sterile deionized water or PBS are prepared into solutions with concentrations of 0.5-10%, 1%-5%, 0.5%-5%, and 0.1-1 mg / ml, respectively.

[0065] In a specific implementation, the tissue engineering blood vessel is placed in the decellularization reagent SDS, and is treated at room temperature for 3-8 hours, then the decellularization reagent is replaced every 0.5-2 hours, and finally the enzyme treatment process is 6-24 hours, preferably 12 hours, and the decellularized tissue engineering blood vessel is obtained by washing with sterile PBS solution.

[0066] In addition, the decellularization step can also be treated with pure chemical reagents. In this embodiment, hydrochloric acid, sodium chloride, EDTA, phenylmethylsulfonyl fluoride (PMSF), butylated hydroxyanisole (BHA), and sterile deionized water or PBS are prepared into solutions with concentrations of 5-20 mM, 0.1-2 M, 1-20 mM, 1-50 μM, and 10-100 μM, respectively.

[0067] In step S300, the tissue engineering blood vessel is placed in the decellularization reagent SDS, and is treated at room temperature for 3-8 hours, then the decellularization reagent is replaced every 0.5-2 hours, and finally the enzyme treatment process is 6-24 hours, preferably 12 hours, and the decellularized tissue engineering blood vessel is obtained by washing with sterile PBS solution.

[0068] The decellularization step can also use a detergent reagent-based treatment method. Preferably, the amphoteric detergent CHAPS, sodium chloride, EDTA, SDS are prepared into 8mM, 1M, 25mM, 1.8mM solutions respectively using PBS.

[0069] Preferably, the tissue engineered blood vessel is placed into a CHAPS, EDTA, sodium chloride mixed decellularization reagent, treated at room temperature for 8-15h, preferably 12h, then treated with SDS decellularization reagent for 8-15h, preferably 12h, and washed with sterile PBS solution to obtain a decellularized tissue engineered blood vessel.

[0070] The above decellularization method can also be treated at 37 degrees Celsius, which is not specifically limited and can be limited according to the actual situation.

[0071] The preparation method of the small-diameter tissue engineered blood vessel provided by the application first mixes seed cells with a first solution to form a seed cell-protein mixed solution, and injects the seed cell-protein mixed solution into a columnar sandwich mold containing a polyethylene glycol film scaffold to form a gel in the middle layer, wherein the first solution is a solution formed by mixing one or more of fibrin, collagen, hyaluronic acid, and gelatin, and then statically cultures the seed cell-protein-polymer composite gel until it is stable, and then continues dynamic perfusion culture under the condition of simulating the pulsatile flow pressure of the body artery to obtain a tissue engineered blood vessel composed of seed cells and extracellular matrix, and finally uses a decellularization method to remove the cell components in the wall of the tissue engineered blood vessel to obtain a tissue engineered blood vessel composed only of extracellular matrix. The small-diameter tissue engineered blood vessel obtained in this way has the structure and composition of a human autologous blood vessel, and has the advantages of good biocompatibility, antithrombosis, rapid endothelialization, and promotion of tissue regeneration when implanted into the body.

[0072] To achieve the above-mentioned purpose, the application also provides a small-diameter tissue engineered blood vessel prepared by the preparation method of the small-diameter tissue engineered blood vessel described above. The embodiments of the small-diameter tissue engineered blood vessel include the embodiments of the preparation method of the small-diameter tissue engineered blood vessel described above. The advantages of the preparation method of the small-diameter tissue engineered blood vessel described above can also be applied to the small-diameter tissue engineered blood vessel.

[0073] The extracellular matrix protein of the small-diameter tissue engineered blood vessel includes collagen I and collagen III. The small-diameter tissue engineered blood vessel is composed of extracellular matrix components secreted by seed cells, and the components have in-situ recellularization effect in the body.

[0074] In this embodiment, the wall of the small-diameter tissue engineered blood vessel is composed of extracellular matrix secreted by cells; the extracellular matrix protein includes but is not limited to collagen I and collagen III.

