Method for constructing small-caliber tissue-engineered blood vessels and artificial blood vessels

Abstract

The application discloses a kind of small-bore tissue engineering blood vessel construction methods and artificial blood vessel.The construction method includes: obtaining ex vivo vascular smooth muscle cells and preparing it into cell suspension;The cell suspension is inoculated on artificial blood vessel support and is carried out segmented culture, and the segmented culture includes: first-stage culture, second-stage culture, third-stage culture;The first-stage culture uses first culture medium;The second-stage culture uses second culture medium;The third-stage culture uses third culture medium;The construction method provided by the application has the advantages of simple equipment, low cost, convenient operation, can realize high-throughput parallel culture and significantly shorten the culture cycle, provides efficient, feasible new scheme for the scale preparation and clinical application of small-bore tissue engineering blood vessel.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a method for constructing small-diameter tissue-engineered blood vessels and artificial blood vessels. Background Technology

[0002] Currently, the mainstream method for constructing small-diameter tissue-engineered blood vessels is to use bioreactors for dynamic pulsatile culture. This method relies on complex equipment and long-term (usually more than 8 weeks) mechanical stimulation to induce cells to secrete matrix and obtain mechanical strength. It has significant drawbacks such as expensive equipment, complex operation, low throughput and long cycle. Summary of the Invention

[0003] In order to solve the above-mentioned technical problems, this application provides a method for constructing small-diameter tissue-engineered blood vessels and artificial blood vessels.

[0004] In a first aspect, this application provides a method for constructing small-diameter tissue-engineered blood vessels, comprising: obtaining isolated vascular smooth muscle cells and preparing them into a cell suspension; seeding the cell suspension onto an artificial blood vessel scaffold and performing segmented culture; wherein the segmented culture includes: a first culture, a second culture, and a third culture; wherein the first culture uses a first culture medium, which includes ascorbic acid, growth factors, and fetal bovine serum; the second culture uses a second culture medium, which includes the first culture medium and copper ions; the third culture uses a third culture medium, which includes the fetal bovine serum; wherein the concentration of fetal bovine serum in the third culture medium is lower than the concentration of fetal bovine serum in the first culture medium.

[0005] In some embodiments of this application, the first culture medium further includes DMEM culture medium, antibiotics, insulin, and amino acids; the amino acids include proline, glycine, and alanine.

[0006] In some embodiments of this application, the concentration of ascorbic acid in the first culture medium is 50 μg / mL, and the concentration of fetal bovine serum in the first culture medium is 20%.

[0007] In some embodiments of this application, the growth factors include TGF-β1, FGF, and EGF; The concentration of TGF-β1 in the first culture medium is 3 ng / mL; the concentration of FGF in the first culture medium is 10 ng / mL; and the concentration of EGF in the first culture medium is 0.5 ng / mL.

[0008] In some embodiments of this application, the concentration of copper ions in the second culture medium is 3 ng / mL, and / or the concentration of fetal bovine serum in the third culture medium is 10%.

[0009] In some embodiments of this application, the first culture period is 0 to 7 days, the second culture period is 7 to 21 days, and the third culture period is 21 to 42 days.

[0010] In some embodiments of this application, the artificial vascular stent is a tubular polyglycolic acid stent.

[0011] In some embodiments of this application, the method for preparing the artificial vascular stent includes: sewing a polyglycolic acid nonwoven sheet onto the surface of a mandrel to form a tubular stent; performing a hydrophilic modification treatment on the tubular stent using an alkaline solution; and cleaning and drying the modified stent.

[0012] In some embodiments of this application, the alkaline solution is a sodium hydroxide solution.

[0013] Secondly, this application provides an artificial blood vessel, which is constructed using the method for constructing small-diameter tissue-engineered blood vessels described in any of the preceding claims.

