Tissue-engineered artificial blood vessel, preparation method therefor, and use thereof

Abstract

A tissue-engineered artificial blood vessel, a preparation method therefor, and use thereof. The preparation method comprises: inoculating vascular endothelial cells and smooth muscle cells in an inner layer of a tubular cell scaffold and culturing in a mixed manner for 7-13 days; changing to a pro-secretory medium and meanwhile applying a perfusion pulse pressure in the tubular cell scaffold, culturing for another 8-13 days, inoculating vascular fibroblasts in an outer layer of the tubular cell scaffold and culturing the cells, and changing to a mixed medium and co-culturing for 8-13 days; and replacing the mixed medium with a pro-secretory medium and co-culturing for 10-16 days and then decellularizing in situ to obtain the tissue-engineered artificial blood vessel. The preparation method can promote extracellular matrix secretion, enabling the prepared artificial blood vessel to have better mechanical strength and elasticity, and shorten culture time.

Description

A tissue engineering artificial blood vessel and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and in particular to a tissue engineering artificial blood vessel and a preparation method and application thereof. Background Art

[0002] From the inside out, human blood vessels are mainly composed of endothelial cells, smooth muscle cells, and fibroblasts, as well as the extracellular matrix secreted by these three cells. The three cells and their extracellular matrix play different roles in maintaining vascular structure and patency. Vascular diseases are currently very common and have a high mortality rate. They are usually caused by stenosis or blockage of blood vessels, resulting in reduced blood flow and lack of nutrients, which in turn damages tissues or organs. Using the patient's own blood vessels for vascular transplantation surgery is a conventional method for treating vascular diseases. However, the collection of the patient's own blood vessels faces problems such as short length of the harvestable blood vessels, mismatched blood vessel caliber, and difficulty in finding blood vessels that meet the requirements. Therefore, artificial blood vessels are a very promising solution.

[0003] The current technology for preparing artificial blood vessels using materials such as polyethylene terephthalate, expanded polytetrafluoroethylene, PCL, PLCL, PU, ​​PGS, PLA, PDO, and collagen still has the disadvantages of difficulty in preparing small-caliber blood vessels with an inner diameter of less than 6 mm, lack of biological activity of the prepared blood vessels, slow vascular regeneration, and poor long-term patency. This is because the lumen of artificial blood vessels prepared with these materials lacks the coverage of physiologically functional vascular endothelial cells. Therefore, researchers have attempted to construct endothelialized artificial blood vessels by planting endothelial cells in vitro through tissue engineering. However, due to the significant difference between in vitro culture conditions and the in vivo environment, endothelial cells lack the support of smooth muscle cells. Therefore, such artificial blood vessels are prone to endothelial shedding, leading to thrombosis.

[0004] In addition, the culture methods of tissue-engineered blood vessels in related technologies mostly involve culturing single cells and single cell culture media in containers such as in vitro reactors to form tissues. The cell types and culture methods do not conform to the actual conditions of blood vessels in the human body. This type of technology also has the problem of poor cell secretion of extracellular matrix such as structural proteins and supporting proteins in the preparation of artificial blood vessels, resulting in poor remodeling ability of vascular tissue and difficulty in forming artificial blood vessels with high elasticity, good mechanical properties and biocompatibility. The use of three types of cells to co-culture to obtain artificial blood vessels also has the problem of requiring different cells to grow in the same environment and obtain the cytokines they need. Moreover, the time it takes to culture artificial blood vessels with tissue engineering in related technologies is as long as 8 weeks or more, which leads to high costs. Long-term cell culture can lead to bacterial and fungal infections, and the risk of microbial invasion is greatly increased.

[0005] Therefore, there is an urgent need for a method for preparing tissue-engineered artificial blood vessels that has high elasticity, good mechanical properties and biocompatibility, as well as good long-term patency, and can prepare small-caliber blood vessels with an inner diameter of less than 6 mm.

[0006] Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a method for preparing a tissue-engineered artificial blood vessel. The method can produce small-caliber artificial blood vessels with an inner diameter of less than 6 mm. During the preparation and culture process, the method promotes the secretion of large amounts of extracellular matrix components such as structural and supporting proteins by cells, resulting in the prepared tissue-engineered artificial blood vessels having high elasticity, good mechanical properties and biocompatibility, and excellent long-term patency, enabling them to replace natural blood vessels in humans or animals. Furthermore, the method shortens the culture time, reduces costs, and reduces the risk of contamination.

[0008] The present invention also provides a tissue engineering artificial blood vessel.

[0009] The present invention also provides the preparation method and application of the tissue engineering artificial blood vessel.

[0010] A first aspect of the present invention provides a method for preparing a tissue engineering artificial blood vessel, comprising the following steps:

[0011] S1. Vascular endothelial cells and smooth muscle cells are seeded on the inner layer of the tubular cell scaffold and mixed cultured for 7-13 days;

[0012] S2, replacing the secretion-promoting medium and applying perfusion pulsatile pressure in the tubular cell scaffold, continuing the culture for 8-13 days, inoculating vascular fibroblasts on the outer layer of the tubular cell scaffold for culture, replacing the mixed medium and co-culturing for 8-13 days;

[0013] S3, replacing the mixed culture medium with a secretion-promoting culture medium, culturing for 10-16 days, and then performing in situ decellularization to obtain a tissue-engineered artificial blood vessel;

[0014] The secretory medium comprises cytokines, amino acids and vitamins, as well as serum or serum replacement.

