Method for preparing artificial blood vessel and artificial blood vessel

Abstract

The invention discloses a method for preparing an artificial blood vessel and the artificial blood vessel. The method comprises the following steps: providing a mixed solution containing polycaprolactone and a human decellularized amnion; mixing and depositing the polycaprolactone and the human decellularized amniotic membrane in the mixed solution on the surface of the core mold to prepare a sediment; and drying the sediment and removing the core mold to obtain the artificial blood vessel. According to the artificial blood vessel prepared through the method, the biocompatibility and mechanical performance of the artificial blood vessel can be improved, organization and functionalization of the artificial blood vessel are promoted, and thrombus can be avoided.

Description

Technical Field

[0001] This application belongs to the technical field of tissue engineering blood vessels, and particularly relates to a method for preparing artificial blood vessels and artificial blood vessels. Background Art

[0002] Cardiovascular diseases refer to a group of diseases that affect the heart and blood vessels, such as coronary heart disease, hypertension, myocardial infarction, heart failure, etc. These diseases usually involve the cardiovascular system, that is, the heart and blood vessels, and cause varying degrees of impact on the body's blood supply and oxygen supply capabilities. When severe vascular lesions occur, vascular transplantation is an effective treatment method.

[0003] Small-caliber artificial blood vessels (d < 6 mm) have been the focus and difficulty of research for many years. However, due to slow local blood flow velocity, small blood vessel diameter, complex blood flow environment, and low blood pressure in the lumen, small-caliber artificial blood vessels are prone to thrombosis. The long-term patency rate of small-caliber artificial blood vessels is poor. Most of the designs and preparations of artificial blood vessels adopt the idea of in-situ tissue-induced regeneration, providing a three-dimensional scaffold platform for vascular regeneration and cell infiltration by implanting artificial blood vessels into the body. By optimizing the pore structure of the three-dimensional scaffold and loading surface active substances, host stem cells, endothelial cells, etc. are induced to infiltrate into the interior of the scaffold, proliferate, differentiate, and migrate within the three-dimensional scaffold to form vascular smooth muscle cells and vascular endothelial cells.

[0004] At present, there is no mature preparation method for small-caliber artificial blood vessels that can achieve long-term use of blood vessels, so improvement is urgently needed. Summary of the Invention

[0005] Embodiments of this application provide a method for preparing artificial blood vessels and artificial blood vessels. The artificial blood vessels prepared by the method of the embodiments of this application can improve the biocompatibility and mechanical properties of artificial blood vessels, promote the tissueization and functionalization of artificial blood vessels, and can also avoid thrombosis.

[0006] In a first aspect, embodiments of this application provide a method for preparing artificial blood vessels, including:

[0007] Providing a mixed solution containing polycaprolactone and human acellular amnion;

[0008] Mixing and depositing the polycaprolactone and the human acellular amnion in the mixed solution on the surface of a mandrel to obtain a deposit;

[0009] Drying the deposit and removing the mandrel to obtain the artificial blood vessel.

[0010] According to an embodiment of one aspect of this application, the mass concentration of polycaprolactone in the mixed solution is 4.5% - 5.5%.

[0011] According to an embodiment of one aspect of the present application, the mass concentration of human acellular amnion in the mixed solution is 4.5%-5.5%.

[0012] According to an embodiment of one aspect of the present application, the diameter of the core mold is 3.5-4.5 mm.

[0013] According to an embodiment of one aspect of the present application, the thickness of the sediment is 0.45-0.55 cm.

[0014] According to an embodiment of one aspect of the present application, the deposition of the polycaprolactone and the human acellular amnion in the mixed solution on the surface of the core mold includes:

[0015] Rotating the core mold at a speed of 150-550 r / min, and applying a voltage of 15-18 kV to the core mold;

[0016] Extruding the mixed solution at a flow rate of 2.5-3.2 ml / h, and the distance between the extrusion port and the surface of the core mold is 13-18 cm, so that the polycaprolactone and the human acellular amnion in the mixed solution are mixed and deposited on the surface of the core mold.

[0017] According to an embodiment of one aspect of the present application, the drying temperature is 50-60 °C, and the drying time is 3 to 5 days.

[0018] In a second aspect, an embodiment of the present application provides an artificial blood vessel prepared by the method of the first aspect.