[0075] To achieve the above object, the present application also provides a small-diameter tissue engineered blood vessel, which is applied to chronic renal failure dialysis blood vessel access, coronary bypass, peripheral blood vessel replacement, and can also be applied to esophagus, trachea, ureter, urethra, biliary tract, fallopian tube or vas deferens replacement; or, applied to preparation of allogeneic heart valve; or, applied to surgical operation patch. In other embodiments, the small-diameter tissue engineered blood vessel can also be applied to other fields, which are not listed here.

[0076] To facilitate the effect description of the small-diameter tissue engineered blood vessel prepared by the present application, the following examples are used for illustration.

[0077] Comparative Example 1

[0078] S1, preparation of seed cell-protein mixture solution: seed cells are mixed with a first solution to prepare a seed cell-protein mixture solution, wherein the first solution is a mixed solution of fibrin and collagen; wherein a fibrin solution with a concentration of 1-10 mg / ml and a collagen solution with a concentration of 1-10 mg / ml are prepared using 20 mM HEPES buffer physiological saline, and the mixing ratio of fibrin and collagen is 1:1-5:1. The density of the seed cell suspension is 10 5 / ml-10 7 / ml, and the mixing ratio of fibrin, collagen and cell suspension is 2:2:1-5:5:1.

[0079] S2, tissue engineering tubular structure forming and in-vitro static culture: the seed cell-protein mixture solution is injected into the middle layer (between 102 and 103) of a columnar sandwich mold, and a gel is formed by standing at 37°C for 1-3 hours. Appropriate culture medium is added to the outer layer of the sandwich mold and the hollow shaft, and static culture is carried out for 10 weeks.

[0080] S3, the tissue engineered blood vessel is subjected to decellularization to remove the cell components in the wall of the blood vessel to obtain a blood vessel structure.

[0081] Comparative Example 2

[0082] S1, preparation of seed cell-protein mixture solution: seed cells are mixed with a first solution to prepare a seed cell-protein mixture solution, wherein the first solution is a mixed solution of fibrin and collagen; wherein a fibrin solution with a concentration of 1-10 mg / ml and a collagen solution with a concentration of 1-10 mg / ml are prepared using 20 mM HEPES buffer physiological saline, and the mixing ratio of fibrin and collagen is 1:1-5:1. The density of the seed cell suspension is 10 5 / ml-10 7 / ml, fibrin, collagen, cell suspension mixed ratio 2:2:1-5:5:1.

[0083] S2, tissue engineering tubular structure forming, in vitro static culture, tissue engineering blood vessel in vitro culture: the seed cell-protein mixed solution is injected into the middle layer of the columnar sandwich mold (between 102 and 103), and a gel is formed at 37 degrees Celsius for 1-3 hours. The outer layer of the sandwich mold and the hollow shaft are added with appropriate culture medium (the culture medium contains high-sugar DMEM or DMEM / F12, mammalian serum or human platelet lysate (5-30%), insulin (0.1-5.0 mg / ml), growth factors bFGF and (or) EGF (1-50 ng / ml), PDGF-BB (1-50 ng / ml), angiogenin (1-100 ng / ml), vasculotensin (1-80 ng / ml), insulin-like growth factor (1-50 ng / ml), platelet-derived endothelial cell growth factor (1-50 ng / ml), endothelial cell growth factor (1-50 ng / ml), angiotensin (1-50 ng / ml), endothelial cell vitality maintenance factor (1-50 ng / ml), bone morphogenetic protein (1-50 ng / ml), penicillin (1-300 U / ml), streptomycin (1-500 mg / ml), serine (1-150 ug / ml), proline (1-150 ug / ml), alanine (1-100 ug / ml), glutamic acid (1-150 ug / ml), aspartic acid (1-150 ug / ml), asparagine (1-150 ug / ml), glycine (1-100 ug / ml), arginine (1-100 ug / ml), cysteine (1-100 ug / ml)), and static culture for 2 weeks; the blood vessels after the static culture period are transferred to a pulse flow bioreactor, and dynamic perfusion culture is continued under the condition of simulating the arterial pulsatile flow pressure of the body for 10 weeks. The seed cells grow, proliferate, and secrete extracellular matrix in the protein gel, while the gel components gradually degrade, and a tissue engineering blood vessel composed of seed cells and extracellular matrix is obtained,

[0084] The simulation of the body's arterial pulsatile flow culture conditions is as follows: under the condition of 0-90 mmHg arterial pulsatile flow, culture for 1-2 weeks, then increase the arterial pulsatile flow to 90-130 mmHg for continuous culture, and in the last week of the culture process, increase the arterial pulsatile flow pressure to 130-180 mmHg for culture.

