A method for constructing a fully biological small-caliber tissue-engineered blood vessel

Abstract

The present invention discloses a method for constructing a fully biological small-caliber tissue-engineered blood vessel, and belongs to the field of bioengineering technology. This method combines biological 3D printing technology and stem cell in situ induction technology for blood vessel construction for the first time, and creatively proposes vascular bionics. Biological 3D printing is used to quickly prepare blood vessels to support customized caliber and length, with a number of vascular cells close to physiological density, internal cells have a good growth environment, rich extracellular matrix types, and excellent mechanical properties. In situ induction technology obtains a large number of vascular smooth muscle cells; mechanical stimulation is applied, and the extracellular matrix is ​​further reshaped to obtain mechanical properties close to natural blood vessels; after in vitro endothelialization, fully biological small-caliber tissue-engineered blood vessels are obtained. The fully biological small-caliber tissue-engineered blood vessels constructed by this method can be used clinically for fully biological small-caliber tissue-engineered blood vessels for peripheral vascular replacement, arteriovenous fistula, and coronary artery bypass.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for constructing a fully biological small-caliber tissue engineering blood vessel. Background Art

[0002] For most patients, vascular transplantation is required to establish a bypass or replace blocked and damaged blood vessels. At this time, the emergence of tissue-engineered blood vessels provides a good solution to the shortage of grafts. After decades of development, artificial blood vessels made of synthetic materials have been successfully used to replace large blood vessels. However, for small-caliber blood vessels (<6mm), complications such as acute thrombosis, intimal hyperplasia of the anastomosis, aneurysms, and long-term calcification limit their further application. Constructing biological small-caliber tissue-engineered blood vessels has become the main research direction in the field.

[0003] The fine structure of natural arteries ensures the performance of their functions, including the intima layer, where endothelial cells exert anticoagulant and vascular regulation functions; the media layer, where smooth muscle cells respond to biochemical signals to regulate vascular tension, and collagen fibers and elastic fibers provide mechanical support against stretching and expansion; the adventitia layer, where afferent nerves and vasculature and progenitor cells regulate and maintain vascular function. Based on this, the construction of biological tissue engineering blood vessels should include major vascular cells and a variety of extracellular matrices. Bio-3D printing technology is considered to be a promising tissue engineering technology, allowing personalized design of printed structures, rapid and large-scale preparation of biological tissues, and better control of the spatial distribution of cells in the structure and the microstructure of the scaffold. Traditional vascular manufacturing methods include vascular molding, cell membrane preparation, secretion of extracellular matrix after scaffold material implantation, subcutaneous embedding of scaffold material, and bio-3D printing of blood vessels, and some studies have been carried out clinical trials. However, these methods all have certain defects. For example, the implantation of scaffold materials, poor material biocompatibility, limited number of cell inoculation, uneven cell dispersion, resulting in prolonged graft generation time and large batch differences, and residual scaffold material leading to foreign body reaction in the body. The graft generated by subcutaneous embedding of scaffold material is loose connective tissue with obviously insufficient mechanical tension.

[0004] In addition to manufacturing technology, another key issue in the construction of bionic tissues is seed cells. In human parenchymal organs, the cell density reaches 3×10 8 / cm 3 In the past, it was difficult to provide such a large number of cells by culturing cells in two dimensions in vitro. Moreover, in recent years, new evidence has continuously emerged to show that cells cultured in traditional ways are far from in vivo cells in terms of phenotype and function. Three-dimensional culture, multicellular sphere culture, and organoid culture that simulate the in vivo environment have gradually become the mainstream.

[0005] In organ construction, people have realized the importance of vascularization, and vascularization is also indispensable for tissue engineering blood vessels. The thickness of engineered tissues lacking blood vessels is often limited to less than 200 μm. For small and medium-sized arteries in the human body, their outer membranes contain a rich network of nutrient vessels that deliver essential nutrients and oxygen to the cells inside. In the construction of tissue engineering blood vessels, while increasing cell density, nutrient vessels should also be taken into consideration to ensure cell survival and give blood vessels excellent mechanical properties based on the number of cells and the thickness and strength of the extracellular matrix. However, this factor has not been mentioned in current vascular research. Summary of the invention

[0006] The first purpose of the present invention is to provide a construction method, which utilizes natural extracellular matrix components and photosensitive biomaterials in combination with stem cells and microvascular fragments to prepare bio-ink for 3D printing, thus opening up a new way to obtain tissue engineering blood vessel construction containing printed "living" tissues containing cells.