[0019] According to an embodiment of one aspect of the present application, the mass ratio of polycaprolactone to human acellular amnion is 1:(0.8-1.3).

[0020] According to an embodiment of one aspect of the present application, the wall thickness of the artificial blood vessel is 0.30-0.45 cm; the inner diameter of the artificial blood vessel is 3.5-4.5 mm.

[0021] The embodiments of the present application have at least the following beneficial effects:

[0022] The method provided by the embodiments of the present application uses human acellular amnion and polycaprolactone to prepare an artificial blood vessel. The prepared artificial blood vessel has good biocompatibility, which is beneficial to cell adhesion and proliferation. During subsequent use, it also avoids the blockage of the artificial blood vessel and is conducive to the long-term and stable use of the artificial blood vessel. At the same time, the prepared artificial blood vessel has good mechanical properties and can be used to prepare small-diameter blood vessels, solving the technical problem of easy blockage of small-diameter blood vessels. Brief Description of the Drawings

[0023] To more clearly illustrate the technical solutions of the embodiments of the present application, the accompanying drawings required for use in the embodiments of the present application will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0024] Figure 1 Shows the preparation schematic diagram of the artificial blood vessels in Example 1 and Comparative Example 1, the micrograph of the artificial blood vessels, the mechanical properties, and the OD values of the co-cultured cells therein.

[0025] Figure 2 Shows the effects of the artificial blood vessels in Example 1 and Comparative Example 1 on the behavior of human umbilical vein endothelial cells.

[0026] Figure 3 Shows the effects of the artificial blood vessels in Example 1 and Comparative Example 1 on the migration ability of human umbilical vein endothelial cells.

[0027] Figure 4 Shows the physical effects of the artificial blood vessels in Example 1 and Comparative Example 1 in the experimental model of rat abdominal aortic vascular transplantation.

[0028] Figure 5 Shows the staining conditions of the artificial blood vessels in Example 1 and Comparative Example 1 after transplantation into rats using hematoxylin and eosin staining.

[0029] Figure 6 Shows the endothelial cell generation on the inner lumen surface of the artificial blood vessels in Example 1 and Comparative Example 1.

[0030] Figure 7 Shows the immunofluorescence staining conditions of the artificial blood vessels in Example 1 and Comparative Example 1 using CD31 staining and α-SMA antibody. Detailed implementation manners

[0031] In order to make the invention purpose, technical solutions and beneficial technical effects of the present application clearer, the present application will be described in detail below in conjunction with specific embodiments. It should be understood that the embodiments described in this specification are only for explaining the present application and not for limiting the present application.

[0032] For simplicity, only some numerical ranges are explicitly disclosed in this text. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with other lower limits to form a range not explicitly recited, and similarly any upper limit can be combined with any other upper limit to form a range not explicitly recited. In addition, although not explicitly recited, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be used as its own lower or upper limit and combined with any other point or single value or with other lower or upper limits to form a range not explicitly recited.

[0033] In the description of this text, it should be noted that unless otherwise specified, "above" and "below" include this number, and in "one or several", the meaning of "several" is two or more.

[0034] The above invention content of this application does not intend to describe every disclosed embodiment or every implementation manner in this application. The following description more specifically illustrates exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments, which can be used in various combinations. In each instance, the enumeration is only a representative group and should not be construed as exhaustive.

[0035] In a first aspect, an embodiment of this application provides a method for preparing an artificial blood vessel, including:

[0036] providing a mixed solution containing polycaprolactone and human acellular amnion;

[0037] mixing and depositing the polycaprolactone and the human acellular amnion in the mixed solution on the surface of a mandrel to obtain a deposit;

[0038] drying the deposit and removing the mandrel to obtain the artificial blood vessel.

[0039] Polycaprolactone (Poly(ε-caprolactone), PCL) is a synthetic polymer with good biocompatibility, capable of interacting with surrounding tissues in vivo without causing obvious immune reactions, having suitable mechanical properties, and can provide sufficient strength and flexibility, enabling the artificial blood vessel to withstand the pressure of blood flow in vivo without being easily broken or deformed. However, when using PCL alone, its hydrophilicity and cell adhesion are poor, which limits its application in artificial blood vessels.