[0085] S3, the tissue engineering blood vessel is decellularized to remove the cell components in the wall, and a tissue engineering blood vessel composed only of extracellular matrix is obtained.

[0086] Example 1

[0087] S1, preparing electrospun polyethylene glycol tubular film and seed cell-protein mixture solution: homogenized polyethylene glycol spinning solution with a concentration of 100-300 mg / ml is directly spun into a tubular film with an inner diameter consistent with the outer diameter of the hollow shaft formed by the third supporting wall 103 in the sandwich mold, the film thickness is 0.1-0.3 um, and the fiber diameter is 100 nm-500 nm, as shown in Figure 3 ; the tubular stent is placed between layers 102 and 103 in the sandwich columnar mold; a seed cell-protein mixture solution is prepared: seed cells are mixed with a first solution to make a seed cell-protein mixture solution, wherein the first solution is a mixed solution of fibrin and collagen; wherein a fibrin solution with a concentration of 1-10 mg / ml and a collagen solution with a concentration of 1-10 mg / ml are prepared using 20 mM HEPES buffer physiological saline, and the mixing ratio of fibrin and collagen is 1:1-5:1. The density of the seed cell suspension is 10 5 / ml-10 7 / ml, and the mixing ratio of fibrin, collagen, and cell suspension is 2:2:1-5:5:1.

[0088] S2, tissue engineering tubular structure forming, in vitro static culture, tissue engineering blood vessel in vitro culture: the seed cell-protein mixed solution is injected into the middle layer of sandwich mold containing polyethylene glycol film stent, and a gel is formed at 37 degrees Celsius for 1-3 hours. Appropriate culture medium (the culture medium contains high-sugar DMEM or DMEM / F12, mammalian serum or human platelet lysate (5-30%), insulin (0.1-5.0 mg / ml), growth factors bFGF and (or) EGF (1-50 ng / ml), PDGF-BB (1-50 ng / ml), angiogenin (1-100 ng / ml), vasculotensin (1-80 ng / ml), insulin-like growth factor (1-50 ng / ml), platelet-derived endothelial cell growth factor (1-50 ng / ml), endothelial cell growth factor (1-50 ng / ml), angiotensin (1-50 ng / ml), endothelial cell vitality maintenance factor (1-50 ng / ml), bone morphogenetic protein (1-50 ng / ml), penicillin (1-300 U / ml), streptomycin (1-500 mg / ml), serine (1-150 ug / ml), proline (1-150 ug / ml), alanine (1-100 ug / ml), glutamic acid (1-150 ug / ml), aspartic acid (1-150 ug / ml), asparagine (1-150 ug / ml), glycine (1-100 ug / ml), arginine (1-100 ug / ml), cysteine (1-100 ug / ml)) is added to the outer layer of the sandwich mold and the hollow shaft, and static culture is carried out for 2 weeks. The blood vessel after the static culture period is transferred to a pulse flow bioreactor, and dynamic perfusion culture is continued for 10 weeks under the condition of simulating the arterial pulsatile flow pressure of the body. The seed cells grow, proliferate and secrete extracellular matrix in the protein gel, and at the same time, the gel components are gradually degraded, and a tissue engineering blood vessel composed of seed cells and extracellular matrix is obtained.

[0089] The simulation of the body's arterial pulsatile flow culture condition is as follows: under the condition of 0-90 mmHg arterial pulsatile flow, culture for 1-2 weeks, then increase the arterial pulsatile flow to 90-130 mmHg for continuous culture, and in the last week of the culture process, increase the arterial pulsatile flow pressure to 130-180 mmHg for culture.

[0090] S3, the tissue engineering blood vessel is decellularized by a decellularization method to obtain a tissue engineering blood vessel composed of only extracellular matrix.

