Bio-ink for promoting microangiogenesis, method for preparing same, and use thereof

Abstract

Disclosed are a bio-ink for promoting microangiogenesis, a method for preparing same, and use thereof. The bio-ink is prepared by the following method: 1) preparing a filamentous microgel with a diameter within 20 microns from a sacrificial-phase material A as a porogenic template; 2) seeding vascular endothelial cells and perivascular cells on the surface of the filamentous microgel to give a hydrogel microfilament with the surface loaded with cells; and 3) mixing the hydrogel microfilament with the surface loaded with cells with a solution of a matrix-phase material B to give the bio-ink for promoting microangiogenesis. The bioprinting using the bio-ink can construct a capillary-like structure and cross-scale microvessels. The present invention can achieve the preparation of a micro-channel porous hydrogel structure with an average diameter of 20 microns or less and controllable porosity and the formation of a capillary-like network structure.

Description

A bio-ink that promotes microangiogenesis and its preparation method and application Technical Field

[0001] The present invention relates to the field of tissue engineering, particularly focusing on the field of vascularization of blood vessel construction, and more specifically to a bio-ink that promotes microvessel formation, and a preparation method and application thereof. Background Art

[0002] Tissue engineering is dedicated to constructing engineered tissue structures in vitro, and has important applications in drug screening, pathological models, tissue repair, and regenerative medicine. Currently, a major challenge facing the construction of larger-scale tissue models is the transport of oxygen, nutrients, and metabolites. The maximum natural diffusion distance of these components is approximately 100-200 microns [TRAORE MA, GEORGE S C. Tissue Engineering the Vascular Tree [J]. Tissue Eng Part B Rev, 2017, 23(6): 505-14.]. When the scale of the tissue model exceeds this size, the cells inside it will die due to the lack of timely and effective material exchange. In the human body, material transport is achieved through capillaries throughout the body. How to achieve vascularization in engineered tissue structures is an important scientific issue in the field of tissue engineering.

[0003] For microvessels with a diameter of less than 20 microns, a common construction method is to utilize the self-assembly behavior of vascular endothelial cells by regulating cell [ LEE VK, LANZI AM, NGO H, et al. Generation of Multi-scale Vascular Network System Within 3D Hydrogel Using 3D Bio-printing Technology [J]. Cell Mol Bioeng, 2014, 7(3): 460-72;JEON JS, BERSINI S, WHISLER JA, et al. Generation of 3D functional microvascular networks with human mesenchymal stem cells in microfluidic systems [J]. Integr Biol (Camb), 2014, 6(5): 555-63;BLINDER YJ, FREIMAN A, RAINDEL N, et al. Vasculogenic dynamics in 3D engineered tissue constructs [J]. Sci Rep, 2015, 5: 17840], biochemical [ KANT RJ, COULOMBE KL K. Integrated approaches to spatiotemporally directing angiogenesis in host and engineered tissues [J]. Acta Biomater, 2018, 69: 42-62, SON J, HONG SJ, LIM JW, et al. Engineering Tissue-Specific, Multiscale Microvasculature with a Capillary Network for Prevascularized Tissue [J]. Small Methods, 2021, 5(10): e2100632], mechanics [KOO MA, KANG JK, LEE MH, et al.Stimulated migration and penetration of vascular endothelial cells into poly (L-lactic acid) scaffolds under flow conditions [J]. Biomaterials research, 2014, 18: 7-; WEI Z, SCHNELLMANN R, PRUITT HC, et al. Hydrogel Network Dynamics Regulate Vascular Morphogenesis [J]. Cell Stem Cell, 2020, 27(5): 798-812 e6; CHEN YC, LIN RZ, QI H, et al. Functional Human Vascular Network Generated in Photocrosslinkable Gelatin Methacrylate Hydrogels [J]. Adv Funct Mater, 2012, 22(10): 2027-39] and other factors promote the formation of microvessels. Limited by the construction accuracy of microchannels in hydrogel environments, there are few studies exploring the influence of topological factors on microvascular formation. However, existing studies have shown that topological structures can effectively induce the spreading of endothelial cells and angiogenesis on two-dimensional surfaces or larger three-dimensional channels [ ARORA S, LIN S, CHEUNG C, et al. Topography elicits distinct phenotypes and functions in human primary and stem cell derived endothelial cells [J]. Biomaterials, 2020, 234: 119747; ARAKAWA C, GUNNARSSON C, HOWARD C, et al. Biophysical and biomolecular interactions of malaria-infected erythrocytes in engineered human capillaries [J]. Sci Adv, 2020, 6(3): 10].