[0040] Human amniotic membrane (HAM) is the innermost layer of the fetal membrane. It is a semi-transparent membrane with a thickness of 0.02 to 0.5 mm, including a basement membrane and avascular stroma. Human acellular amniotic membrane contains various bioactive substances, such as cytokines, growth factors, and anti-inflammatory proteins, etc. The artificial blood vessel in the embodiment of the present application contains human acellular amniotic membrane, which can regulate the inflammatory response, reduce tissue inflammation, and contribute to promoting the regeneration and healing of damaged tissues. It can activate the proliferation and migration of surrounding cells, promote the formation of new blood vessels in the artificial blood vessel, and the attachment of respective cells, and contribute to reconstructing the structure and function of damaged vascular tissues. In addition, human acellular amniotic membrane reduces immunogenicity, reduces the rejection reaction of allogeneic transplantation, and improves the biocompatibility of biomaterials.

[0041] The method provided in the embodiment of the present application uses an artificial blood vessel prepared from human acellular amniotic membrane (HAAM) and polycaprolactone. The prepared artificial blood vessel has good biocompatibility, which is beneficial to the adhesion and proliferation of cells. During subsequent use, it also avoids the blockage of the artificial blood vessel, which is beneficial to the long-term and stable use of the artificial blood vessel. At the same time, the prepared artificial blood vessel has good mechanical properties, promotes the organization and functionalization of the artificial blood vessel, and can also avoid thrombosis.

[0042] In some embodiments, providing a mixed solution containing polycaprolactone and human acellular amniotic membrane includes: providing a solution containing human acellular amniotic membrane.

[0043] In some embodiments, the solution containing human acellular amniotic membrane contains PBS buffer. Human acellular amniotic membrane can be obtained by entrusting a relevant company to purchase it, or it can be prepared.

[0044] In some embodiments, the preparation method of the solution containing human acellular amniotic membrane includes: placing human acellular amniotic membrane in a ball mill and pulverizing it to obtain human acellular amniotic membrane particles (the diameter can be about 40um);

[0045] Under room temperature conditions, treating the human acellular amniotic membrane particles with pepsin under acidic conditions, then neutralizing, and adding a buffer solution to achieve the preparation of the solution containing human acellular amniotic membrane.

[0046] In some embodiments, at room temperature, human acellular amniotic membrane particles are treated with pepsin under acidic conditions and then neutralized. The buffer solution added includes: in a 0.01 M HCl solution containing 1 mg / ml pepsin for 24 h, with continuous stirring by a magnetic stirrer. The concentration is 10 mg / ml, that is, 1 g of human acellular amniotic membrane is dissolved in 100 ml of HCl solution containing 100 mg of pepsin. After complete dissolution, the human acellular amniotic membrane solution is placed on an ice plate. Take 1 / 10 volume of 0.1 M NaOH solution and 1 / 9 volume of 10×PBS solution of the volume of the human acellular amniotic membrane solution, and then add 1×PBS solution, mix well, and adjust the pH value to 7.4.

[0047] In some embodiments, the preparation method of human acellular amniotic membrane includes: obtaining fresh amniotic membrane within 6 hours after cesarean section of a healthy parturient, and performing the next acellular treatment under sterile conditions. The amniotic membrane is repeatedly rinsed with sterile PBS to wash away the attached blood and mucus on the amniotic membrane, and the surface impurities and sponge layer of the amniotic membrane are further removed with sterile gauze, and then placed in 0.25% trypsin-EDTA and shaken at 37°C for 6 hours, and repeatedly rinsed with sterile PBS. After that, a cell scraper is used to scrape off the surface cells, and shaken with 0.5% SDS for 4 hours, and then repeatedly rinsed with sterile PBS until the odor disappears. The completely acellular amniotic membrane is cut into 3.0×3.0 cm with an ophthalmic scissors 2 , sterilized by ethylene oxide for subsequent experiments.

[0048] In some alternative embodiments, the mass ratio of polycaprolactone to human acellular amniotic membrane is 1:(0.8 - 1.3). Optionally, the mass ratio of polycaprolactone to human acellular amniotic membrane is any value among 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3 or the range composed of them.

[0049] According to the embodiments of the present application, by controlling the mass ratio of polycaprolactone to human acellular amniotic membrane within the above range, it is beneficial to control the components in the artificial blood vessel, make it have good biocompatibility, promote the attachment of endothelial cells and other cells inside and outside the artificial blood vessel, and make its physiological function closer to that of a normal blood vessel.