[0091] Example 2

[0092] S1, preparing electrospun polyethylene glycol tubular film and seed cell-protein mixture solution: homogenized polyethylene glycol spinning solution with a concentration of 100-300 mg / ml is directly spun into a tubular film with an inner diameter consistent with the outer diameter of the hollow shaft formed by the third support wall 103 of the sandwich mold, the film thickness is 0.1-0.3 um, and the fiber diameter is 100 nm-500 nm, as shown in Figure 3 ; the tubular stent is placed between the middle layers 102 and 103 of the sandwich tubular mold; a seed cell-protein mixture solution is prepared: seed cells are mixed with a first solution to make a seed cell-protein mixture solution, wherein the first solution is a stable mixed solution of fibrinogen, thrombin, and collagen; wherein a fibrin solution with a concentration of 1-10 mg / ml, a thrombin solution with a concentration of 0.1-2 U / ml, and collagen with a concentration of 1-10 mg / ml are prepared using 20 mM HEPES buffer physiological saline, and the mixing ratio of fibrinogen, thrombin, and collagen is 2:1:1-8:1:1. The density of the seed cell suspension is 10 5 / ml-10 7 / ml, and the mixing ratio of fibrinogen, thrombin, collagen, and seed cell suspension is 2:1:1:1-8:1:1:1.

[0093] S2, tissue engineering tubular structure forming, in vitro static culture, tissue engineering blood vessel in vitro culture: the seed cell-protein mixed solution is injected into the middle layer of the sandwich mold, and a gel is formed at 37 degrees Celsius for 1-3 hours. The outer layer of the sandwich mold and the hollow shaft are added with appropriate culture medium (the culture medium contains high-sugar DMEM or DMEM / F12, mammalian serum or human platelet lysate (5-30%), insulin (0.1-5.0 mg / ml), growth factors bFGF and (or) EGF (1-50 ng / ml), PDGF-BB (1-50 ng / ml), angiogenin (1-100 ng / ml), vasculotensin (1-80 ng / ml), insulin-like growth factor (1-50 ng / ml), platelet-derived endothelial cell growth factor (1-50 ng / ml), endothelial cell growth factor (1-50 ng / ml), angiotensin (1-50 ng / ml), endothelial cell vitality maintenance factor (1-50 ng / ml), bone morphogenetic protein (1-50 ng / ml), penicillin (1-300 U / ml), streptomycin (1-500 mg / ml), serine (1-150 ug / ml), proline (1-150 ug / ml), alanine (1-100 ug / ml), glutamic acid (1-150 ug / ml), aspartic acid (1-150 ug / ml), asparagine (1-150 ug / ml), glycine (1-100 ug / ml), arginine (1-100 ug / ml), cysteine (1-100 ug / ml)), and static culture for 2 weeks; the blood vessels after the static culture period are transferred to a pulse flow bioreactor, and dynamic perfusion culture is continued for 10 weeks under the condition of simulating the arterial pulsatile flow pressure of the body. The seed cells grow, proliferate, and secrete extracellular matrix in the protein gel, while the gel components are gradually degraded, and a tissue engineering blood vessel composed of seed cells and extracellular matrix is obtained,

[0094] The simulated body arterial pulsatile flow culture condition is: under the condition of 0-90 mmHg arterial pulsatile flow, culture for 1-2 weeks, then increase the arterial pulsatile flow to 90-130 mmHg for continuous culture, and increase the arterial pulsatile flow pressure to 130-180 mmHg for culture in the last week of the culture process;

[0095] S3, the tissue engineering blood vessel is decellularized by a decellularization method to obtain a tissue engineering blood vessel composed of only extracellular matrix.