[0004] Currently, existing biomanufacturing technologies can already achieve the construction of blood vessels on a larger scale. The core idea is to construct tubular pores in a matrix phase environment suitable for cell growth (mainly a hydrogel environment), and then deliver vascular endothelial cells through perfusion and other methods, combined with appropriate biochemical factors or cell co-culture to induce the formation of vascular structures. The scale of blood vessels prepared by this method depends on the construction accuracy of the three-dimensional microchannels in the hydrogel.

[0005] The construction of small-diameter pores is limited by manufacturing techniques: the template method is difficult to construct small-diameter, three-dimensional network-shaped pore templates; stereolithography is difficult due to its limited depth of structure and range of applicable materials, and it is also difficult to achieve multi-material printing; direct construction methods such as bio-3D printing are difficult to construct branched tubular structures and have limited printing accuracy. In summary, using existing methods to construct complex three-dimensional microchannel structures with controllable porosity below 20 microns remains a challenge. SUMMARY OF THE INVENTION

[0006] The present invention prepares a filamentous microgel with a diameter of less than 20 microns from a sacrificial phase material A as a pore-forming template; vascular endothelial cells and perivascular cells are planted on the surface of the filamentous microgel to obtain hydrogel microfilaments with surface-loaded cells; and the hydrogel microfilaments with surface-loaded cells are mixed with a solution of a matrix phase material B to obtain a bio-ink that promotes microvessel formation. Technical issues

[0007] Using existing methods, constructing complex three-dimensional microchannel structures with controllable porosity and diameters below 20 microns remains a challenge. Furthermore, self-assembly-based microvascularization methods have high requirements for the properties of the hydrogel environment and lack versatility. Technical Solutions

[0008] The purpose of the present invention is to propose a method for promoting microvascularization by utilizing micron-scale tubular pores of hydrogels to realize the construction of capillary-like structures in three-dimensional tissues.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] In one aspect, the present invention provides a bio-ink that promotes microvessel formation and a preparation method thereof.

[0011] The bio-ink for promoting microvessel formation provided by the present invention is prepared by a method comprising the following steps:

[0012] 1) Prepare filamentous microgels with a diameter of less than 20 μm from sacrificial phase material A as pore-forming templates;

[0013] 2) planting vascular endothelial cells and perivascular cells on the surface of the filamentous microgel to obtain surface-cell-loaded hydrogel microfilaments;

[0014] 3) Mixing the surface-loaded cell-containing hydrogel microfilaments with a solution of matrix material B to obtain a bio-ink that promotes microvessel formation.

[0015] In step 1) of the above method, the sacrificial phase material A has suitable temperature-sensitive properties, maintaining a gel state at a relatively low temperature (4-20°C) and sacrificially dissolving at a relatively high temperature (25-37°C), while supporting the adhesion of vascular endothelial cells and perivascular cells;

[0016] In one embodiment of the present invention, the sacrificial phase material A is gelatin.

[0017] In step 1), the steps for preparing a filamentous microgel having a diameter of less than 20 microns from a sacrificial phase material A are as follows: preparing a granular microgel of the sacrificial phase material A; in situ shearing of the microgel in a continuous phase to prepare a hydrogel structure, wherein microfilaments (i.e., filamentous microgels having a diameter of less than 20 microns) are oriented and arranged in a fiber-bundle-like manner in the continuous phase material; and removing the continuous phase material while maintaining the microfilaments in a gel state to obtain a filamentous microgel.

[0018] The preparation method of the granular microgel of the sacrificial phase material A is not limited, and can include microfluidics, emulsion, complex coacervation, mechanical crushing, etc.

[0019] The diameter of the resulting granular microgel can be 10-5000 microns;

[0020] The method for preparing a hydrogel structure by in-situ shearing of microgels in a continuous phase comprises the following steps: mixing granular microgels of sacrificial phase material A with a continuous phase material and then loading the mixture into a syringe; and, under conditions exceeding the sol temperature of material A, continuously applying shear force to the granular microgels during extrusion to cause them to undergo directional deformation, thereby producing filamentous microgels (microfilaments). The microfilaments are directionally arranged in a fiber-like manner within the extruded continuous phase material.