[0015] According to a specific embodiment of the present invention, the method for preparing a tissue-engineered artificial blood vessel of the present invention can prepare small-caliber artificial blood vessels with an inner diameter of less than 6 mm. During the culture process, it can promote the interaction between cells and promote the cells to secrete a large amount of extracellular matrix components, so that the prepared tissue-engineered artificial blood vessels have high elasticity, good mechanical properties and biocompatibility. The artificial blood vessels prepared by the present invention have high content of structural proteins and supporting proteins, can maintain good elasticity and mechanical properties, and have good long-term patency. In addition, the method for preparing artificial blood vessels of the present invention uses three types of cells to culture simultaneously, which shortens the culture time, reduces costs, and reduces the risk of contamination.

[0016] In some embodiments of the present invention, sources of the endothelial cells include the aorta and the umbilical artery vein.

[0017] In some preferred embodiments of the present invention, the endothelial cells are derived from umbilical arteries.

[0018] In some embodiments of the present invention, sources of the smooth muscle cells include the aorta and the umbilical cord vein.

[0019] In some preferred embodiments of the present invention, the smooth muscle cells are derived from umbilical arteries.

[0020] In some embodiments of the present invention, sources of the fibroblasts include the aorta and the umbilical artery vein.

[0021] In some preferred embodiments of the present invention, the fibroblasts are derived from umbilical cord arteries.

[0022] In some embodiments of the present invention, the seeding density of the endothelial cells is 100,000-1.5 million / cm.

[0023] In some preferred embodiments of the present invention, the seeding density of the endothelial cells is 200,000-1,000,000 / cm.

[0024] In some embodiments of the present invention, the smooth muscle cells are seeded at a density of 1 to 7 million cells / cm.

[0025] In some preferred embodiments of the present invention, the seeding density of the smooth muscle cells is 2-6 million / cm.

[0026] In some more preferred embodiments of the present invention, the seeding density of the smooth muscle cells is 2-5 million / cm.

[0027] In some embodiments of the present invention, the seeding density of the fibroblasts is 500,000-7,000,000 / cm.

[0028] In some preferred embodiments of the present invention, the seeding density of the fibroblasts is 1-4 million / cm.

[0029] In some more preferred embodiments of the present invention, the seeding density of the fibroblasts is 1 to 3 million / cm.

[0030] In some embodiments of the present invention, the tubular cell scaffold is made of a degradable polymer material.

[0031] In some embodiments of the present invention, the component of the tubular cell scaffold is at least one of polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA), polyhydroxyalkanoate (PHA), and collagen.

[0032] In some preferred embodiments of the present invention, the component of the tubular cell scaffold is polyglycolic acid (PGA).

[0033] In some embodiments of the present invention, the wall thickness of the tubular cell scaffold is 0.1-2.5 mm.

[0034] In some preferred embodiments of the present invention, the wall thickness of the tubular cell scaffold is 0.5-1.5 mm.

[0035] In some more preferred embodiments of the present invention, the wall thickness of the tubular cell scaffold is 0.8-1 mm.

[0036] In some embodiments of the present invention, the application of perfusion pulsatile pressure treatment in step S2 is specifically: first applying 1-10 mmHg column, and then increasing 3-20 mmHg column every day.

[0037] In some preferred embodiments of the present invention, the application of perfusion pulsatile pressure treatment in step S2 is specifically: first applying 1-10 mmHg column, and then increasing it by 5-15 mmHg column every day.

[0038] In some preferred embodiments of the present invention, the application of perfusion pulsatile pressure treatment in step S2 is specifically: first applying 1-10 mmHg column, and then increasing 5-15 mmHg column every day, and the range of applied perfusion pulsatile pressure is between 1-300 mmHg.

[0039] During the cultivation and preparation process of the artificial blood vessels, the present invention applies pulsatile pressure and gradually increases the applied pulsatile pressure value within an appropriate range. Applying pulsatile pressure helps promote the growth of cells and the secretion of extracellular matrix on the artificial blood vessels, promotes the formation of vascular tissue, can shorten the cultivation time to a certain extent, and effectively improve the mechanical strength, elasticity and other properties of the artificial blood vessels.

[0040] In some embodiments of the present invention, the mixed culture medium in step S1 is a mixture of vascular endothelial cell culture medium and smooth muscle cell culture medium in a volume ratio of 1:2-10.

[0041] In some preferred embodiments of the present invention, the culture medium for the mixed culture in step S1 is a mixture of vascular endothelial cell culture medium and smooth muscle cell culture medium in a volume ratio of 1:4.

[0042] In some embodiments of the present invention, the culture medium for the mixed culture in step S1 contains serum or serum substitute HPL at a mass concentration of 10%-25%.

[0043] In some preferred embodiments of the present invention, the culture medium for the mixed culture in step S1 contains serum at a mass concentration of 15%-25% or a serum substitute at a mass concentration of 10%-20%.