[0014] Compared to existing methods that rely on complex bioreactors for dynamic pulsatile culture, this application provides a method for constructing small-diameter tissue-engineered blood vessels. By employing a static culture strategy that adjusts the culture medium composition in stages, this method successfully constructs artificial blood vessels with sufficient mechanical strength, dense structure, and smooth intima within a shorter culture cycle, without the need for expensive and complex pulsatile stimulation equipment. This method reduces equipment dependence and preparation costs, simplifies the operation process, and enables high-throughput parallel culture, providing a simpler and more efficient solution for the large-scale preparation and clinical application of tissue-engineered blood vessels. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 Photographs of the long axis and cross-section of tissue-engineered blood vessels cultured in vitro for 6 weeks; Figure 2 H&E staining and Masson trichrome staining of tissue-engineered vessels with aortic wall smooth muscle cells cultured in vitro for 6 weeks; Figure 3 Stress-strain curves for testing the tensile strength of tissue-engineered blood vessels after 6 weeks of in vitro culture; Figure 4 This image shows the results of compressive strength testing of tissue-engineered blood vessels after 6 weeks of in vitro culture. Figure 5 Doppler ultrasound image of blood flow signal in the transplanted vessel 4 weeks post-surgery; Figure 6 Comparison of the morphology of tissue-engineered blood vessels on the day of implantation and 4 weeks post-operation; Figure 7 This is a histological staining comparison of tissue-engineered vessels 4 weeks post-operation and vessels before implantation. Figure 8 Immunohistochemical staining image of tissue-engineered blood vessels 4 weeks post-surgery.

[0017] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Currently, the mainstream technology for constructing small-diameter tissue-engineered blood vessels (typically less than 6 mm in diameter) generally relies on bioreactor-assisted pulsatile dynamic culture systems. The core of this technology involves seeding vascular smooth muscle cells onto biodegradable scaffolds such as polyglycolic acid (PEG) and placing them in a closed-loop system connected to a peristaltic pump. During a culture period of up to 8 weeks or longer, the system generates periodic radial pulsations simulating a heartbeat frequency through fluid, applying continuous mechanical stress to the vessel wall. This mechanical stimulation is considered a key necessary condition for inducing smooth muscle cells to secrete large amounts of collagen and promoting extracellular matrix cross-linking, thereby enabling the engineered blood vessel to acquire sufficient mechanical strength to resist blood pressure in vivo.

[0020] However, this dynamic culture technology has several significant drawbacks: First, it is highly dependent on equipment, requiring dedicated bioreactors, high-precision peristaltic pumps, pressure-resistant pipelines, and a matching control system, resulting in high purchase and operating costs and hindering widespread application. Second, the system is complex, involving sterile pipeline connections and long-term circulation maintenance, making operation difficult, prone to leakage or contamination, and demanding on personnel skills. Third, the culture throughput is extremely low; a large-scale device can typically only culture a single or a few blood vessels simultaneously, severely limiting the feasibility of large-scale preparation. Finally, the lengthy culture cycle further increases time and economic costs. Therefore, developing a simple, low-cost, convenient, and efficient method for constructing small-diameter tissue-engineered blood vessels has become an urgent technical problem to be solved in this field.

[0021] Therefore, in a first aspect, this application provides a method for constructing small-diameter tissue-engineered blood vessels, comprising: Step S1: Obtain isolated vascular smooth muscle cells and prepare a cell suspension. This step, by obtaining isolated vascular smooth muscle cells and preparing a high-density cell suspension, provides a source of seed cells with good activity and sufficient quantity for subsequent inoculation.

[0022] Step S2: The cell suspension is seeded onto the artificial vascular scaffold for segmented culture. This step employs a static drip seeding and segmented culture strategy, achieving efficient adhesion and uniform distribution of cells on the scaffold surface, and laying the foundation for subsequent directed differentiation induction.

[0023] The phased culture includes three stages: a first stage, a second stage, and a third stage. The first stage uses a first culture medium containing ascorbic acid, growth factors, and fetal bovine serum. This culture system, by adding ascorbic acid and growth factors under static conditions, promotes the proliferation of smooth muscle cells and the secretion of extracellular matrix, providing a material basis for the initial formation of the blood vessel wall.

[0024] In some embodiments of this application, the concentration of ascorbic acid in the first culture medium is 50 μg / mL, and the concentration of fetal bovine serum in the first culture medium is 20%. This concentration setting helps to enhance collagen synthesis efficiency while maintaining cell viability and accelerating early matrix deposition.

[0025] In some embodiments of this application, the growth factors include TGF-β1, FGF, and EGF; the concentration of TGF-β1 in the first culture medium is 3 ng / mL; the concentration of FGF in the first culture medium is 10 ng / mL; and the concentration of EGF in the first culture medium is 0.5 ng / mL. This combination of growth factors can synergistically regulate the proliferation, migration, and matrix synthesis of smooth muscle cells, thereby improving tissue construction efficiency.

[0026] In some embodiments of this application, the first culture medium further includes DMEM medium, antibiotics, insulin, and amino acids; the amino acids include proline, glycine, and alanine. This nutrient formulation provides cells with a stable living environment and synthetic substrates, which is beneficial for maintaining cell activity and substrate yield during long-term culture.