[0021] Wherein, the continuous phase material maintains extrudability within the operating temperature range and has poor miscibility with the sacrificial phase material A;

[0022] Specifically, the continuous phase material may be: Pluronic;

[0023] In the solution of the continuous phase material, the mass concentration of Pluronic is 10%-50%, preferably 30%;

[0024] The ratio of the granular microgel of the sacrificial phase material A to the continuous phase material is: 1g:2ml to 1g:32ml, preferably 1g:8ml;

[0025] The continuous phase material was removed by gentle immersion washing with phosphate buffer at 4°C for 10 min, and the filamentous microgels were collected;

[0026] The process of preparing the filamentous microgel with a diameter of less than 20 microns from the sacrificial phase material A further includes repeatedly blowing the obtained filamentous microgel to fully disperse it, and storing it in the form of a microfilament suspension.

[0027] In step 2), the operation of planting vascular endothelial cells and perivascular cells on the surface of the filamentous microgel includes: preparing a cell suspension containing vascular endothelial cells and perivascular cells, transferring the filamentous microgel into the cell suspension, and incubating.

[0028] The incubation condition is 3-15 hours at a temperature lower than the sol temperature of material A;

[0029] In step 3), the matrix phase material B has good biocompatibility, can be stably cross-linked and solidified, and has certain mechanical properties;

[0030] The matrix phase material B may be selected from at least one of: methacrylated gelatin (GelMA), alginic acid, hyaluronic acid, Matrigel, fibrinogen, and the like.

[0031] If the matrix phase material B is a photo-crosslinked polymer, the solution also contains a photoinitiator.

[0032] In one embodiment of the present invention, the matrix phase material B is methacrylated gelatin (GelMA);

[0033] The photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate;

[0034] The mass concentration of the matrix phase material B in the solution of the matrix phase material B is 1%-10%; the mass concentration of the photoinitiator is 0.05%-0.5%;

[0035] The solution of the matrix phase material B uses phosphate buffer as solvent;

[0036] The ratio of the surface-loaded cell hydrogel microfilaments to the solution of matrix material B can be: 1 g: 2 ml to 1 g: 20 ml.

[0037] The application of the above-mentioned bio-ink that promotes microvessel formation in the preparation of capillary-like structures and the construction of cross-scale microvessels also falls within the scope of protection of the present invention.

[0038] In another aspect, the present invention further provides a capillary-like structure.

[0039] The capillary-like structure is prepared by a method comprising the following steps: mixing the filamentous microgel with a matrix phase material B for embedding vascular endothelial cells and perivascular cells, and after shaping and solidifying, allowing the sacrificial phase material A therein to dissolve and flow out to form tubular pores. During subsequent culture, cells migrate into the pores to form a capillary-like structure.

[0040] The specific operation is as follows: a certain amount of vascular endothelial cells and perivascular cells are resuspended in a solution of matrix material B, mixed with the filamentous microgel prepared from sacrificial material A, mixed evenly, and cast into a template to fix the shape. The matrix material B is solidified by photocrosslinking, and then immersed in culture medium and cultured at a high temperature to dissolve the sacrificial material A therein and flow out.

[0041] In order to achieve more efficient cell delivery, the capillary-like structure can be prepared using the bio-ink that promotes microvascular formation. After the matrix phase material B is shaped and solidified, the sacrificial phase material A is dissolved and released to form tubular pores while achieving in situ cell delivery, thereby obtaining a capillary-like structure.

[0042] The specific operation is as follows: the bio-ink promoting microvascular formation is cast in a template to fix the shape, the matrix phase material B in the bio-ink is solidified by photocrosslinking, and the sacrificial phase material A is dissolved and flowed out by immersing in a culture medium and culturing at a high temperature;

[0043] The higher temperature is 30-37°C, specifically 37°C; the sacrificial phase material A is dissolved and flowed out by immersing in the culture medium and culturing at the higher temperature for 12-48 hours, specifically 24 hours.

[0044] On the other hand, the present invention also provides a method for constructing cross-scale microvessels using the above-mentioned biological ink by 3D printing.