[0044] In some more preferred embodiments of the present invention, the culture medium for the mixed culture in step S1 contains serum or serum substitute at a mass concentration of 18%-22%%.

[0045] In some embodiments of the present invention, the mixed culture medium in step S2 is a mixture of vascular endothelial cell culture medium, smooth muscle cell culture medium and fibroblast culture medium in a volume ratio of 1:4-10:2-5.

[0046] In some preferred embodiments of the present invention, the mixed culture medium in step S2 is a culture medium obtained by mixing endothelial cell culture medium, smooth muscle cell culture medium and fibroblast culture medium in a volume ratio of 1:6:3.

[0047] In some embodiments of the present invention, the mixed culture medium in step S2 contains serum or serum substitute at a mass concentration of 5-15%.

[0048] In some preferred embodiments of the present invention, the mixed culture medium in step S2 contains serum or serum substitute with a mass concentration of 8-12%.

[0049] In some embodiments of the present invention, the serum substitute is human platelet lysate HPL.

[0050] The present invention uses a scheme for culturing several types of vascular cells simultaneously. By screening and optimizing different culture medium ratios, the above-mentioned culture medium formula suitable for multi-cell co-culture is obtained, which optimizes the growth rate of each cell and enhances the effect of cell-cell interaction, and also shortens the culture time to a certain extent.

[0051] In some embodiments of the present invention, the secretory medium in step S2 comprises: cytokines, amino acids and vitamins, and 8%-12% serum or serum replacement.

[0052] In some embodiments of the present invention, the cytokine includes at least one of VEGF, PDGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, and BMP.

[0053] In some embodiments of the present invention, the cytokine comprises at least one of VEGF, PDGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, and BMP, and the cytokine concentration is independently 1-20 ng / mL;

[0054] In some preferred embodiments of the present invention, the cytokines include 1-5 cytokines among VEGF, PDGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, and BMP, and the cytokine concentration is independently 1-20 ng / mL.

[0055] In some more preferred embodiments of the present invention, the cytokines include 1-5 cytokines among VEGF, PDGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, and BMP, and the cytokine concentration is 5-15 ng / mL.

[0056] In some more preferred embodiments of the present invention, the cytokines include VEGF, TGF-β1, and TGF-β2, and the concentration of each cytokine is 5-15 ng / mL.

[0057] In some more preferred embodiments of the present invention, the cytokines are VEGF, TGF-β1, and TGF-β2, and the concentration of the cytokines is independently 5-15 ng / mL.

[0058] In some embodiments of the present invention, the vitamins include vitamin B and vitamin C.

[0059] In some preferred embodiments of the present invention, the vitamins include vitamin B and vitamin C, and the concentrations thereof are independently 30-100 mg / mL.

[0060] In some embodiments of the invention, the amino acids include glycine, alanine, glutamic acid and proline.

[0061] In some preferred embodiments of the present invention, the amino acids include glycine, alanine, glutamic acid and proline, and the concentrations thereof are independently 20-60 mg / mL.

[0062] In some embodiments of the present invention, the serum substitute is human platelet lysate HPL.

[0063] In some embodiments of the present invention, the secretion-promoting medium in step S3 comprises: cytokines, amino acids and vitamins, and 3%-7% serum or serum replacement.

[0064] In some embodiments of the present invention, the cytokine includes at least one of bFGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, and BMP.

[0065] In some embodiments of the present invention, the cytokine comprises at least one of bFGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, and BMP, and the cytokine concentration is independently 1-20 ng / mL;

[0066] In some preferred embodiments of the present invention, the cytokines include 1-5 cytokines selected from bFGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, and BMP, and the cytokine concentrations are independently 1-20 ng / mL.

[0067] In some more preferred embodiments of the present invention, the cytokines include 1-5 cytokines among bFGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, and BMP, and the cytokine concentration is 5-15 ng / mL.

[0068] In some more preferred embodiments of the present invention, the cytokines include bFGF, VEGF, TGF-β1, and TGF-β2, and the concentration of each cytokine is 5-15 ng / mL.

[0069] In some more preferred embodiments of the present invention, the cytokines are bFGF, VEGF, TGF-β1, TGF-β2, and the concentration of the cytokines is independently 5-15 ng / mL.

[0070] In some embodiments of the present invention, the vitamins include vitamin B and vitamin C.

[0071] In some preferred embodiments of the present invention, the vitamins include vitamin B and vitamin C, and the concentrations thereof are independently 30-100 mg / mL.

[0072] In some embodiments of the invention, the amino acids include glycine, alanine, glutamic acid and proline.

[0073] In some preferred embodiments of the present invention, the amino acids include glycine, alanine, glutamic acid and proline, and the concentrations thereof are independently 20-60 mg / mL.

[0074] In some embodiments of the present invention, the serum substitute is human platelet lysate HPL.

[0075] The present invention utilizes optimized, screened cytokines and their added concentrations and combinations to obtain a preferred secretory-stimulating culture medium, which effectively promotes cell-cell interactions and the secretion of structural and supporting proteins, thereby facilitating the rapid formation of vascular tissue while enhancing the elasticity and mechanical strength of vascular tissue. This results in better long-term patency of the blood vessels produced by the present invention.