[0050] In some alternative embodiments, the mass concentration of polycaprolactone in the mixed solution is 4.5% - 5.5%. The mass concentration of polycaprolactone in the mixed solution can be any value among 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5% or the range composed of them.

[0051] According to the embodiments of the present application, by controlling the mass concentration of polycaprolactone in the mixed solution, on the basis of ensuring the uniform dispersion of polycaprolactone in the mixed solution, it is beneficial to control the thickness of the deposit, so as to control the wall thickness of the prepared artificial blood vessel, and it is beneficial to control the components in the artificial blood vessel, making it have good biocompatibility, promoting the attachment of cells such as endothelial cells inside and outside the artificial blood vessel, and making its physiological function closer to that of a normal blood vessel.

[0052] In some alternative embodiments, the mass concentration of human acellular amnion in the mixed solution is 4.5% - 5.5%. Optionally, the mass concentration of human acellular amnion in the mixed solution can be any value among 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5% or the range composed of them.

[0053] According to the embodiments of the present application, by controlling the mass concentration of human acellular amnion in the mixed solution, on the basis of ensuring the uniform dispersion of human acellular amnion in the mixed solution, it is beneficial to control the thickness of the deposit, so as to control the wall thickness of the prepared artificial blood vessel, and it is beneficial to control the components in the artificial blood vessel, making it have good biocompatibility, promoting the attachment of cells such as endothelial cells inside and outside the artificial blood vessel, and making its physiological function closer to that of a normal blood vessel.

[0054] In some alternative embodiments, the diameter of the core mold is 3.5 - 4.5 mm.

[0055] Optionally, the diameter of the core mold can be any value among 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm or the range composed of them.

[0056] Controlling the diameter of the core mold within the above range can thus control the inner diameter of the artificial blood vessel, which is beneficial for preparing an artificial blood vessel with a smaller pore size.

[0057] In some alternative embodiments, the thickness of the deposit is 0.45 - 0.55 cm.

[0058] Controlling the thickness of the deposit within the above range can thus control the wall thickness of the artificial blood vessel, which is beneficial for the artificial blood vessel to have certain mechanical properties and can also meet the wall thickness requirements of an artificial blood vessel with a smaller pore size.

[0059] In some alternative embodiments, the deposition of the polycaprolactone and the human acellular amnion in the mixed solution on the surface of the core mold includes:

[0060] Rotate the core mold at a speed of 150 - 550 r / min, and apply a voltage of 15 - 18 kV to the core mold;

[0061] Extrude the mixed solution at a flow rate of 2.5 - 3.2 ml / h, and the distance between the extrusion port and the surface of the core mold is 13 - 18 cm, so that the polycaprolactone and the human acellular amnion in the mixed solution are mixed and deposited on the surface of the core mold.

[0062] According to the embodiments of the present application, rotating the core mold at a suitable speed and extruding the mixed solution at a suitable flow rate are beneficial to preparing an artificial blood vessel with uniform wall thickness, improving the wall thickness uniformity of the artificial blood vessel, and increasing the service life of the artificial blood vessel.

[0063] In some alternative embodiments, the drying temperature is 50 - 60 °C, and the drying time is 3 to 5 days.

[0064] According to the embodiments of the present application, drying at a suitable temperature for a suitable time can prepare an artificial blood vessel with good performance.

[0065] In a second aspect, the embodiments of the present application provide an artificial blood vessel prepared by the method of the first aspect.

[0066] In some alternative embodiments, the mass ratio of polycaprolactone to human acellular amnion is 1:(0.8 - 1.3).

[0067] Optionally, the mass ratio of polycaprolactone to human acellular amnion is any value or the range composed of 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3.

[0068] According to the embodiments of the present application, by controlling the mass ratio of polycaprolactone to human acellular amnion within the above range, it is beneficial to control the components in the artificial blood vessel, endow it with good biocompatibility, promote the adhesion of cells such as endothelial cells inside and outside the artificial blood vessel, and make its physiological function closer to that of a normal blood vessel.

[0069] In some alternative embodiments, the wall thickness of the artificial blood vessel is 0.30 - 0.45 cm; the inner diameter of the artificial blood vessel is 3.5 - 4.5 mm. Optionally, the wall thickness of the artificial blood vessel can be any value or the range composed of 0.30 cm, 0.31 cm, 0.32 cm, 0.33 cm, 0.34 cm, 0.35 cm, 0.36 cm, 0.37 cm, 0.38 cm, 0.39 cm, 0.40 cm, 0.41 cm, 0.42 cm, 0.43 cm, 0.44 cm, 0.45 cm.