[0096] According to the provisions of the national standard YYT-0500-2021, the blood vessels prepared in Example 1 and Example 2, and the comparative example are tested for circumferential tensile strength, axial tensile strength, pressure rupture strength, and suture pulling strength. The test method and results are as follows:

[0097] (1) Circumferential tensile strength

[0098] A 10 mm long tissue engineered blood vessel sample was cut and two pins or metal rods were inserted into the center of the sample along the axial direction. The two pins or metal rods were fixed to the upper and lower clamps of the tensile tester in a special way. The sample was stretched at a stable rate of 50 mm / min until the breaking point was reached. The breaking force, maximum load, and tensile rate were recorded. The maximum load of each sample was divided by its original sample length to calculate the circumferential tensile strength, as follows: -1

[0099] Circumferential tensile strength = Tmax / 2L

[0100] (2) Axial tensile strength

[0101] The two ends of the tissue engineered blood vessel sample were fixed to the clamps of the tensile tester, with a distance of 50 mm between the two clamps. The sample was stretched at a stable rate of 50 mm / min until it broke. The maximum load and tensile rate at the yield or breaking point were measured. -1

[0102] (3) Burst strength

[0103] The tissue engineered blood vessel sample was directly connected to the pressurization instrument, and liquid was supplied into the sample at a controlled flow rate of 10 kPa / s and 70 kPa / s, ensuring a stable increase in pressure. The pressure measurement device recorded the change in pressure within the sample, the rate of pressure increase, and the pressure value at the time of rupture of the sample, which was the burst strength.

[0104] (4) Suture pull-out strength

[0105] A 20 mm long tissue engineered blood vessel sample was cut along the axial direction. A 6-0 Prolene suture was inserted through one layer of the vessel wall 2 mm from the straightened end of the sample and sutured into a half-loop. The suture-free section and the suture were fixed to the upper and lower clamps of the tensile tester, respectively. The sample was stretched at a rate of 50 mm / min until the suture was pulled out of the sample, and the strength at which the suture was pulled out of the sample was recorded. -1

[0106] Table 1: Test results of the mechanical properties of the blood vessels of the examples and comparative examples

[0107]

[0108]

[0109] As can be seen from Table 1, the tissue engineered blood vessels prepared according to the present application have good tensile strength, burst strength, and high suture strength, meeting the requirements for clinical applications. ​​​

[0110] In addition, according to the provisions of the national standard YYT-0500-2021, the decellularized tissue engineered blood vessels in Examples 1 to 3 were subjected to preclinical in vivo experiments to verify the safety and reliability of the tissue engineered blood vessels in hemodialysis arteriovenous vascular access, vascular replacement and coronary artery bypass surgery. The experimental methods are as follows:

[0111] Arteriovenous vascular access: The decellularized tissue engineered blood vessels with an inner diameter of 6 mm were cut to an appropriate length, and the tissue engineered blood vessels were implanted by end-to-side anastomosis between the common carotid artery and the external jugular vein of 6 sheep weighing 70 kg on the same side through surgery. The implanted specimens were removed by surgery at 4 weeks, 12 weeks and 24 weeks after the operation.

[0112] Vascular replacement: The decellularized tissue engineered blood vessels with an inner diameter of 6 mm were cut to an appropriate length, and the tissue engineered blood vessels were implanted by end-to-end anastomosis in the common carotid artery of 6 sheep weighing 70 kg through surgery. The implanted specimens were removed by surgery at 4 weeks, 12 weeks and 24 weeks after the operation.

[0113] Coronary artery bypass surgery: The decellularized tissue engineered blood vessels with an inner diameter of 3.5 mm were cut to an appropriate length, and the tissue engineered blood vessels were implanted by end-to-side anastomosis in the ascending aorta and right coronary artery trunk of 6 sheep weighing 70 kg through surgery. The implanted specimens were removed by surgery at 4 weeks, 12 weeks and 24 weeks after the operation.

[0114] After the implantation of the tissue engineered blood vessels, the patency of the implanted tissue engineered blood vessels was observed and recorded by ultrasound and angiography. As shown in FIGS. 1 to 3, the tissue engineered blood vessels remained patent after 24 weeks of implantation in the common carotid artery. Figure 4 and Figure 5

[0115] Obviously, the above-described examples are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, other different forms of changes or modifications can be made by those skilled in the art without creative labor, and all should belong to the protection scope of the present application.​