[0027] The second culture stage uses a second culture medium, which includes the first culture medium and copper ions. This stage introduces copper ions as a coenzyme for lysyl oxidase, catalyzing the cross-linking reaction between collagen and elastin, thereby enhancing the mechanical strength of the blood vessel wall.

[0028] In some embodiments of this application, the concentration of copper ions in the second culture medium is 3 ng / mL, and / or the concentration of fetal bovine serum in the third culture medium is 10%. The copper ion concentration of 3 ng / mL can reduce the risk of cytotoxicity while promoting cross-linking; the 10% fetal bovine serum concentration in the third culture medium is beneficial for inducing the transformation of cells to a shrinking phenotype.

[0029] The third culture stage uses a third culture medium containing fetal bovine serum (FBS); the FBS concentration in the third culture medium is lower than that in the first culture medium. This downregulation of serum concentration induces the transformation of smooth muscle cells from a synthetic phenotype to a contractile phenotype, promoting tissue maturation and reducing excessive proliferation.

[0030] 1) This construction method eliminates the need for complex equipment such as bioreactors and peristaltic pumps required for traditional dynamic culture. Through static culture in ordinary culture dishes and adjustment of the phased culture medium composition, small-diameter tissue-engineered blood vessels with intact tubular structures, dense tissue morphology, and good mechanical properties can be obtained in a short time. The resulting blood vessels exhibit burst pressure exceeding 500 mmHg, stress-strain behavior similar to natural blood vessels, high patency rate after in vivo transplantation, and good host integration. This helps reduce the dependence on equipment in existing technologies, simplifies the operation process, increases throughput, and shortens the culture cycle.

[0031] In some embodiments of this application, the first culture period is 0 to 7 days, the second culture period is 7 to 21 days, and the third culture period is 21 to 42 days. This cycle division allows cell proliferation, matrix cross-linking, and tissue maturation to proceed sequentially, completing angiogenesis in about 6 weeks, which is shorter than the 8 weeks or more typically required for traditional dynamic culture.

[0032] In some embodiments of this application, the artificial vascular stent is a tubular polyglycolic acid stent. This stent material possesses biocompatibility, biodegradability, and high porosity, providing a three-dimensional support structure for cell growth and matrix deposition.

[0033] In some embodiments of this application, the method for preparing an artificial vascular stent includes: sewing a polyglycolic acid nonwoven sheet onto the surface of a mandrel to form a tubular stent; performing a hydrophilic modification treatment on the tubular stent using an alkaline solution; and cleaning and drying the modified stent. This preparation process is simple to operate and has good repeatability. The hydrophilic modification treatment can improve the adhesion rate and survival rate of cells on the surface of polyglycolic acid fibers.

[0034] In some embodiments of this application, the alkaline solution is a sodium hydroxide solution. Using sodium hydroxide solution for hydrolysis is low-cost, allows for controllable reactions, and can expose the carboxyl and hydroxyl groups on the fiber surface, enhancing the hydrophilicity of the scaffold.

[0035] Secondly, this application provides an artificial blood vessel constructed using any of the aforementioned methods for constructing small-diameter tissue-engineered blood vessels. This artificial blood vessel has a dense structure, smooth inner wall, and a burst pressure exceeding 500 mmHg. It exhibits high patency after in vivo transplantation and good host integration, making it suitable for preclinical studies of small-diameter arterial replacement.

[0036] Example 1 A static construction method for small-diameter tissue-engineered blood vessels, comprising the following steps: (1) Preparation and pretreatment of polyglycolic acid tubular scaffolds; Step 1: Take a 2mm thick polyglycolic acid nonwoven fabric sheet with a porosity of 97% and cut it into a rectangle 5cm long and 2cm wide. Tightly wrap it around the surface of a 2mm outer diameter medical silicone tube (as a temporary mandrel) and suture the long side continuously with 7-0PGA absorbable sutures to make a tubular support.

[0037] Step 2: Immerse the tubular scaffold in a 1 mol / L sodium hydroxide solution for 1 minute to perform surface hydrophilic modification. The purpose of this step is to hydrolyze the ester bonds on the PGA fiber surface, expose carboxyl and hydroxyl groups, increase hydrophilicity, and significantly improve the adsorption rate for subsequent cell seeding.

[0038] Step 3: Remove the support, wash it three times with PBS buffer to remove residual alkali, and air dry it thoroughly in a fume hood or clean bench.