[0045] The method for constructing cross-scale microvessels provided by the present invention comprises the following steps:

[0046] 1) preparing two or more bio-inks respectively, wherein the two or more bio-inks contain hydrogel microfilaments having different diameters;

[0047] 2) Bio-3D printing: A non-porous printing method is used to construct complex three-dimensional structures by alternately printing two or more bio-inks and filling the gaps between them. After the shapes are fixed and solidified, the sacrificial phase material A is dissolved and flows out, thus realizing cross-scale microvascular construction. Beneficial effects

[0048] The present invention has the following advantages over the prior art:

[0049] 1. It can realize the preparation of microchannel porous hydrogel structures with an average diameter of less than 20 microns and controllable porosity;

[0050] 2. Pre-microvascularized bio-inks can be prepared for extrusion or photocuring 3D bio-printing;

[0051] 3. Widely applicable to a variety of matrix materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 is a flowchart of preparing capillary-like structures using the bio-ink of the present invention.

[0053] FIG2 is a schematic diagram of a process for preparing capillary-like structures using the bio-ink of the present invention.

[0054] FIG3 is a schematic diagram of the microvascularization principle using the bio-ink of the present invention.

[0055] FIG4 is a statistical diagram of the diameter of sacrificial phase filamentous microgels.

[0056] FIG5 is a three-dimensional reconstruction of the sacrificial phase microwires mixed with the continuous phase material.

[0057] FIG6 is an observation diagram of gelatin microfilaments adhered to cells.

[0058] FIG7 is a staining diagram of capillary-like structures induced in a GelMA environment.

[0059] FIG8 is a cross-sectional view of capillary-like structures induced in a GelMA environment. Modes for Carrying Out the Invention

[0060] Example 1: Using gelatin microfilaments to prepare micron-sized pores in a GelMA environment and promote microvascular formation

[0061] According to the flow charts shown in Figures 1, 2, and 3, micron-sized pores were prepared and microvessel formation was promoted.

[0062] (1) Preparation of gelatin microspheres: Granular microgels were prepared using a microfluidic chip. The faster flowing oil phase sheared the lower flowing water phase, shearing the water phase into spherical droplets. After cooling and solidification, gel microspheres were obtained. The water phase used a gelatin solution with a mass concentration of 10%, and the solvent was PBS buffer; the oil phase used mineral oil, and a 2% volume concentration of Span80 was mixed into the oil phase as a surfactant to optimize the shearing effect. The water phase flow rate was controlled to 1 ml / h and the oil phase flow rate was 8 ml / h to prepare gelatin microspheres with a diameter of 180-220 microns. The microspheres were separated from the liquid phase by centrifugation. After removing the liquid phase containing the oil phase, PBS buffer was added again for washing. The washing was repeated several times to remove the residual oil phase on the surface of the microspheres.

[0063] (2) Preparation of gelatin microfilaments: Gelatin microfilaments with a diameter of less than 20 μm were prepared by in situ shearing of microgels. The gelatin microspheres were mixed with 30% Pluronic (solvent: PBS buffer) at a ratio of 1 g:8 ml and then loaded into a syringe. The mixture was steadily extruded at 32°C. The inner diameter of the syringe needle was 2 mm and the extrusion speed was 10 mm / min. The continuous phase continuously applied shear force to the gel microspheres to cause them to undergo directional deformation, thereby obtaining filamentous microgels. The microfilaments were oriented in a fiber bundle-like state in the extruded continuous phase material. The microfilaments were transferred to 4°C PBS buffer and gently washed to remove the continuous phase, thereby achieving the collection of filamentous microgels. The gel microfilaments were repeatedly blown to fully disperse them, and repeatedly washed with 4°C PBS to remove the Pluronic residue on the surface of the microfilaments. After washing, the microfilaments were stored in a 4°C refrigerator as a microfilament suspension. When taking it for use, use the screen centrifugation method to separate the microfilaments and the liquid phase. The specific method is to transfer the high-concentration microfilament suspension to a cell screen with a pore size of 40 microns, place the screen on a centrifuge tube and centrifuge it. The liquid phase will seep out of the screen as the centrifuge is carried out, and the microfilaments will remain on the surface of the screen. After scraping and weighing, it can be used. After mixing with the liquid phase, blow it thoroughly and resuspend it.

[0064] FIG4 is a statistical diagram of the diameter of sacrificial phase filamentous microgels.

[0065] FIG5 is a three-dimensional reconstruction of the sacrificial phase microwires mixed with the continuous phase material.