[0070] Optionally, the inner diameter of the artificial blood vessel can be any value among 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm or the range composed thereof.

[0071] Embodiment

[0072] The following embodiments more specifically describe the content disclosed in the present application. These embodiments are only for illustrative purposes, because various modifications and changes within the scope of the present application disclosure are obvious to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on weight, and all reagents used in the embodiments are commercially available or synthesized according to conventional methods, and can be used directly without further treatment, and all instruments used in the embodiments are commercially available.

[0073] Example 1

[0074] Preparation of human acellular amniotic membrane:

[0075] With the consent of the hospital and the parturient, fresh amniotic membrane obtained within 6 hours after cesarean section of a healthy parturient was used for the next acellular treatment under aseptic conditions. The amniotic membrane was repeatedly rinsed with sterile PBS to wash away the attached blood and mucus, and the surface impurities and sponge layer of the amniotic membrane were further removed with sterile gauze. Then it was placed in 0.25% trypsin-EDTA and shaken at 37°C for 6 hours, and repeatedly rinsed with sterile PBS. After that, a cell scraper was used to scrape off the surface cells, and shaken with 0.5% SDS for 4 hours, and then repeatedly rinsed with sterile PBS until the odor disappeared. The completely acellular amniotic membrane was cut into 3.0×3.0 cm with ophthalmic scissors 2 , sterilized by ethylene oxide, and prepared for subsequent experiments.

[0076] Preparation of human acellular amniotic membrane solution: The prepared human acellular amniotic membrane was placed in a ball mill for grinding to prepare human acellular amniotic membrane particles with a diameter of about 40 μm. At room temperature, the human acellular amniotic membrane was dissolved in 0.01M HCl solution containing 1 mg / ml pepsin for 24 h, and continuously stirred with a magnetic stirrer. The concentration was 10 mg / ml, that is, 1 g of human acellular amniotic membrane was dissolved in 100 ml of HCl solution containing 100 mg of pepsin. After complete dissolution, the human acellular amniotic membrane solution was placed on an ice plate. Take 1 / 10 volume of 0.1M NaOH solution and 1 / 9 volume of 10×PBS solution of the volume of the human acellular amniotic membrane solution, and then add 1×PBS solution, and mix well to adjust the pH value to 7.4. The mass concentration of the human acellular amniotic membrane solution is 10%.

[0077] Preparation of polycaprolactone solution: Weigh polycaprolactone and dissolve it in hexafluoroisopropanol solvent with a mass concentration of 10%.

[0078] Preparation of artificial blood vessel: Mix the human acellular amniotic membrane solution and the polycaprolactone solution, and stir evenly to make its mass concentration distribution in the mixed solution be 5%. Use the electrospinning technology to prepare artificial blood vessels. Suck the mixed solution (spinning solution) into a syringe and fix it on an injection pump. Replace the syringe needle with a 21-G metal needle-shaped nozzle and connect it to a high-voltage electrostatic generator with a conducting wire. After setting the voltage of the high-voltage generator, the flow rate of the injection pump, and the distance between the needle and the receiver, carry out electrospinning. Place the prepared artificial blood vessels in a vacuum drying oven and dry them at 55 degrees Celsius for more than three days. Before performing animal surgery, sterilize the artificial blood vessels under ultraviolet light irradiation.

[0079] Among them, Figure 1 Figure A in [ID] shows a schematic flow chart of preparing artificial blood vessels.

[0080] Example 2

[0081] The difference between this example and Example 1 is that in the mixed solution, the mass concentration of human acellular amniotic membrane is 4.5%, and the mass concentration of polycaprolactone is 5.5%.

[0082] Example 3

[0083] The difference between this example and Example 1 is that in the mixed solution, the mass concentration of human acellular amniotic membrane is 5.5%, and the mass concentration of polycaprolactone is 4.5%.

[0084] Comparative Example 1

[0085] The difference between this comparative example and Example 1 is that no human acellular amniotic membrane is added, and the mass concentration of polycaprolactone is 10%.