[0076] The present invention optimizes the secretory culture medium and adds vitamins and amino acids of appropriate concentrations and types, thereby promoting cell growth and the secretion of extracellular matrix such as structural proteins and supporting proteins. It can also synergize with cytokines to promote the maturation of vascular tissue and enhance the elasticity and mechanical strength of artificial blood vessels.

[0077] The solution of the present invention can also effectively increase the growth rate of vascular cells and shorten the preparation time of artificial blood vessels by replacing serum with human platelet lysate HPL, a serum substitute.

[0078] In some embodiments of the present invention, the vascular fibroblasts in step S2 are inoculated by cyclic inoculation, and the inoculation time is 1-4 days.

[0079] In some preferred embodiments of the present invention, the vascular fibroblasts are inoculated in step S2 by cyclic inoculation, and the inoculation time is 2-4 days.

[0080] The second aspect of the present invention provides a tissue engineering artificial blood vessel produced by the preparation method according to the first aspect of the present invention.

[0081] Since the tissue engineering artificial blood vessel of the second aspect of the present invention adopts all the technical solutions of the preparation method of the above embodiment, it has at least all the beneficial effects brought by the technical solutions of the above embodiment.

[0082] In some embodiments of the present invention, the mass proportion of the stent material in the artificial blood vessel is 0%-10%.

[0083] In some preferred embodiments of the present invention, the mass proportion of the stent material in the artificial blood vessel is 0%-6%.

[0084] In some embodiments of the present invention, the inner diameter of the artificial blood vessel is 1.5-15 mm.

[0085] In some preferred embodiments of the present invention, the inner diameter of the artificial blood vessel is 2-10 mm.

[0086] In some more preferred embodiments of the present invention, the inner diameter of the artificial blood vessel is 2-6 mm.

[0087] In some embodiments of the present invention, the wall thickness of the artificial blood vessel is 0.1-2 mm.

[0088] In some preferred embodiments of the present invention, the wall thickness of the artificial blood vessel is 0.2-1.5 mm.

[0089] In some more preferred embodiments of the present invention, the wall thickness of the artificial blood vessel is 0.2-1 mm.

[0090] The third aspect of the present invention provides the preparation method of the first aspect of the present invention and the application of the tissue engineering artificial blood vessel of the second aspect in A1)-A5):

[0091] A1) Vascular products and their preparation;

[0092] A2) Products for the treatment of vascular diseases and their preparation;

[0093] A3) Artificial intestines, artificial esophagus and other tubular tissue products and their preparation;

[0094] A4) In vitro blood circulation simulation device and its preparation;

[0095] A5) In vivo vascular implantation device and its preparation.

[0096] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0098] FIG1 is a photographic appearance diagram of a tissue-engineered artificial blood vessel according to Example 1 of the present invention;

[0099] FIG2 is a diagram showing HE staining results of the tissue-engineered artificial blood vessel of Example 1 of the present invention;

[0100] FIG3 is a photographic appearance diagram of the tissue-engineered artificial blood vessel of Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0101] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0102] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0103] The endothelial cell culture medium used in the examples is brand: Lonza, item number: CC-3156; the smooth muscle cell culture medium used is brand: Lonza, item number: CC-3182; and the fibroblast culture medium used is brand: Sciencell, item number: 2301.

[0104] The biodegradable polymer tubular cell scaffold used in the examples is a high-porosity tubular material made of polyglycolic acid polymer, purchased from Suzhou Genin Instrument Technology Co., Ltd., product model BIOFELT / PGA, and the product can be purchased at http: / / www.genintech.com / content / ?164.html.

[0105] The vascular endothelial cells, smooth muscle cells and fibroblasts in this example were purchased from Wuhan Procell Life Science Co., Ltd. The product purchase website is https: / / www.procell.com.cn / xbzy / ydxb / .

[0106] Example 1

[0107] This embodiment provides a method for preparing a small-caliber artificial blood vessel with an inner diameter of 2 mm. The specific steps are as follows:

[0108] (1) Prepare endothelial cells and smooth muscle cells isolated from human umbilical arteries in vitro, mix endothelial cell culture medium and smooth muscle cell culture medium in a ratio of 1:4 to obtain mixed culture medium 1, wherein the serum mass concentration is 20%, and 1% double antibody (penicillin and streptomycin) is added. Resuspend the endothelial cells and smooth muscle cells in the mixed culture medium 1 so that the density ratio of endothelial cells to smooth muscle cells is about 1:10 to obtain a cell suspension. The preparation operations of the cell suspension and the culture medium used are all prepared in a sterile isolator.

[0109] (2) Prepare a tubular cell scaffold made of a degradable polymer material with a wall thickness of 0.8 mm and an inner diameter of 2 mm, place it in a vascular culture reactor, and pass the cell suspension of step (1) into the cell scaffold of the vascular culture reactor by aseptically connecting the liquid perfusion system. Inoculate cells on the inner layer of the cell scaffold, wherein the inoculation density of endothelial cells is 200,000 / cm, and the inoculation density of smooth muscle cells is 2 million / cm. The culture environment is: culture in a tissue culture incubator at a temperature of 37°C, a carbon dioxide concentration of 5%, and a humidity of more than 90%.