[0007] The second purpose of the present invention is to provide a method of using cytokines to promote cell proliferation and differentiation, thereby improving the mechanical properties of printed blood vessels and solving the technical problem of poor strength of biologically printed tissues.

[0008] The third object of the present invention is to provide a method of in vitro culture, in which microvessel fragments are combined and used as the main material for printing the adventitia of the blood vessel, thereby constructing a real "living" tissue.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for constructing a fully biological small-caliber tissue engineering blood vessel comprises the following steps:

[0011] S1. Construction of tissue-engineered blood vessels: using bio-ink containing photosensitive biomaterials, stem cells and microvessels that have been digested and centrifuged are prepared into a cell suspension;

[0012] Using software to model engineered blood vessels;

[0013] The cell suspension is filled into a biological 3D printer for printing, and then cross-linked with blue light to obtain tissue-engineered blood vessels;

[0014] The bio-ink is prepared from a photosensitive biomaterial solution and an extracellular matrix protein solution;

[0015] S2. In situ differentiation induction: The tissue-engineered blood vessels obtained by printing in step S1 are transferred to a culture dish for cell proliferation culture and differentiation induction treatment to obtain tissue-engineered blood vessels surrounded by multiple layers of smooth muscle;

[0016] The differentiation induction treatment is performed by adding cytokines;

[0017] S3, beating culture: fixing the tissue-engineered blood vessels surrounded by the multi-layer smooth muscle obtained in step S2 into a bioreactor to obtain tissue-engineered blood vessels with liquid perfusion function and mechanical tension;

[0018] S4, endothelial implantation: Add endothelial cell solution into the lumen of the tissue-engineered blood vessel processed in step S3, rotate 360 ​​degrees, disperse evenly, and then culture statically, using the blood flow flushing speed to make the endothelial cells adhere stably and arrange along the blood flow direction.

[0019] In the above-mentioned method for constructing the fully biological small-caliber tissue engineering blood vessels, the bio-ink is first synthesized, including the main supporting component photosensitive gelatin, which can quickly form a stable printing structure and provide a three-dimensional environment for cells; collagen, fibronectin, and laminin provide active sites to promote the proliferation and differentiation of stem cells. Then the bio-ink is characterized, including photocrosslinking time, porosity, rheological properties, elastic modulus, and cytotoxicity. Finally, the printing parameters are optimized, the printing structure is designed by software, the suspended gel is selected as the support, and the barrel temperature, printing speed, and printing pressure are adjusted to obtain high fidelity and high cell viability.

[0020] After the pluripotent stem cells are digested into single cells, they are prepared into cell suspensions with bio-ink for printing, and in situ induction is performed directly after in vitro amplification. Unlike traditional two-dimensional induction, the stem cells here are implanted in a three-dimensional microenvironment, and the induction process is more complicated and closer to the real in vivo environment. Through the stimulation of different cytokines in the culture medium, the interaction between cells and the extracellular matrix, and the interaction between cells, the proliferation and differentiation of stem cells into specific cells are promoted. Taking into account the changes in the environment, we increased the concentration and action time of the cytokines. In order to ensure the survival of the internal cells, we added microvascular fragments to the cell suspension to achieve pre-vascularization of the printed tissue. In addition, dynamic culture is used to promote the formation of vascular networks and the exchange of internal nutrients.

[0021] After two weeks of induction, stem cells differentiated into vascular smooth muscle cells and retained some progenitor cells. To further promote cell secretion of extracellular matrix and extracellular matrix remodeling, the engineered blood vessels were fixed in the bioreactor and stimulated by gradually increasing pulsating pressure to improve the mechanical properties of the blood vessels. The suture strength, burst strength, tensile properties, drug stimulation response, etc. of the engineered blood vessels were further tested and compared with natural arteries and veins.

[0022] Endothelial implantation: For small-caliber tissue-engineered blood vessels, the incidence of thrombosis is extremely high due to the small blood flow and slow flow rate. In vitro endothelialization is the most biomimetic and effective solution. Here, we use pluripotent stem cells from the same source to induce vascular endothelial cells for cell implantation. Endothelial cells are added to the lumen of the blood vessel, and the cells are automatically rotated 360 degrees to disperse the cells evenly, and then the blood flow flushing speed is gradually increased to allow the endothelial cells to stably adhere and arrange in the direction of blood flow.