[0086] Test section

[0087] 1) Characterization of artificial blood vessels: Transversely and longitudinally cut the artificial blood vessels prepared in Example 1 (HAM+PCL) and Comparative Example 1 (PCL) in liquid nitrogen. Fix the cut samples on the sample stage with conductive glue. After evenly spraying gold, observe the fiber morphology of the tube wall and cross-section of the artificial blood vessels through a scanning electron microscope. Measure the fiber diameter and pore size through Image J software. Test the longitudinal mechanical properties of the artificial blood vessels with a mechanical tester. After calibrating the tensile sensor, fix the upper and lower ends of the 3-cm-long artificial blood vessels on the clamps respectively and perform longitudinal stretching at a speed of 10 mm / min until fracture to obtain the stress-strain curve. Calculate the Young's modulus, maximum stress, and elongation at break.

[0088] The inner diameter of the blood vessel prepared in Test Example 1 was detected to be 3 mm, and the average wall thickness was about 0.5 mm. The microscopic display results are as Figure 1 shown in Figure C therein, which is composed of randomly distributed fibers with a diameter of 1-4 microns. Figure 1 Figure B therein shows the mechanical property curve of the artificial blood vessel. Among them, the abscissa is the deformation rate of the artificial blood vessel, and the ordinate is the tensile pressure, indicating that the artificial blood vessels prepared in Example 1 and Comparative Example 1 have comparable mechanical properties, good mechanical properties, and high reliability of the artificial blood vessel. Compared with natural arteries, it shows that the mechanical properties of these prepared artificial blood vessels are strong enough to withstand the stresses from adjacent blood vessels and blood pressure.

[0089] Cell proliferation test: Human umbilical vein endothelial cells were seeded in a 48-well plate at a density of 2×10 3 . The culture medium was changed every other day. The wells in the experimental group were HAAM / PCL artificial blood vessels, and the control group was PCL artificial blood vessels. The number of cells in each well was measured using an MTT kit at 1, 3, and 5 days respectively. The effect of the artificial blood vessel on the in vitro proliferation ability of human umbilical vein endothelial cells was observed.

[0090] The results of detecting the proliferation of human umbilical vein endothelial cells (HUVECs) in the artificial blood vessel are as Figure 1 shown in Figure E. Cells were cultured on the artificial blood vessels of Example 1 and Comparative Example 1, and the OD value was measured using the MTT assay. The growth of HUVECs in the artificial blood vessel of Example 1 was significantly faster than that of the artificial blood vessel prepared in Comparative Example 1, indicating that it helps to promote the organization and functionalization of the artificial blood vessel and is beneficial to reducing rejection.

[0091] 2) Effect of the artificial blood vessel on the behavior of human umbilical vein endothelial cells:

[0092] Culture of human umbilical vein endothelial cells: The cryopreserved cells were thawed. The cryotube was centrifuged at 1000 rpm for 1 min, and the upper cryopreservation solution after centrifugation was discarded. Then 1 ml of complete culture medium was added thereto, and the liquid in the cryotube was aspirated repeatedly to gently resuspend the cells in the tube completely. The completely mixed cell suspension was all added to the culture flask, and then a certain amount of complete culture medium was slowly added to each culture flask. The culture medium was changed every 2 days until the cell density reached about 90%, and then passage was carried out. The cells of the 3rd passage were used for subsequent experiments.

[0093] Cell angiogenesis assay: Place Matrigel in a 4°C refrigerator overnight to allow it to melt. Under low-temperature conditions, add 200 μl of Matrigel to a 24-well plate, place it in a 4°C refrigerator overnight to spread it evenly, eliminate air bubbles, and then place it in a 37°C environment for 30 min to form a gel. Cut the artificial blood vessel into an appropriate size and spread it evenly in the Transwell chamber for standby. Seed human umbilical vein endothelial cells on the surface of the successfully gelled Matrigel at a cell density of 1x104 cells / well, gently shake, and after the cells are mixed evenly, place the Transwell chamber containing the artificial blood vessel above. Observe the tube formation of human umbilical vein endothelial cells using a microscope at 0, 3, 6, and 12 h, and use ImageJ to record the number of reticular circles, branch points, and total tube length of tube formation.