[0039] Step 4: Immerse the dried scaffold in 70% ethanol for 30 minutes for sterilization, wash it three times with PBS, place it in a sterile petri dish and air dry it overnight in a laminar flow hood for later use, thus preparing a polyglycolic acid tubular scaffold.

[0040] (2) Obtaining vascular smooth muscle cells and preparing high-density cell suspension; Step 5: Obtain aortic tissue, remove the adventitia and intima, and isolate primary smooth muscle cells using a median tissue block via adherent culture. Expand and passage the cells to passage 3 (P3).

[0041] Step Six: Preparation of Polyethylene Glycol (PEG) Cell Encapsulation Gel Solution: Using the Michael addition reaction, the thiol groups of the polypeptide are covalently bound to the unsaturated double bonds of tetrabranched acryloyl polyethylene glycol (4armed-PEG-DA) to form a cell encapsulation gel. Prepare a 10 mg / 45 μL solution of 4armed-PEG-DA in triethanolamine-hydrochloric acid buffer (TEA).

[0042] Based on the thiol content of matrix metalloproteinase (MMP) substrate peptides, a TEA solution of the MMP substrate peptides was prepared according to a molar ratio of thiol groups to unsaturated double bonds of 1:1.

[0043] Preparation of high-density cell suspension: When the confluence of P3 generation cells reaches 60%-70%, digest with 0.25% trypsin, centrifuge at 200g for 5 minutes and collect the cell pellet.

[0044] Step 7: Aspirate the supernatant and mix the cell pellet with 20% FBS (fetal bovine serum) medium, 10 mg / 45 μL of 4-armed-PEG-DA TEA solution, and MMP substrate peptide TEA solution in a 1:1:1 ratio to prepare a 7× concentration. 106 A homogeneous, high-density cell suspension (cell gel suspension) with cells / ml.

[0045] (3) Static inoculation and phased culture; Step 8: Slowly and evenly drop the above high-density cell suspension onto the surface of the dried PGA tubular scaffold prepared in Step 4 until the scaffold fiber network is saturated and no liquid overflows.

[0046] Step 9: Subsequently, the inoculated stent is transferred to a temperature of 37°C and a concentration of 5%. CO2 Incubate in a static temperature incubator for 30 minutes to allow the cells to initially adhere to the scaffold fibers.

[0047] Step 10, First Stage Culture (6 days, cell proliferation and matrix secretion phase): Add 50 ml of culture medium to a 100 mm culture dish, completely immersing the vascular scaffold, and perform static culture. Culture medium formula A: DMEM high-glucose medium + 20% FBS + 1% penicillin-streptomycin + growth factor combination (TGF-β1 3 ng / mL, FGF 10 ng / mL, EGF 0.5 ng / mL) + insulin 0.13 U / mL + proline 50 μg / mL + glycine 50 μg / mL + alanine 20 μg / mL + ascorbic acid 50 μg / mL. Medium change frequency: Change the culture medium every 3 days.

[0048] Step 11, Second Stage Culture (This stage lasts 15 days, matrix cross-linking enhancement period): Starting from day 7 (day 7 refers to the first day after the end of the first 6-day culture stage), add an additional 3 ng / mL of copper sulfate to the above-mentioned culture medium formulation A. Medium change frequency: Change the medium twice a week. Purpose of this step: Copper ions are a coenzyme of lysyl oxidase (LOX). Adding copper ions can catalyze the cross-linking of collagen and elastin, significantly improving the mechanical strength of blood vessels.

[0049] Step 12, Third Stage Culture (This stage lasts 3 weeks, tissue maturation period): Starting from week 3 (referring to the third week after the end of the second stage culture), reduce the FBS concentration in the culture medium to 10%. The purpose of this stage is to reduce the serum concentration, which can induce smooth muscle cells to transform from a "synthetic phenotype" to a "contractile phenotype," promote tissue maturation, and stop excessive proliferation.

[0050] Cycle: Continue static culture until the end of week 6 (a total of 42 days across three stages) to obtain mature tissue-engineered blood vessels.

[0051] (4) Evaluation of engineered blood vessels; Figure 1 These are tissue-engineered blood vessels cultured in vitro, among which Figure 1 Image A in the image shows the long axis view of a tissue-engineered blood vessel cultured in vitro for 6 weeks. Figure 1 Image B in the image is a cross-sectional view.