[0023] Preferably, the stem cells are derived from any one of different hiPSC cell lines, different ESC cell lines and MSCs from different sources;

[0024] The microvessels are derived from any one of a fat pad, a progenitor cell-derived vascular network, and a stem cell-derived vascular organoid.

[0025] Preferably, the photosensitive biomaterial comprises photosensitive gelatin and / or photosensitive collagen;

[0026] The photosensitive biomaterial solution includes a photosensitive gelatin solution and / or a photosensitive collagen solution.

[0027] Preferably, the photosensitive gelatin solution is prepared by heating the mTeSR1 complete culture medium to 50-60°C, stirring continuously until the photosensitive gelatin is completely dissolved, and then preparing a 20% solution, followed by adding 0.5% LAP photocrosslinker and 20 μM Y27632 factor to mix;

[0028] The photosensitive collagen solution is obtained by dissolving the photosensitive collagen in a 0.2N acetic acid solution at 4°C.

[0029] Preferably, the cytokines include any one of glycogen synthase kinase-3 inhibitor (CHIR99021), bone morphogenetic protein 4 (BMP4), activin A (ActivinA), platelet-derived growth factor BB (PDGF-BB), transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF165).

[0030] Preferably, the extracellular matrix protein solution is prepared by dissolving the extracellular matrix protein in a 2X PBS solution to obtain a solution with a concentration of 375-400 g / mL;

[0031] The extracellular matrix proteins include laminin and fibronectin.

[0032] Preferably, the endothelial cells are any one of primary endothelial cells, progenitor cell-derived endothelial cells and stem cell-derived endothelial cells;

[0033] The endothelial cell content in the endothelial cell solution is 1×10 7 / mL-2×10 7 / mL.

[0034] Preferably, the static culture is 24 hours;

[0035] The blood flow flushing rate is 5-100 mL / min.

[0036] A fully biological small-caliber tissue engineering blood vessel is obtained by the construction method of the fully biological small-caliber tissue engineering blood vessel.

[0037] A fully biological small-caliber tissue-engineered blood vessel can be used clinically in peripheral vascular replacement, arteriovenous fistula devices, and coronary artery bypass grafting devices.

[0038] Compared with the prior art, the present invention has at least the following technical effects:

[0039] The present invention provides a method for constructing a fully biological small-caliber tissue engineering blood vessel. This method is aimed at the current huge demand for small-caliber tissue engineering blood vessels and the difficulty in constructing them. It combines biological 3D printing technology and stem cell in situ induction technology for blood vessel construction for the first time, and creatively proposes vasodilation bionics. The blood vessels quickly prepared using biological 3D printing technology support personalized customization of caliber and length, have a number of vascular cells close to physiological density, and the internal cells have a good growth environment, and the extracellular matrix is ​​rich in variety and has excellent mechanical properties. A large number of vascular smooth muscle cells are obtained through in situ induction technology; after applying mechanical stimulation, the extracellular matrix is ​​further reshaped to obtain mechanical properties close to those of natural blood vessels; finally, after in vitro endothelialization, a fully biological small-caliber tissue engineering blood vessel is obtained. The present invention solves the difficulty in constructing small-caliber tissue engineering blood vessels, and can also provide reference significance for the construction of more complex tissues and organs.

[0040] The fully biological small-caliber tissue-engineered blood vessels constructed by this method can be clinically used for peripheral vascular replacement, arteriovenous fistula, and coronary artery bypass grafting. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of light microscopic images of cells in the three-dimensional culture group of Experimental Example 1 on day 0, day 7 and day 14 (scale bar 100 μm);

[0042] Figure 2 Schematic diagram of light microscopy images of in situ induced smooth muscle cells on day 0, day 7 and day 14 of culture in Experimental Example 1 (scale bar 100 μm);

[0043] Figure 3 Schematic diagram of immunofluorescence staining of two-dimensional induced smooth muscle cells in Experimental Example 1 (scale bar 50 μm);

[0044] Figure 4 Schematic diagram of immunofluorescence staining of three-dimensional cultured smooth muscle cells in Experimental Example 1 (scale bar 50 μm);