[0094] The detection results are as Figure 2 shown. Figure A shows a schematic diagram of its culture design. Figure B shows an optical image of the tubular network between the two groups of Example 1 and Comparative Example 1. Figure C evaluates the formation of the tubular network by the number of nodes, the number of rings, and the wall length of the tube. *p<0.05. The tube length and the number of nodes of the HAAM+PCL artificial blood vessel at 6 hours are significantly better than those of the PCL artificial blood vessel. In addition, at 12 hours, the number of nodes in the HAAM / PCL artificial blood vessel is significantly higher than that of the PCL artificial blood vessel transplantation. The peak of tube formation in both artificial blood vessels occurs at 6 hours, indicating that the artificial blood vessel of Example 1 can improve the function of HUVEC, such as tube formation.

[0095] 3) Cell migration experiment: Using a cell culture and cell migration device Transwell, insert a Transwell chamber with a filter membrane pore size of 8 μm into a 24-well culture plate. Add 200 μL of serum-free medium containing 5 human umbilical vein blood endothelial cells above the chamber, and add 800 μL of serum-free medium containing the artificial blood vessel to the lower well plate. After 12 h of cell migration, fix it with 4% paraformaldehyde for 15 min, and then stain it with crystal violet for 30 min. Randomly select five different fields of view for photography and statistics. Cell scratch: First, seed 4 human umbilical vein blood endothelial cells into a 6-well plate. When the cell confluence reaches 90%, use a 1 mL pipette tip to draw a straight line at the midline position of the well plate. Then add the medium containing the artificial blood vessel. After 12 h of migration, the cell fixation, staining, and statistical methods are the same as above. Evaluate the effect of the artificial blood vessel on the migration ability of human umbilical vein blood endothelial cells.

[0096] In the cell migration assay, the results are as Figure 3, Figure A is an optical image of the cell migration experiment. Figure B shows that the cells in the HAAM+PCL and PCL artificial blood vessels migrated 48.76±16.19% and 38.33±14.41% of the original wound distance, respectively. The calculation method is: migration percentage = (distance migrated / distance of the initial scratch). It shows that the artificial blood vessel containing HAAM improved cell migration.

[0097] 4) Rat abdominal aortic vascular transplantation model experiment: After the experimental rats were anesthetized, low molecular weight heparin (100 U / kg) was intravenously injected for anticoagulation. The abdominal aorta was dissected free, and the two ends of the artery were clamped with artery clamps, and then the abdominal aorta was cut off. The artificial blood vessels prepared in Example 1 and Comparative Example 1 were sutured respectively by end-to-end anastomosis, and the other experimental conditions in Example 1 and Comparative Example 1 were the same in the experiment. After anastomosis, the artery clamp was removed to restore blood flow. The abdominal organs were replaced and the abdominal cavity was rinsed with gentamicin sulfate solution, and the muscle and skin were sutured to close the abdominal cavity. After disinfection with iodophor, it was placed in an incubator waiting for the rats to wake up. Samples were taken and analyzed at different time points such as 1 month and 3 months after transplantation. This animal experiment was reviewed by the Experimental Animal Ethics Committee of Fuwai Hospital, Chinese Academy of Medical Sciences and met the animal ethics requirements. Observe the situation of the artificial blood vessels prepared in the examples and comparative examples in the rats.

[0098] After the artificial blood vessels were transplanted into the rat abdominal aortic transplantation model, no thrombosis or aneurysm was found in both groups at 4 weeks. The shapes of the two groups of artificial blood vessels were maintained before and after implantation, and the results are as Figure 4 shown in Figures A and B.

[0099] Ultrasonic examination at 3 months after transplantation showed no thrombus in the artificial blood vessels, and the results are as Figure 4 shown in Figures C and D.

[0100] The situation of the artificial blood vessels in Example 1 and Comparative Example 1 in the rats was stained with hematoxylin and eosin, and the results are as Figure 5 .

[0101] Figure 5 It shows the regeneration of the vascular media in the artificial blood vessels in Example 1 and Comparative Example 1 during the implantation time. Among them, Figure A is the cross-section of the artificial blood vessel of the PCL group stained with H&E; Figure B is the enlarged part of Figure A. Figure C is the cross-section of the artificial blood vessel of the HAAM+PCL group stained with H&E. Figure D is the enlarged part of Figure C, and more endothelial layers were found in the HAAM+PCL group

[0102] It was found that the human acellular amniotic membrane in the artificial blood vessel of Example 1 played an important role in maintaining vascular homeostasis. At 4 weeks, H&E and SEM images showed that the HAAM+PCL group ( Figure 5 and Figure 6 ) had more endothelial cells than the PCL group.