[0052] Figure 2 These are tissue-engineered vascular histological staining images of aortic wall smooth muscle cells cultured in vitro for 6 weeks. Figure 2 Figure A in the middle and Figure 2 Image B in the diagram shows H&E staining, observed under 4x and 20x magnification, respectively. Figure 2 Figure C in the middle and Figure 2 Image D in the diagram shows Masson's trichrome staining, observed under 4x and 20x magnification, respectively.

[0053] Figure 3 The stress-strain curves of tissue-engineered blood vessels after 6 weeks of in vitro culture show that they exhibit typical nonlinear viscoelastic characteristics, similar to the mechanical behavior of natural blood vessels of the same diameter.

[0054] Figure 4 The results show the compressive strength of the tissue-engineered blood vessels after 6 weeks of in vitro culture, with a burst pressure of 579.77 mmHg.

[0055] Figure 5 The image is a Doppler ultrasound image taken 4 weeks post-surgery, showing unobstructed blood flow, no thrombus obstruction, and good morphology of the implanted blood vessel.

[0056] Figure 6 This is a morphological comparison of tissue-engineered blood vessels before and after implantation into the rabbit carotid artery. Figure 6 Image A in the image shows the day of implantation. Figure 6 Figure B in the middle and Figure 6 Image C in the figure shows the results 4 weeks post-surgery, with no obvious morphological changes observed.

[0057] Figure 7 For the comparison of histological staining before and after tissue-engineered blood vessel implantation, among which Figure 7 Figure A in the middle and Figure 7 Figure B in the image shows H&E staining of blood vessels 4 weeks post-surgery, observed under 4x and 20x magnification. Figure 7 Figure C in the middle and Figure 7 Figures D in the image show Masson's trichrome staining of blood vessels 4 weeks post-surgery, observed under 4x and 20x magnification. Figure 7 E diagram and Figure 7 The F images in the figure show the H&E staining of the blood vessel before implantation, observed under 4x and 20x magnification. Figure 7 G-graph and Figure 7 The H images show Masson's trichrome staining of blood vessels before implantation, observed under 4x and 20x magnification.

[0058] Figure 8 These are immunohistochemical staining images of tissue-engineered blood vessels 4 weeks post-surgery. Figure 8 Figure A in the image shows HLA staining with human-specific protein antibodies. Figure 8 Figure B in the image shows staining with the smooth muscle cell-specific protein antibody MYH11, all observed under a 20x microscope.

[0059] Experimental Results: After 6 weeks of culture, the engineered blood vessels were removed, and the internal silicone tubing was taken out. Macroscopic observation revealed that the vessels were milky white, with dense walls, smooth inner walls, and a stable inner diameter of 2 mm. Their macroscopic morphology is as follows: Figure 1 Figure A (major axis plane) and Figure 1 Figure B (cross-section) is shown in the diagram.

[0060] Histological staining showed that smooth muscle cells grew uniformly within the scaffold and secreted a large amount of collagen matrix, forming a dense tubular wall with a thickness of about 300-400 μm.

[0061] H&E staining results ( Figure 2 Figures A and B show that the cells are arranged in a tightly packed, layered manner, and Masson's trichrome staining (Figure A and Figure B) shows that the cells are arranged in a tightly packed, layered manner. Figure 2 Figures C and D show abundant blue collagen fiber deposits.

[0062] Mechanical property tests show that the stress-strain curve of the blood vessel exhibits nonlinear viscoelastic characteristics similar to those of a natural blood vessel. Figure 3 ), and its burst pressure can reach 579.77 mmHg ( Figure 4 The mechanical strength meets the requirements for transplantation.

[0063] The vessel was transplanted in situ into a rabbit carotid artery model. Four weeks post-surgery, ultrasound examination showed patent vessels, good blood flow signals, and no thrombotic obstruction. Figure 5 ).

[0064] The vascular morphology remained good after transplantation, with no significant changes observed. Figure 6 (Figures B and C in the middle).

[0065] Histological analysis showed that the postoperative vascular wall structure was denser than that before implantation. Figure 7 AD comparison Figure 7 EH). Further immunohistochemical staining confirmed that the implanted human smooth muscle cells survived in vivo (HLA positive signal). Figure 8 (Figure A) and maintains the contraction phenotype (MYH11 positive expression, Figure 8 (Figure B in the middle)

[0066] In summary, the small-diameter tissue-engineered blood vessel static construction method provided in this application successfully constructs fully functional artificial blood vessels within 6 weeks without the need for a complex pulsating bioreactor by implementing a core process combining three-stage culture medium composition regulation and high-density cell encapsulation and seeding under ordinary static culture conditions. This method promotes cell proliferation and matrix secretion through the following steps: the first stage (0-7 days) uses a culture medium containing high concentrations of ascorbic acid, growth factors, and 20% fetal bovine serum; the second stage (7 days-3 weeks) adds copper ions to catalyze collagen cross-linking and strengthening; and the third stage (3-6 weeks) reduces serum concentration to induce cell maturation and transformation, thereby achieving autonomous development and strengthening of vascular tissue without mechanical stimulation.