[0045] Figure 5 Schematic diagram of immunofluorescence staining of smooth muscle cells induced in situ in Experimental Example 1 (scale bar 50 μm);

[0046] Figure 6 This is a schematic diagram of the RT-qPCR results of smooth muscle cells in Experimental Example 1;

[0047] Figure 7 Schematic diagram of immunofluorescence staining of cell patches in Experimental Example 1;

[0048] Figure 8 A schematic diagram of the cross-section of the 3D bioprinted modeled vascular structure in Experimental Example 2;

[0049] Fig. 9 Stereoscopic image of the vascular structure modeled by 3D bioprinting in Experimental Example 2

[0050] Fig.10 This is a schematic diagram of the appearance of the 3D printed blood vessel in Experimental Example 2;

[0051] Fig.11 This is a light microscopic image of the in situ induction of printed cells in Experimental Example 2 2 days later.

[0052] Fig.12 This is a light microscopic image of the in situ induction of printed cells in Experimental Example 2 14 days later.

[0053] Fig.13 This is a schematic diagram of D14 HE staining of blood vessels in Experimental Example 2;

[0054] Fig.14 This is a schematic diagram of the beating culture process in Experimental Example 2;

[0055] Fig.15 Schematic diagram of immunofluorescence staining of iPSC-EC in Experimental Example 4;

[0056] Fig.16 This is a schematic diagram of the scanning electron microscopy results of Experimental Example 4 at different time periods after endothelial cell implantation. DETAILED DESCRIPTION

[0057] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0058] Embodiment 1:

[0059] A method for constructing a fully biological small-caliber tissue engineering blood vessel comprises the following steps:

[0060] 1. Biological 3D printing:

[0061] 1) Synthesize bio-ink: Dissolve photosensitive gelatin in mTeSR1 complete medium heated to 60°C, stir continuously until completely dissolved, and prepare a 20% solution, wherein 0.5% photocrosslinker LAP and 20uM Y27632 factor are added.

[0062] The photosensitive collagen was dissolved in 0.2N acetic acid solution at 4°C and continuously shaken on a low-temperature shaker to prepare a 1% solution.

[0063] Dissolve laminin and fibronectin in 2X PBS solution at room temperature to adjust the concentration to 375ug / mL. Then mix the fibronectin solution and collagen solution in a 1:1 ratio and keep on ice.

[0064] Finally, the mixed solution in the previous step was mixed with the gelatin solution at 37° C. in a 1:1 ratio with vigorous shaking, and 0.5% of the total volume of 1 mol NaOH solution was added to adjust the pH to obtain bio-ink.

[0065] After aliquoting, store at -20°C until use.

[0066] 2) Materialise Magic software was used to model blood vessels, with an inner diameter of 2-6 mm and a length of 1-4 cm.

[0067] 3) The bio-ink was rewarmed to 37°C, and the stem cells were digested and prepared into a single cell suspension using the bio-ink. The cell concentration was adjusted to 2×10 7 / mL, in which 10000 blood vessel fragments / mL or 2×10 induced endothelial cells were added 6 / mL.

[0068] Then transfer the cell suspension into a 5 ml tube and precool it in a 4°C refrigerator for 5 min.

[0069] The cartridge is then filled into the envisionTEC bioprinter for printing.

[0070] 4) Printing parameter settings: use 27G 1-inch needle, gelatin suspension bath, print structure layer height 200-300um, extrusion pressure 0.5-1bar, barrel temperature 25℃, and print speed 15-30mm / s.

[0071] 5) Irradiate with 405nm blue light in six directions for 20s each, then wash away the suspended gel and transfer the printed blood vessels to a culture dish for culture.

[0072] 2. In situ differentiation induction: The printed blood vessels in step 1) were transferred to a culture dish and expanded for 3 days by adding stem cell culture medium. Then, N2B27 culture medium (DMEM / F12:Neurobasal 1:1, 1X N2 additive, 1X less vitamin A B27 additive, 0.012mMβ-mercaptoethanol, 2mM L-glutamine, P / S) containing 12uM CHIR99021, 30ng / ml BMP4 was added to induce 3d to the mesoderm. Then, N2B27 culture medium containing 2ng / mL ActivinA and 20ng / ml PDGF-BB was added to induce smooth muscle cells, and the medium was changed every other day for a total of 4d. Finally, SmGM culture medium containing 10% fetal bovine serum and 1ng / mL TGF-β was added to expand for 7d. During this period, the culture dish was placed on a shaker and continuously shaken at 40rpm to obtain tissue engineering blood vessels surrounded by multiple layers of smooth muscle.