[0103] Figure 6 The endothelial cell generation on the inner lumen surface of the artificial blood vessels in Example 1 and Comparative Example 1 is shown. Figure A is a scanning electron microscope image of the longitudinally cut inner surface of the artificial blood vessel in the PCL group. Figure B is a high-magnification image of the inner lumen surface in Figure A. Figure C is a scanning electron microscope image of the longitudinally cut inner surface of the artificial blood vessel in the HAAM+PCL group. Figure D is a high-magnification image of the inner lumen surface in Figure C.

[0104] Figure 7 The immunofluorescence staining of the artificial blood vessels in Example 1 and Comparative Example 1 using CD31 staining and α-SMA antibody is shown. Figure A is the staining image of the artificial blood vessel in the PCL group with CD31 antibody. Figure B is the staining image of the artificial blood vessel in the HAAM+PCL group with CD31 antibody. Figure C is the immunofluorescence staining image of the artificial blood vessel in the PCL group using α-SMA antibody. Figure D is the immunofluorescence staining image of the artificial blood vessel in the HAAM+PCL group using α-SMA antibody. (E) Four weeks after implantation, the area covered by CD31-positive cells on the surface of the HAAM+PCL artificial blood vessel was 79.87±10.59%, while that of the PCL artificial blood vessel was 46.43±6.96%. The results show that the HAAM+PCL artificial blood vessel promoted in vivo endothelialization. (CD) Immunofluorescence image of α-SMA. (F) In the HAAM / PCL artificial blood vessel, the average thickness of α-SMA was 30.24±7.52 μm, while in the PCL artificial blood vessel it was 13.18±4.11 μm. Immunofluorescence staining using α-SMA antibody was used to detect the effect of the HAAM+PCL artificial blood vessel on the regeneration of smooth muscle cells (SMCs), indicating that the HAAM+PCL group artificial blood vessel promoted the regeneration of vascular SMCs.

[0105] It should also be noted that in the exemplary embodiments mentioned in the present application, some methods or systems are described based on a series of steps or devices. However, the present application is not limited to the order of the above steps. That is, the steps can be executed in the order mentioned in the embodiments, or different from the order in the embodiments, or several steps can be executed simultaneously.

[0106] As mentioned above, it is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should all be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing an artificial blood vessel, comprising: Providing a mixed solution containing polycaprolactone and human acellular amniotic membrane; Mixing and depositing the polycaprolactone and the human acellular amniotic membrane in the mixed solution on the surface of a mandrel to obtain a deposit; Drying the deposit and removing the mandrel to obtain the artificial blood vessel.

2. The method according to claim 1, wherein The mass concentration of the polycaprolactone in the mixed solution is 4.5%-5.5%.

3. The method according to claim 1, wherein The mass concentration of the human acellular amniotic membrane in the mixed solution is 4.5%-5.5%.

4. The method according to claim 1, characterized in that, The diameter of the mandrel is 3.5-4.5 mm.

5. The method according to claim 1, wherein The thickness of the deposit is 0.45-0.55 cm.

6. The method according to claim 1, wherein Mixing and depositing the polycaprolactone and the human acellular amniotic membrane in the mixed solution on the surface of a mandrel includes: Rotating the mandrel at a speed of 150-550 r / min, and applying a voltage of 15-18 kV to the mandrel; Extruding the mixed solution at a flow rate of 2.5-3.2 ml / h, and the distance between the extrusion port and the surface of the mandrel is 13-18 cm, so that the polycaprolactone and the human acellular amniotic membrane in the mixed solution are mixed and deposited on the surface of the mandrel.

7. The method according to claim 1, characterized in that, The temperature of the drying is 50-60 °C, and the time of the drying is 3 to 5 days.

8. An artificial blood vessel prepared by the method according to any one of claims 1-7.

9. The artificial blood vessel according to claim 8, wherein, The mass ratio of the polycaprolactone to the human acellular amniotic membrane is 1:(0.8-1.3).

10. The artificial blood vessel according to the claim, characterized in that, The wall thickness of the artificial blood vessel is 0.30-0.45 cm; the inner diameter of the artificial blood vessel is 3.5-4.5 mm.

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