[0066] (3) Cell seeding: Prepare a cell suspension containing human umbilical vein endothelial cells and human lung fibroblasts, where the concentration of vascular endothelial cells is 5*10 6 / ml, the concentration of fibroblasts is 2.5*10 6 / ml, sterile gelatin microfilaments and cell suspension were mixed at a ratio of 1g:4ml and pipetted evenly, and incubated in a metal bath at 20°C for 10 hours to achieve cell adhesion on the gelatin microfilaments.

[0067] FIG6 is an observation diagram of gelatin microfilaments adhered to cells.

[0068] (4) Preparation of capillary-like structures: A 5% GelMA solution was prepared using PBS buffer as the matrix phase, and a 0.15% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator was added. The cell-loaded gelatin microfilaments were mixed with the GelMA solution at a ratio of 1 g:10 ml and blown evenly. After being cast into the template and fixed, the GelMA was solidified by cross-linking with UV light at a wavelength of 405 nm for 180 seconds. The microfilaments were immersed in culture medium and cultured in a 37°C incubator. The culture medium was changed every day. After 24 hours of culture, more than 80% of the gelatin microfilaments were dissolved. Capillary-like structures were observed on the third day of culture, and the structure remained stable on the fifth day of culture.

[0069] FIG7 is a staining diagram of capillary-like structures induced in a GelMA environment.

[0070] FIG8 is a cross-sectional view of capillary-like structures induced in a GelMA environment.

[0071] Example 2: Preparation of bio-ink using cell-laden gelatin microfilaments for application in 3D bio-printing of porous structures

[0072] (1) Preparation of cell-loaded gelatin microfilaments: The same steps (1) to (3) as in Example 1 were followed to prepare gelatin microfilaments with vascular endothelial cells and fibroblasts loaded on their surfaces.

[0073] (2) Preparation of bio-ink: Cell-laden gelatin microfilaments were mixed with a 7.5% mass concentration of GelMA solution at a ratio of 1 g:10 ml to prepare bio-ink. The mixture was kept at 25°C to prevent the gelatin microfilaments from dissolving.

[0074] (3) Extrusion bio-3D printing: The mixed bio-ink was transferred into a syringe and loaded onto an extrusion bio-3D printer. The sleeve temperature was controlled at 17°C and the base plate temperature was controlled at 10°C. After printing, the GelMA was solidified by UV cross-linking and then immersed in culture medium and cultured in a 37°C incubator. The culture medium was changed daily and cultured for 3-7 days to obtain a porous hydrogel sample containing a capillary-like structure.

[0075] (4) Photocuring printing: In addition, when using GelMA and other photocrosslinkable materials as the matrix phase to prepare bio-ink, photocuring printing can also be used. The specific method is: transfer the mixed bio-ink to the printing container, place it in a 10℃ environment for pre-cooling for 5 minutes to prevent the material from flowing during the printing process; load the pre-cooled ink into the photocuring printer and print it. After completion, place it at 37℃ to remove the unphotocrosslinked material and take out the printed structure. Use ultraviolet light post-crosslinking for 1 minute to fully solidify the structure. Soak it in culture medium and place it in a 37℃ incubator for culture. Change the culture medium every day. After culturing for 3-7 days, a porous hydrogel sample containing a capillary-like structure is obtained.

[0076] Example 3: Combining biological 3D printing to achieve cross-scale microvascular construction

[0077] (1) Preparation of bio-ink: The same steps (1)-(2) as in Example 2 were followed to prepare bio-ink A containing cell-laden gelatin microfilaments; a gelatin solution with a mass concentration of 5% was mixed with a concentration of 1*10 6 -1*10 7 Bio-ink B was prepared by adding vascular endothelial cells at a concentration of 100 cells / ml.

[0078] (2) Bio-3D printing: A non-porous printing method using two inks alternately printed and filled with each other's gaps to construct complex three-dimensional structures. After printing, the structures were UV-crosslinked and immersed in culture medium and cultured in a 37°C incubator for 3-7 days. The GelMA in ink A solidifies to provide mechanical support for the overall structure, and the gelatin microfilaments inside are sacrificially dissolved to construct vascular structures with a diameter of 15-20 microns in situ. The gelatin in ink B is sacrificially dissolved to construct vascular structures with a diameter of 100-200 microns in situ. The two inks are used to achieve cross-scale microvascular construction. Industrial Applicability

[0079] The present invention can realize the preparation of microchannel porous hydrogel structures with an average diameter of less than 20 microns and controllable porosity; it can be used to prepare pre-microvascularized bio-ink for extrusion or photocuring bio-3D printing; and it is widely applicable to a variety of matrix phase materials.