[0073] 3. Pulsating culture: After two weeks of induction, stem cells differentiate into vascular smooth muscle cells and retain some progenitor cells. To further promote cell secretion of extracellular matrix and extracellular matrix remodeling, the engineered blood vessels are fixed in the bioreactor, and the electrospun membrane is attached to provide initial mechanical support. The mechanical properties of the blood vessels are improved through gradually increasing pulsating pressure stimulation.

[0074] The flow rate was set at 100-200 mL / min, the pulsation frequency was 60-200 bmp, and the radial strain was 0.5%-3%. During this period, the culture medium was replaced with DMEM supplemented with 20% fetal bovine serum, 1% NEAA, 2 mM L-glutamine, 0.012 mM β-mercaptoethanol, 1 ng / mL TGF-β and P / S.

[0075] After four weeks of culture, tissue-engineered blood vessels were obtained after extracellular matrix remodeling.

[0076] 4. Endothelial implantation: For small-caliber tissue-engineered blood vessels, the incidence of thrombosis is extremely high due to the small blood flow and slow flow rate. In vitro endothelialization is the most bionic and effective solution.

[0077] Pluripotent stem cells from the same source are selected and induced into vascular endothelial cells for cell implantation.

[0078] Add 1×10 7 / mL of endothelial cells, automatically rotate 360 ​​degrees to evenly disperse the cells, and culture them statically for 24 hours. Then connect them to the bioreactor and flush them with a peristaltic pump, with the flow rate gradually increasing from 5mL / min to 100mL / min, so that the endothelial cells can stably adhere and align along the blood flow direction.

[0079] Experimental Example 1: Bio-3D printing combined with in situ induction of hiPSC-SMC to construct cell patches.

[0080] According to step 1, the bio-ink was synthesized and prepared into a single cell suspension, which was then printed into a 20X20X1mm cell patch.

[0081] Perform in situ induction according to step 2 for 14 days.

[0082] Stretch culture was performed according to step 3 for 7 days.

[0083] like Figure 1 As shown, cells numbered 1, 2, and 3 in the figure are light microscopic images of the three-dimensional culture group cells on the 0th day, the 7th day, and the 14th day of culture. The results show that after culturing for 14 days, the cells in the three-dimensional culture group are in a dispersed state.

[0084] like Figure 2 As shown, the images marked 1, 2, and 3 in the figure are light microscopic images of the in situ induced smooth muscle cells at day 0, day 7, and day 14. The results showed that the cells in the in situ induced group were densely arranged with no gaps between the cells.

[0085] The above results indicate that this method can obtain high-density smooth muscle cells for constructing tissue engineering blood vessels.

[0086] like Figure 3 Shown is a schematic diagram of immunofluorescence staining of two-dimensional induced smooth muscle cells;

[0087] like Figure 4 Shown is a schematic diagram of immunofluorescence staining of three-dimensional cultured smooth muscle cells;

[0088] like Figure 5 Shown is a schematic diagram of immunofluorescence staining of in situ induced smooth muscle cells.

[0089] The results in the attached figure show that, as identified by immunofluorescence staining, stem cells differentiated into smooth muscle cells after in situ induction, and expressed smooth muscle cell-specific markers CNN1 and α-SMA, just like two-dimensional induction and three-dimensional culture.

[0090] like Figure 6 The figure shows the RT-qPCR results of smooth muscle cells. RT-qPCR detected the gene expression of smooth muscle cells obtained by in situ induction. From the results, it can be seen that smooth muscle cells do not express stem cell pluripotency transcription factors OCT3 / 4. Compared with two-dimensional induced smooth muscle-like cells, they express more smooth muscle markers MYH11 and α-SMA, and can synthesize and secrete collagen (COL1) and elastin (ELN), which is more conducive to the subsequent extracellular matrix remodeling of tissue engineering blood vessels.

[0091] like Figure 7The figure shows the immunofluorescence staining of the cell patch. After in situ induction, a dense smooth muscle cell layer with a thickness of 200um can be obtained.