[0110] (3) After 10 days of culture, the endothelial cells and smooth muscle cells proliferate rapidly and form endothelial cell layers and smooth muscle cell layers. The culture medium is replaced with secretory medium 1, which is a medium containing a 10% lower serum concentration than mixed medium 1 and the addition of growth factors VEGF, TGF-β1, and TGF-β2 at a final concentration of 10 ng / mL, glycine, alanine, glutamic acid, and proline at a final concentration of 40 mg / mL, and vitamin B and vitamin C at a final concentration of 70 mg / mL.

[0111] (4) The culture was continued for 10 days. At the same time, an external peristaltic pump was used to control the flow rate to give a radial amplitude stimulation cycle of 1%-10% to the tubing and stent to apply a perfusion pulsation pressure of 1-300 mmHg. The perfusion pulsation pressure was increased by 5 mmHg daily from the initial 1 mmHg column. At the same time, a fibroblast suspension resuspended in fibroblast culture medium was introduced into the outer culture medium of the vascular stent for inoculation. The fibroblasts were also isolated from human umbilical arteries in vitro. The inoculation density was 1 million / cm. The circulation time of the outer layer fibroblast suspension was 3 days.

[0112] (5) Prepare mixed culture medium 2, in which the volume ratio of culture medium is endothelial cell culture medium: smooth muscle cell culture medium: fibroblast culture medium = 1:6:3, and the mass concentration of serum is 10%. Replace the culture medium of both the inner and outer layers with mixed culture medium 2 and co-culture the three cells for 10 days, and the outer layer of the scaffold forms a fibroblast layer with a thickness of 0.05-0.1mm. Replace the culture medium with secretion-promoting culture medium 2, which reduces the serum concentration to 5% based on secretion-promoting culture medium 1 and adds the growth factor bFGF at a concentration of 10ng / mL. At the same time, gradually increase the perfusion pulsation pressure by 10-15mmHg per day. The cell scaffold gradually degrades, and the degraded part is replaced by cells and extracellular matrix such as structural proteins and supporting proteins secreted by the cells.

[0113] (6) After culturing for 2 weeks in the secretory culture medium 2 system, a tubular tissue rich in cells and extracellular matrix such as structural proteins and supporting proteins and a small amount of scaffold material is formed, and in situ decellularization treatment is performed to obtain a tissue-engineered artificial blood vessel.

[0114] The specific steps of the in situ decellularization treatment are as follows: after the culture is completed, the culture medium is replaced with a decellularization solution, wherein the decellularization solution contains SDS with a mass concentration of 1%, TritonX-100 with a mass concentration of 1%, and CHAPS with a molar concentration of 8mM. The decellularization treatment is completed at 37°C, 25 rpm stirring or perfusion with the decellularization solution for 48 hours.

[0115] The artificial blood vessel of this embodiment took approximately 7 weeks to prepare. The inner diameter of the tissue-engineered artificial blood vessel was measured to be 2 mm, and the wall thickness was 0.3 mm. The residual rate of polymer materials in the artificial blood vessel was tested to be 5.3%. The tissue-engineered artificial blood vessel prepared in this embodiment was photographed, as shown in Figure 1. Sections of the tissue-engineered artificial blood vessel prepared in this embodiment were stained using the HE staining method, as shown in Figure 2. After HE staining, the vessel wall was primarily stained red, indicating that the main component of the tissue-engineered artificial blood vessel prepared in this embodiment is an extracellular matrix composed of structural proteins and support proteins.

[0116] Example 2

[0117] This embodiment provides a method for preparing a medium-to-large caliber artificial blood vessel with an inner diameter of 6 mm. The specific steps are as follows:

[0118] (1) Prepare endothelial cells and smooth muscle cells isolated from human umbilical arteries in vitro, mix endothelial cell culture medium and smooth muscle cell culture medium in a ratio of 1:4 to obtain mixed culture medium 1, wherein the serum mass concentration is 20%, and 1% double antibody (penicillin and streptomycin) is added. Resuspend the endothelial cells and smooth muscle cells in the mixed culture medium 1 so that the density ratio of endothelial cells to smooth muscle cells is about 1:6 to obtain a cell suspension. The preparation operations of the cell suspension and the culture medium used are all prepared in a sterile isolator.

[0119] (2) Prepare a tubular cell scaffold made of a biodegradable polymer material with a wall thickness of 1 mm and an inner diameter of 6 mm, place it in a vascular culture reactor, and aseptically connect the cell suspension of step (1) to the cell scaffold of the vascular culture reactor through a liquid perfusion system. Inoculate cells on the inner layer of the cell scaffold, wherein the inoculation density of endothelial cells is 800,000 / cm, and the inoculation density of smooth muscle cells is 5 million / cm. The culture environment is: culture in a tissue culture incubator at a temperature of 37°C, a carbon dioxide concentration of 5%, and a humidity of more than 90%.