[0067] Furthermore, this method offers significant advantages such as simple equipment, low cost, and convenient operation, completely eliminating reliance on expensive bioreactor systems. The constructed blood vessels exhibit excellent mechanical properties, with burst pressure exceeding 500 mmHg, and stress-strain behavior similar to natural blood vessels, fully meeting the requirements for transplantation. In vivo transplantation experiments further confirmed that the blood vessels maintain long-term patency in the blood flow environment, host cells integrate well, and transplanted human smooth muscle cells survive and maintain their functional phenotype.

[0068] In summary, this application not only provides a new, efficient, economical, and scalable approach to the preparation of tissue-engineered blood vessels, shortening the traditional dynamic culture cycle of more than 8 weeks to 6 weeks, but also provides an important technical reference for the static construction of other tubular tissue engineering products, and has clear prospects for clinical translation and industrial application value.

[0069] This embodiment demonstrates that by optimizing the culture medium composition and cell seeding process using a static culture method, without the need for a complex pulsatile bioreactor, small-diameter tissue-engineered blood vessels with suitable mechanical properties, good tissue structure, and in vivo transplantation function can be successfully constructed within 6 weeks.

Claims

1. A method for constructing small-diameter tissue-engineered blood vessels, characterized in that, include: Isolated vascular smooth muscle cells were obtained and prepared into a cell suspension; The cell suspension was seeded onto an artificial blood vessel stent and cultured in segments. The phased cultivation includes: first stage cultivation, second stage cultivation, and third stage cultivation; The first culture segment uses a first culture medium, which includes ascorbic acid, growth factors and fetal bovine serum. The second culture segment uses a second culture medium, which includes the first culture medium and copper ions; The third culture segment uses a third culture medium, which includes the fetal bovine serum. The concentration of fetal bovine serum in the third culture medium is lower than that in the first culture medium.

2. The method for constructing small-diameter tissue-engineered blood vessels according to claim 1, characterized in that, The first culture medium also includes DMEM medium, antibiotics, insulin, and amino acids; The amino acids include proline, glycine, and alanine.

3. The method for constructing small-diameter tissue-engineered blood vessels according to claim 1, characterized in that, The concentration of ascorbic acid in the first culture medium is 50 μg / mL, and the concentration of fetal bovine serum in the first culture medium is 20%.

4. The method for constructing small-diameter tissue-engineered blood vessels according to claim 1, characterized in that, The growth factors include TGF-β1, FGF, and EGF; The concentration of TGF-β1 in the first culture medium was 3 ng / mL; The concentration of FGF in the first culture medium is 10 ng / mL; The concentration of EGF in the first culture medium was 0.5 ng / mL.

5. The method for constructing small-diameter tissue-engineered blood vessels according to claim 1, characterized in that, The concentration of copper ions in the second culture medium is 3 ng / mL, and / or The concentration of the fetal bovine serum in the third culture medium was 10%.

6. The method for constructing small-diameter tissue-engineered blood vessels according to claim 1, characterized in that, The first stage of cultivation lasts from 0 to 7 days, the second stage lasts from 7 to 21 days, and the third stage lasts from 21 to 42 days.

7. The method for constructing small-diameter tissue-engineered blood vessels according to claim 1, characterized in that, The artificial vascular stent is a tubular polyglycolic acid stent.

8. The method for constructing small-diameter tissue-engineered blood vessels according to claim 7, characterized in that, The method for preparing the artificial vascular stent includes: Polyglycolic acid nonwoven fabric sheets are sewn onto the surface of the mandrel to form a tubular support; The tubular scaffold was hydrophilically modified using an alkaline solution. The modified scaffold is cleaned and dried.

9. The method for constructing small-diameter tissue-engineered blood vessels according to claim 8, characterized in that, The alkaline solution is a sodium hydroxide solution.

10. An artificial blood vessel, characterized in that, The artificial blood vessel is constructed using the method for constructing small-diameter tissue-engineered blood vessels according to any one of claims 1 to 9.

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