[0092] Figure (a) shows the low-power field of view, with a scale bar of 200 μm. Figures (b), (c), and (d) show single-channel and superimposed images of the partially enlarged field of view, with a scale bar of 100 μm.

[0093] Experimental Example 2: Bio-3D printing combined with in situ induction of hiPSC-SMC to construct small-caliber tissue engineering blood vessels.

[0094] According to step 1, the bio-ink was synthesized and prepared into a single cell suspension, which was then 3D printed into a small-caliber tissue engineering blood vessel with an inner diameter of 4 mm, an outer diameter of 5 mm, and a length of 20 mm.

[0095] Perform in situ induction according to step 2 for 14 days.

[0096] Incubate the culture for 4 weeks according to step 3.

[0097] like Figure 8 Shown is a schematic diagram of the cross-section of the 3D bioprinted vascular structure modeled.

[0098] like Fig. 9 Shown is a stereoscopic image of the 3D bioprinted modeled vascular structure.

[0099] The vascular structure is designed to be double-layer printed with the media and adventitia, with a diameter of 4 mm and a length of 2-6 cm.

[0100] like Fig.10 As shown, this is a schematic diagram of the appearance of a 3D printed blood vessel, which has good fidelity and a scale of 1 cm.

[0101] like Fig.11 The following are light microscopy images of in situ induced cells 2 days after printing. The scale bar in (a) is 500 μm, and the scale bar in (b) is 200 μm.

[0102] like Fig.12 The following are light microscopy images of in situ induced printed cells 14 days after printing. The scale bar in (a) is 500 μm, and the scale bar in (b) is 200 μm.

[0103] From the above Fig.11 and 12 It can be seen that after printing, the stem cells are initially scattered as single cells. After induced differentiation, the cells are spindle-shaped smooth muscle-like and densely arranged.

[0104] like Fig.13The figure shows HE staining of blood vessels at D14. HE staining was performed at 14D after the blood vessels were printed and induced to differentiate. It can be seen that a dense smooth muscle layer was induced in situ, with cells arranged neatly and a thickness of 100-200um. Compared with the control group, the survival rate of cells inside the vascular wall increased after the vasa vasorum was added to the outer membrane.

[0105] like Fig.14 The figure shows a schematic diagram of the beating culture process. In (a) and (b), the printed blood vessels are surrounded by multiple layers of electrospun membranes to increase their mechanical properties, and in (c), they are transferred to a vascular incubator for long-term beating culture to promote extracellular matrix remodeling.

[0106] Experimental Example 3: Bio-3D printing combined with in situ induction of hESC-SMC to construct small-caliber tissue engineering blood vessels.

[0107] According to step 1, the bio-ink was synthesized and prepared into a single cell suspension, which was then 3D printed into a small-caliber tissue engineering blood vessel with an inner diameter of 4 mm, an outer diameter of 5 mm, and a length of 20 mm.

[0108] Perform in situ induction according to step 2 for 14 days.

[0109] Incubate the culture for 4 weeks according to step 3.

[0110] Experimental Example 4: Bio-3D printing combined with in situ induction to construct decellularization and endothelialization of small-caliber tissue engineering blood vessels.

[0111] According to step 1, the bio-ink was synthesized and prepared into a single cell suspension, which was then 3D printed into a small-caliber tissue engineering blood vessel with an inner diameter of 4 mm, an outer diameter of 5 mm, and a length of 20 mm.

[0112] Perform in situ induction according to step 2 for 14 days.

[0113] Incubate the culture for 4 weeks according to step 3.

[0114] Conventional SDS method for decellularization.

[0115] Finally, endothelial cells were implanted according to step 4, and the endothelial cells were umbilical vein endothelial cells, iPSC-EC, and ESC-EC.

[0116] like Fig.15 The following are immunofluorescence staining images of iPSC-EC. (a), (b), and (c) are single-channel staining light microscopy images. (d) is an overlay image.

[0117] The diagram shows that iPSCs were successfully induced to differentiate into vascular endothelial cells. Immunofluorescence staining showed that the induced cells highly expressed endothelial-specific markers vwF and CD144.