[0120] (3) After 8 days of culture, the endothelial cells and smooth muscle cells proliferate rapidly and form endothelial cell layers and smooth muscle cell layers. The culture medium is replaced with secretory medium 1, which is a medium containing a 10% reduction in serum concentration based on mixed medium 1, and the addition of growth factors VEGF, TGF-β1, and TGF-β2 at a concentration of 10 ng / mL, glycine, alanine, glutamic acid, and proline at a concentration of 40 mg / mL, and vitamin B and vitamin C at a concentration of 70 mg / mL.

[0121] (4) The culture was continued for 10 days. At the same time, an external peristaltic pump was used to control the flow rate to give a radial amplitude stimulation cycle of 1%-10% to the pipeline and the stent to apply a perfusion pulsation pressure of 1-300 mmHg. The perfusion pulsation pressure was increased by 5 mmHg in proportion every day from the initial 1 mmHg column. At the same time, a fibroblast suspension resuspended in fibroblast culture medium was introduced into the outer culture medium of the vascular stent for inoculation. The fibroblasts were also isolated from the human umbilical cord vein in vitro. The inoculation density was 3 million / cm. The circulation time of the outer layer fibroblast suspension was 3 days.

[0122] (5) Prepare mixed culture medium 2, wherein the volume ratio of the culture medium is endothelial cell culture medium: smooth muscle cell culture medium: fibroblast culture medium = 1:6:3, wherein the mass concentration of serum is 10%. Replace the culture medium of the inner layer and the outer layer with mixed culture medium 2 and co-culture the three cells for 10 days, and the scaffold as a whole forms a cell layer with a thickness of 0.8-1mm. Replace the culture medium with secretion-promoting culture medium 2, which reduces the serum concentration to 5% based on secretion-promoting culture medium 1 and adds the growth factor bFGF at a concentration of 10ng / mL. At the same time, gradually increase the perfusion pulsation pressure by 10-15mmHg per day. After 1 week of culture, the cell scaffold begins to gradually degrade, and the degraded part is replaced by cells and extracellular matrix such as structural proteins and supporting proteins secreted by the cells.

[0123] (6) After culturing for 2 weeks in the secretory culture medium 2 system, a tubular tissue rich in cells and extracellular matrix such as structural proteins and supporting proteins and a small amount of scaffold material is formed, and in situ decellularization treatment is performed to obtain a tissue-engineered artificial blood vessel.

[0124] The artificial blood vessel of this embodiment was prepared in less than 7 weeks. The inner diameter of the tissue engineering artificial blood vessel was measured to be 6 mm and the wall thickness was 1 mm.

[0125] Example 3

[0126] This embodiment provides a preparation method for a small-caliber artificial blood vessel with an inner diameter of 2 mm:

[0127] The difference between this embodiment and embodiment 1 is that the serum in all culture media of embodiment 1 is replaced with serum substitute HPL, wherein the serum mass concentration of 20% in mixed culture medium 1 is replaced with serum substitute HPL concentration of 15%, the serum concentration of 10% in secretagogue culture medium 1 is replaced with serum substitute HPL concentration of 10%, the serum concentration of 10% in mixed culture medium 2 is replaced with serum substitute HPL concentration of 10%, and the serum concentration of 5% in secretagogue culture medium 2 is replaced with serum substitute HPL concentration of 5%; the culture time of 10 days in step (3) is replaced with 6 days, the culture time of 10 days in step (4) is replaced with 8 days, the cycle time of 2-4 days in step (4) is replaced with 2 days, and the other culture times remain unchanged. The overall culture time is shortened by about 1 week compared with embodiment 1.

[0128] Example 4

[0129] This embodiment provides a preparation method for a small-caliber artificial blood vessel with an inner diameter of 2 mm:

[0130] The difference between this embodiment and embodiment 1 is that the seeding concentrations of the three cells in embodiment 1 are increased, the seeding density of endothelial cells is replaced from 200,000 cells / cm to 400,000 cells / cm, the seeding density of smooth muscle cells is replaced from 2 million cells / cm to 4 million cells / cm, and the seeding density of fibroblasts is replaced from 1 million cells / cm to 2 million cells / cm; the culture time in step (3) is replaced from 10 days to 6 days, the culture time in step (4) is replaced from 10 days to 8 days, and the circulation time in step (4) is replaced from 2 to 4 days to 2 days. The remaining culture times remain unchanged, and the overall culture time is shortened by about 1 week compared with embodiment 1.

[0131] Example 5

[0132] This embodiment provides a preparation method for a small-caliber artificial blood vessel with an inner diameter of 2 mm:

[0133] The difference between this embodiment and embodiment 1 is that the volume ratio of the endothelial cell and smooth muscle cell suspension in step (1) is replaced by a volume ratio of the endothelial cell and smooth muscle cell suspension of 1:4 to a volume ratio of the endothelial cell and smooth muscle cell suspension of 1:10, without changing the density ratio and seeding density of the two cells; and the ratio of endothelial cell culture medium: smooth muscle cell culture medium: fibroblast culture medium = 1:6:3 in step (5) is replaced by endothelial cell culture medium: smooth muscle cell culture medium: fibroblast culture medium = 1:10:5.

[0134] The growth rate of each cell layer and the interaction effect between different cells in the artificial blood vessel prepared in this embodiment are slightly worse than those in Example 1. Under the same culture time, the vascular wall thickness of the artificial blood vessel in this embodiment is reduced compared with that in Example 1.