Claims

1. A method for preparing a bioink for promoting microvascular formation, comprising the following steps: 1) Preparing filamentous microgels with a diameter within 20 microns from a sacrificial phase material A as a pore-forming template; 2) Culturing vascular endothelial cells and perivascular cells on the surface of the filamentous microgels to obtain hydrogel microfilaments with cells loaded on the surface; 3) Mixing the hydrogel microfilaments with cells loaded on the surface with a solution of a matrix phase material B to obtain a bioink for promoting microvascular formation.

2. The method according to claim 1, characterized in that: In step 1), the sacrificial phase material A has suitable thermosensitive properties, remains in a gel state at a lower temperature and can be sacrificed and dissolved at a higher temperature, and at the same time supports the adhesion of vascular endothelial cells and perivascular cells; In step 1), the operation of preparing filamentous microgels with a diameter within 20 microns from a sacrificial phase material A is as follows: preparing particulate microgels of the sacrificial phase material A; preparing a hydrogel structure by in-situ shearing of the microgels in a continuous phase, in which the microfilaments are arranged directionally in the continuous phase material in a state similar to a fiber bundle; Removing the continuous phase material under the condition of keeping the microfilaments in a gel state to obtain filamentous microgels.

3. The method according to claim 2, characterized in that: The diameter of the obtained particulate microgels is 10 - 5000 microns; The operation of preparing a hydrogel structure by in-situ shearing of the microgels in a continuous phase is as follows: mixing the particulate microgels of the sacrificial phase material A with the continuous phase material and loading them into a syringe, and under the condition of a temperature higher than the sol temperature of material A, continuously applying a shearing force to the particulate microgels during the extrusion process to cause them to undergo directional deformation, thereby obtaining filamentous microgels, and the microfilaments are arranged directionally in the extruded continuous phase material in a state similar to a fiber bundle; Wherein, the continuous phase material maintains extrudability within the operating temperature range and has poor miscibility with the sacrificial phase material A.

4. The method according to claim 1, characterized in that: In step 2), the operation of culturing vascular endothelial cells and perivascular cells on the surface of the filamentous microgels includes: preparing a cell suspension containing vascular endothelial cells and perivascular cells, transferring the filamentous microgels into the cell suspension, and incubating, that's all, wherein, the incubation condition is to incubate for 3 - 15 hours at a temperature lower than the sol temperature of material A.

5. The method according to claim 1, characterized in that: In step 3), the matrix phase material B has good biocompatibility, can be stably crosslinked and cured and has certain mechanical properties; The matrix phase material B is selected from at least one of: methacrylated gelatin, alginic acid, hyaluronic acid, Matrigel, fibrinogen; If the matrix phase material B is a photo-crosslinkable polymer, the solution also contains a photoinitiator.

6. The method according to claim 1, characterized in that: The mass concentration of the matrix phase material B in the solution of the matrix phase material B is 1% - 10%; the mass concentration of the photoinitiator is 0.05% - 0.5%; The ratio of the hydrogel microfilaments with cells loaded on the surface to the solution of the matrix phase material B is: 1 g : 2 ml to 1 g : 20 ml.

7. A bioink for promoting microvascular formation prepared by the method according to any one of claims 1-6.

8. Use of the bioink according to claim 7 in the preparation of capillary-like structures and the construction of multi-scale microvessels.

9. A capillary-like structure prepared by a method comprising the following steps: after shaping and curing the matrix phase in the bioink according to claim 8, melting and flowing out the sacrificial phase material A therein, forming tubular pores while achieving in-situ delivery of cells, to obtain a capillary-like structure; or Mixing filamentous microgels with a matrix phase material B embedding vascular endothelial cells and perivascular cells, shaping and curing, then melting and flowing out the sacrificial phase material A therein to form tubular pores, and during subsequent culture, the cells migrate into the pores to form a capillary-like structure.

10. A method for constructing multi-scale microvessels, comprising the following steps: 1) Respectively preparing two or more bioinks by the method according to any one of claims 1-6, wherein the diameters of the hydrogel microfilaments contained in the two or more bioinks are different; 2) Bio 3D printing: Using a poreless printing method of alternately printing two or more bioinks and filling voids with each other to construct a complex three-dimensional structure, shaping and curing, then melting and flowing out the sacrificial phase material A therein to achieve the construction of multi-scale microvessels.

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