[0118] like Fig.16As shown in the figure, the scanning electron microscopy results of endothelial cells at different time periods after implantation, the scale bar is 20μm. Endothelial cells were implanted on the surface of the vascular endothelium, and the cells began to spread after 24 hours. After flow flushing culture, the cells further spread. At 72 hours, the endothelial cells were tightly connected and arranged along the direction of blood flow, eventually forming an endothelial barrier.

[0119] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a fully biological small-caliber tissue-engineered blood vessel, characterized in that: The steps include: S1. Construction of tissue-engineered blood vessels: using bio-ink containing photosensitive biomaterials, stem cells and microvessels that have been digested and centrifuged are prepared into a cell suspension; Using software to model engineered blood vessels; The cell suspension is filled into a biological 3D printer for printing, and then cross-linked with blue light to obtain tissue-engineered blood vessels; The bio-ink is prepared from a photosensitive biomaterial solution and an extracellular matrix protein solution; S2. In situ differentiation induction: The tissue-engineered blood vessels obtained by printing in step S1 are transferred to a culture dish for cell proliferation culture and differentiation induction treatment to obtain tissue-engineered blood vessels surrounded by multiple layers of smooth muscle; The differentiation induction treatment is performed by adding cytokines; S3, beating culture: fixing the tissue-engineered blood vessels surrounded by the multi-layer smooth muscle obtained in step S2 into a bioreactor to obtain tissue-engineered blood vessels with liquid perfusion function and mechanical tension; S4, endothelial implantation: Add endothelial cell solution into the lumen of the tissue-engineered blood vessel processed in step S3, rotate 360 ​​degrees, disperse evenly, and then culture statically, using the blood flow flushing speed to make the endothelial cells adhere stably and arrange along the blood flow direction.

2. The method for constructing a fully biological small-caliber tissue engineering blood vessel according to claim 1, characterized in that: The stem cells are derived from any one of a hiPSC cell line, an ESC cell line and a MSC cell line; The microvessels are derived from any one of a fat pad, a progenitor cell-derived vascular network, and a stem cell-derived vascular organoid.

3. The method for constructing a fully biological small-caliber tissue engineering blood vessel according to claim 1, characterized in that: The photosensitive biomaterial includes photosensitive gelatin and / or photosensitive collagen; The photosensitive biomaterial solution includes a photosensitive gelatin solution and / or a photosensitive collagen solution.

4. The method for constructing a fully biological small-caliber tissue engineering blood vessel according to claim 3, characterized in that: The photosensitive gelatin solution is prepared by heating the mTeSR1 complete culture medium to 50-60° C., stirring continuously until the photosensitive gelatin is completely dissolved, and then preparing a 20% solution, followed by adding 0.5% LAP photocrosslinker and 20 μM Y27632 factor to mix; The photosensitive collagen solution is obtained by dissolving the photosensitive collagen in a 0.2N acetic acid solution at 4°C.

5. The method for constructing a fully biological small-caliber tissue engineering blood vessel according to claim 1, characterized in that: The cytokine comprises any one of glycogen synthase kinase-3 inhibitor, bone morphogenetic protein 4, activin A, platelet-derived growth factor BB, transforming growth factor β and vascular endothelial growth factor.

6. The method for constructing a fully biological small-caliber tissue engineering blood vessel according to claim 1, characterized in that: The extracellular matrix protein solution is prepared by dissolving the extracellular matrix protein in a 2X PBS solution to obtain a solution with a concentration of 375-400 g / mL; The extracellular matrix proteins include laminin and fibronectin.

7. The method for constructing a fully biological small-caliber tissue engineering blood vessel according to claim 1, characterized in that: The endothelial cells are any one of primary endothelial cells, progenitor cell-derived endothelial cells and stem cell-derived endothelial cells; The endothelial cell content in the endothelial cell solution is 1×10 7 / mL-2×10 7 / mL.

8. The method for constructing a fully biological small-caliber tissue engineering blood vessel according to claim 1, characterized in that: The static culture is carried out for 24 hours; The blood flow flushing rate is 5-100 mL / min.

9. A fully biological small-caliber tissue-engineered blood vessel, characterized in that: Obtained by the method for constructing a fully biological small-caliber tissue engineering blood vessel as described in any one of claims 1-8.

10. A fully biological small-caliber tissue-engineered blood vessel as claimed in claim 9 which can be used clinically in peripheral vascular replacement, arteriovenous fistula devices, and coronary artery bypass graft devices.

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