[0135] Comparative Example 1

[0136] This comparative example provides a preparation method for a small-caliber artificial blood vessel with an inner diameter of 2 mm:

[0137] The difference between this comparative example and Example 1 is that no secretion-promoting medium was used during the culture process, secretion-promoting medium 1 was replaced by mixed medium 1, and secretion-promoting medium 2 was replaced by mixed medium 2. All other culture parameters and intervention parameters were exactly the same as those in Example 1.

[0138] The artificial blood vessel product finally produced in this comparative example still formed a relatively complete tubular white tissue, but the formed tissue lacked sufficient elasticity and mechanical properties, and did not secrete sufficient extracellular matrix such as structural proteins and supporting proteins.

[0139] Comparative Example 2

[0140] This comparative example provides a preparation method for a small-caliber artificial blood vessel with an inner diameter of 2 mm:

[0141] The only difference between this comparative example and Example 1 is that no pulsatile pressure stimulation was given during the culture process, and the culture was static throughout the entire process.

[0142] The artificial blood vessels produced in this comparative example formed intact vascular tissue after decellularization. However, the secretion of extracellular matrix components, such as structural and supportive proteins, within the vascular tissue was insufficient, and the resulting vascular tissue lacked sufficient elasticity and mechanical strength. Photographs of the tissue-engineered artificial blood vessels in this comparative example, as shown in Figure 3, show that the artificial blood vessels in this comparative example were easily broken and had poor elasticity and mechanical properties.

[0143] Comparative Example 3

[0144] This embodiment provides a preparation method for a small-caliber artificial blood vessel with an inner diameter of 2 mm:

[0145] The difference between this embodiment and embodiment 1 is that the culturing time of 10 days in step (3) is replaced by 6 days, the culturing time of 10 days in step (4) is replaced by 8 days, the cycle time of 2-4 days in step (4) is replaced by 2 days, and the other culturing times remain unchanged.

[0146] Since the culture time in multiple time periods is shorter than that in Example 1, the number of cell proliferation in each layer and the secretion of extracellular matrix in the artificial blood vessel prepared in this comparative example are insufficient, the artificial blood vessel prepared is thinner, and the elasticity and mechanical strength properties are poor.

[0147] Test Case

[0148] Physical performance tests were performed on the artificial blood vessels prepared in Examples 1-4 and Comparative Examples 1-2 to characterize their elasticity and strength. The specific test methods are as follows: According to the methods in the industry standard YY / T0500-2021 "Cardiovascular Implants", the axial tensile strength, circumferential tensile strength, pressurized burst strength, and suture pull strength of the artificial blood vessels were tested:

[0149] 1) Axial Tensile Strength: Axial tensile strength measures the axial tensile strength of a tubular vascular graft in its tubular state. During the test, the vessel is secured at each end to the clamps of a materials testing machine, with the clamps spaced 50 mm apart. Care is taken to ensure that the specimen is not stretched, twisted, or damaged by the clamps, maintaining its natural state as much as possible. The specimen is then stretched at a constant rate of 150 mm / min until it breaks. The load at break is recorded as the axial tensile strength.

[0150] 2) Circumferential tensile strength: Circumferential tensile strength is the measurement of the tensile strength of a tubular vascular graft in the circumferential direction in the tubular state. Cut a test specimen of 40 mm in length from the sample, recorded as the length of the specimen (L), in mm. Place the specimen on the two pins of the universal material testing machine fixture, ensuring that the specimen is not stretched or twisted and remains in a natural state. Stretch the specimen at a rate of 150 mm / min until it reaches the breaking point. Record the maximum load (Tmax). Calculate the circumferential tensile strength by dividing the maximum load (Tmax) of each specimen by its original specimen length. The calculation method is: Circumferential tensile strength = Tmax / 2L.

[0151] 3) Pressurized rupture strength: Connect the vascular tissue to a pressurized perfusion instrument and pump liquid into the blood vessels at a flow rate of 10kPa / s-25kPa / s. Keep the pressure increasing steadily and record it in real time. When the pressure reaches the intensity at which the blood vessel ruptures, it is the pressurized rupture strength.

[0152] 4) Suture Pull Strength: This test measures the force required to pull a suture out of a tubular vascular graft. A 20 mm long specimen is cut axially. A 6-0 nylon suture (0.16 mm diameter) is inserted through a layer of the vessel wall 2 mm from one end of the straightened specimen, forming a semicircular loop. The suture is stretched at a rate of 150 mm / min, and the force required to pull the suture out of the vessel or cause damage to the vessel wall is recorded.

[0153] The test results are shown in Table 1.

[0154] Table 1 Physical performance data of artificial blood vessels prepared in Examples 1-4 and Comparative Examples 1-2

[0155] From the data of the above test results, it can be seen that the artificial blood vessels prepared in Examples 1-4 of the present invention have significantly higher axial tensile strength, circumferential tensile strength, pressurized burst strength and suture tensile strength than the artificial blood vessels prepared in Comparative Examples 1-2. Each value is basically one order of magnitude higher than that of the comparative example, indicating that the scheme for preparing artificial blood vessels of the present invention has obvious advantages in promoting the secretion of extracellular matrix such as structural and supporting proteins by cells, and in forming tissues with higher elasticity and strength. Therefore, the artificial blood vessel preparation method of the present invention and the artificial blood vessels prepared have good application value.