Claims

1. A method for preparing a small diameter tissue engineered blood vessel, characterized by, The application relates to a method for preparing a tissue-engineered blood vessel. The method comprises the following steps: A static culture is carried out on the seed cell-protein-polymer composite gel until the seed cell-protein-polymer composite gel is stable, and then a dynamic perfusion culture is carried out on the seed cell-protein-polymer composite gel under a simulated arterial pulsatile flow pressure condition to obtain a tissue-engineered blood vessel composed of seed cells and extracellular matrix. The tissue-engineered blood vessel is subjected to a decellularization treatment to obtain a tissue-engineered blood vessel composed of extracellular matrix. The cylindrical sandwich mold comprises a first supporting wall, a second supporting wall and a hollow shaft structure composed of a third supporting wall from outside to inside. The first supporting wall forms an outer layer with an inner diameter of 8-30 mm, the second supporting wall forms a middle layer with an inner diameter of 3-10 mm, and the third supporting wall forms a hollow shaft structure with a diameter of 2-6 mm.

2. The method for preparing small-diameter tissue-engineered blood vessels as described in claim 1, characterized in that, The first protein solution is a mixture of fibrin, collagen, hyaluronic acid and gelatin, and the proportion of each component is 1%-90%.

3. The method for preparing small-diameter tissue-engineered blood vessels as described in claim 1, characterized in that, The simulated arterial pulsatile flow pressure condition comprises the following steps: The blood vessel is transferred to a pulse flow bioreactor after a static in-vitro culture for 1-2 weeks, and then cultured under an arterial pulsatile flow with a pressure of 0-90 mmHg for 1-2 weeks.

4. The method for preparing small-diameter tissue-engineered blood vessels as described in claim 1, characterized in that, The arterial pulsatile flow pressure is increased to 130-180 mmHg in the last week of the culture process. The culture conditions for the static culture include a culture medium, a culture medium supplement and active cytokines. The culture medium includes one or more of low-sugar or high-sugar DMEM, DMEM / F-12, RPMI 1640 and MEM. The culture medium supplement includes one or two of fetal bovine serum (FBS or FCS), bovine serum (BS), calf serum (CS), newborn calf serum (NCS), goat serum, horse serum, human serum, pig serum, chicken serum, rabbit serum and serum substitutes, and the proportion of the culture medium supplement in the total culture medium is 5%-50%. The active cytokines include one or more of fibroblast growth factor, angiogenin, angiotensin, endothelin, AcSDKP polypeptide, angiostatin, vasculotensin, endothelial cell growth factor, endothelioma-derived motility factor, epidermal growth factor, endothelial cell viability maintenance factor, insulin-like growth factor, heparin-binding neurotrophic factor, human uterine angiogenic factor, platelet-derived endothelial cell growth factor, platelet-derived growth factor, placental growth factor / vascular permeability factor, transforming growth factor TGF, bone morphogenetic protein BMP, interleukin, chemokine ligand CCL and CXCL, and growth hormone, in combination, and the content of the active cytokines is 1 ng-100 ug / ml.

5. The method for preparing small-diameter tissue-engineered blood vessels as described in claim 1, characterized in that, The seed cells include one or more of adult human or mammalian vascular fibroblasts, subcutaneous connective tissue fibroblasts, skin fibroblasts, vascular smooth muscle cells, vascular endothelial cells, mesenchymal stem cells, embryonic stem cells, and fibroblasts, smooth muscle cells, and endothelial cells differentiated from induced pluripotent stem cells.

6. A small diameter tissue engineered blood vessel, characterized in that, The small-diameter tissue engineered blood vessels are prepared by the preparation method of the small-diameter tissue engineered blood vessels according to any one of claims 1 to 5.

7. The small diameter tissue engineered blood vessel of claim 6, wherein, The extracellular matrix proteins of the small-diameter tissue engineered blood vessels include collagen I and collagen III.

8. Use of small bore tissue engineered blood vessels, characterized in that, The small-diameter tissue engineered blood vessels are prepared by the preparation method of the small-diameter tissue engineered blood vessels according to any one of claims 1 to 5, and are applied to esophageal, tracheal, ureteral, urethral, biliary, fallopian tube, or vas deferens substitutes. Alternatively, the small-diameter tissue engineered blood vessels are applied to preparation of allogeneic heart valve. Alternatively, the small-diameter tissue engineered blood vessels are applied to surgical operation patches.

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