[0156] In summary, the preparation method of the tissue-engineered artificial blood vessel of the present invention can be used to prepare small-caliber artificial blood vessels with an inner diameter of less than 6 mm. During the culture and preparation process, it can promote cells to secrete a large amount of extracellular matrix components, so that the prepared tissue-engineered artificial blood vessels have high elasticity, good mechanical properties and biocompatibility. The artificial blood vessels prepared by the present invention have high content of structural proteins and supporting proteins, which can enable the blood vessel wall to maintain good elasticity and mechanical properties, thereby having good long-term patency in actual use. In addition, the method for preparing artificial blood vessels of the present invention uses three types of cells to culture simultaneously, shortening the culture time, reducing costs, and reducing the risk of contamination. The invented tissue-engineered artificial blood vessels and preparation methods have good application prospects in the artificial preparation of vascular products and vascular disease products, as well as artificial intestines, artificial esophagus and other tubular tissues.

[0157] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A method for preparing a tissue engineering artificial blood vessel, characterized in that: The following steps are involved: S1, inoculating vascular endothelial cells and smooth muscle cells in the inner layer of the tubular cell scaffold and co-culturing for 7-13 days; S2, replacing the secretion-promoting medium, applying perfusion pulsating pressure in the tubular cell scaffold, and continuing to culture for 8-13 days, inoculating vascular fibroblasts on the outer layer of the tubular cell scaffold for culture, and replacing the mixed medium for co-culture for 8-13 days; S3, replacing the mixed culture medium with a secretion-promoting culture medium, culturing for 10-16 days, and performing in situ decellularization to obtain a tissue-engineered artificial blood vessel; The secretory medium comprises cytokines, amino acids and vitamins, as well as serum or serum replacement.

2. The preparation method according to claim 1, characterized in that: The sources of the endothelial cells, the smooth muscle cells and the fibroblasts include the aorta and the umbilical cord vein; Preferably, the seeding density of the endothelial cells is 0.1-1.5 million / cm, the seeding density of the smooth muscle cells is 1-7 million / cm, and the seeding density of the fibroblasts is 0.5-7 million / cm.

3. The preparation method according to claim 1, characterized in that: The tubular cell scaffold is made of degradable polymer material; The wall thickness of the tubular cell scaffold is 0.1-2.5 mm; Preferably, the tubular cell scaffold is composed of at least one of polylactic acid, polyglycolic acid, polycaprolactone, polylactic-co-glycolic acid, polyhydroxyalkanoate, and collagen.

4. The preparation method according to claim 1, characterized in that: The step S2 of applying the perfusion pulsatile pressure is specifically: first applying 1-10 mmHg column, and then increasing it by 3-20 mmHg column every day.

5. The preparation method according to claim 1, characterized in that: The mixed culture medium in step S1 is a medium obtained by mixing vascular endothelial cell culture medium and smooth muscle cell culture medium in a volume ratio of 1:2-10, and the mixed culture medium in step S2 is a medium obtained by mixing vascular endothelial cell culture medium, smooth muscle cell culture medium and fibroblast culture medium in a volume ratio of 1:4-10:2-5; Preferably, the mixed culture medium in step S1 contains serum or serum substitute HPL at a mass concentration of 10%-25%; Preferably, the mixed culture medium in step S2 contains serum or serum substitute HPL at a mass concentration of 5%-15%.

6. The preparation method according to claim 1, characterized in that: The secretion-promoting medium in step S2 comprises: cytokines, amino acids and vitamins, and 8%-12% serum or serum substitute; The cytokines include VEGF, PDGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, BMP At least one; The amino acids include glycine, alanine, glutamic acid and proline; The vitamins include vitamin B and vitamin C.

7. The preparation method according to claim 1, characterized in that: The secretion-promoting medium in step S3 comprises: cytokines, amino acids and vitamins, and 3%-7% serum or serum substitute; The cytokines include at least one of bFGF, IGF, HGF, TGF-β1, TGF-β2, TGF-β3, and BMP; The amino acids include glycine, alanine, glutamic acid and proline; The vitamins include vitamin B and vitamin C.

8. An artificial blood vessel for tissue engineering obtained by the preparation method according to any one of claims 1 to 7.

9. The tissue engineering artificial blood vessel according to claim 8, characterized in that: The mass proportion of the stent material in the artificial blood vessel is 0-10%; Preferably, the inner diameter of the artificial blood vessel is 1.5-15 mm; More preferably, the wall thickness of the artificial blood vessel is 0.1-2 mm.

10. Use of the preparation method according to any one of claims 1 to 7 or the artificial blood vessel according to claim 9 in A1) to A5): A1) Vascular products and their preparation; A2) Products for the treatment of vascular diseases and their preparation; A3) Artificial intestine, artificial esophagus and other tubular tissue products and their preparation; A4) In vitro blood circulation simulation device and its preparation; A5) In vivo vascular implantation device and its preparation.

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