WO2025107170A1 - Bio-ink for promoting microangiogenesis, method for preparing same, and use thereof - Google Patents

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 microvessel formation and its preparation method and application Technical Field

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

Background Art

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, an important challenge facing the construction of larger-scale tissue models is the transport of oxygen, nutrients and metabolites. The maximum distance of natural diffusion of these components is about 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.

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 V K, LANZI A M, 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 J S, BERSINI S, WHISLER J A, 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 Y J, FREIMAN A, RAINDEL N, et al. Vasculogenic dynamics in 3D engineered tissue constructs [J]. Sci Rep, 2015, 5: 17840], biochemical [KANT R J, COULOMBE K L K. Integrated approaches to spatiotemporally directing angiogenesis in host and engineered tissues [J]. Acta Biomater, 2018, 69: 42-62, SON J, HONG S J, LIM J W, et al. Engineering Tissue-Specific, Multiscale Microvasculature with a Capillary Network for Prevascularized Tissue [J]. Small Methods, 2021, 5(10): e2100632], mechanics [KOO M-A, KANG J K, LEE M H, 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 H C, et al. Hydrogel Network Dynamics Regulate Vascular Morphogenesis [J]. Cell Stem Cell, 2020, 27(5): 798-812 e6；CHEN Y C, LIN R Z, 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. Due to the limitations of the construction accuracy of microchannels in hydrogel environments, there are few studies exploring the impact of topological factors on microvessel formation. However, existing studies have shown that on two-dimensional surfaces or larger three-dimensional channels, topological structures can effectively induce the spreading of endothelial cells and angiogenesis [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].

Currently, existing biomanufacturing technology 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 (mainly a hydrogel environment) suitable for cell growth, 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.

The construction of small-diameter pores is limited by manufacturing technology: the difficulty of the template method lies in the construction of a small-diameter, three-dimensional network-structured pore-forming template; the difficulty of stereolithography lies in the limited depth of the structure and the range of applicable materials, and it is difficult to achieve multi-material printing; the difficulty of direct construction methods represented by biological 3D printing lies in the construction of branched tubular structures and the limited printing accuracy. In summary, it is still a challenge to use existing methods to construct three-dimensional complex microchannel structures with a diameter of less than 20 microns and controllable porosity.

SUMMARY OF THE INVENTION

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; 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

It is still a challenge to construct three-dimensional complex microchannel structures with a diameter of less than 20 microns and controllable porosity using existing methods. At the same time, the microvascularization method based on the self-assembly principle has high requirements on the properties of the hydrogel environment and lacks versatility.

Technical Solutions

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

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

In one aspect, the present invention provides a bio-ink for promoting microvessel formation and a preparation method thereof.

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

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

2) planting vascular endothelial cells and perivascular cells on the surface of the filamentous microgel to obtain hydrogel microfilaments loaded with cells on the surface;

3) The surface-loaded cell hydrogel microfilaments are mixed with a solution of matrix phase material B to obtain a bio-ink that promotes microvessel formation.

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

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

In step 1), the operation of preparing a filamentous microgel with a diameter of less than 20 microns from a sacrificial phase material A is as follows: preparing a granular microgel of the sacrificial phase material A; preparing a hydrogel structure by an in-situ shearing method of the microgel in a continuous phase, in which the microfilaments (i.e., the filamentous microgel with a diameter of less than 20 microns) are oriented and arranged in the continuous phase material in a state similar to a fiber bundle; removing the continuous phase material while keeping the microfilaments in a gel state to obtain a filamentous microgel;

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

The diameter of the obtained particulate microgel can be 10-5000 microns;

The operation of preparing the hydrogel structure by the method of in-situ shearing of microgels in the continuous phase is as follows: the granular microgel of the sacrificial phase material A is mixed with the continuous phase material and loaded into a syringe, and under the condition of being higher than the sol temperature of the material A, the continuous phase continuously applies shear force to the granular microgel during the pushing process to cause it to undergo directional deformation, thereby obtaining filamentous microgels (microfilaments), and the microfilaments are directional arranged in a state similar to fiber bundles in the extruded continuous phase material;

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

Specifically, the continuous phase material may be: Pluronic;

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

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;

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;

The process of preparing the filamentous microgel with a diameter of less than 20 micrometers 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.

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.

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

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

The matrix phase material B may be selected from at least one of: methacryloyl gelatin (GelMA), alginate, hyaluronic acid, Matrigel, fibrinogen, and the like.

If the matrix phase material B is a photo-crosslinkable polymer, the solution also contains a photoinitiator.

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

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

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 solution of the matrix phase material B uses phosphate buffer as solvent;

The ratio of the hydrogel microfilaments loaded with cells on the surface to the solution of the matrix phase material B can be: 1g:2ml to 1g:20ml.

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.

In another aspect, the present invention also provides a capillary-like structure.

The capillary-like structure is prepared by a method comprising the following steps: mixing the filamentous microgel with the matrix phase material B for embedding vascular endothelial cells and perivascular cells, dissolving and solidifying 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.

The specific operation is as follows: a certain amount of vascular endothelial cells and perivascular cells are taken, resuspended in a solution of matrix phase material B, mixed with the filamentous microgel prepared by sacrificial phase material A, mixed evenly and cast in a template to fix the shape, photocrosslinked to solidify the matrix phase material B, immersed in a culture medium and cultured at a high temperature to dissolve the sacrificial phase material A therein and flow out, and the microgel is obtained;

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

The specific operation is as follows: the bio-ink promoting microvessel formation is cast in a template to be fixed, the matrix phase material B in the bio-ink is solidified by photo-crosslinking, and the sacrificial phase material A therein is dissolved and flowed out by immersing in a culture medium and culturing at a relatively high temperature;

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.

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

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

1) preparing two or more bio-inks respectively, wherein the two or more bio-inks contain hydrogel microfilaments with different diameters;

2) Bio-3D printing: Use a non-porous printing method that uses two or more bio-inks to alternately print and fill the gaps between them to build complex three-dimensional structures. After shaping and curing, the sacrificial phase material A is dissolved and flows out to achieve cross-scale microvascular construction.

Beneficial Effects

The present invention has the following advantages over the prior art:

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

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

3. Widely applicable to a variety of matrix phase materials.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG. 2 is a schematic diagram of a process for preparing a capillary-like structure using the bio-ink of the present invention.

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

FIG4 is a statistical diagram of the diameters of sacrificial phase filamentous microgels.

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

FIG. 6 is an observation diagram of gelatin microfilaments adhering to cells.

FIG. 7 is a staining image of capillary-like structures induced in a GelMA environment.

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

Embodiments of the present invention

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

According to the flow charts shown in Figures 1, 2 and 3, micron-sized pores are prepared and microvessel formation is promoted.

(1) Preparation of gelatin microspheres: Granular microgels were prepared by microfluidic chips. The faster oil phase sheared the lower 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 Span80 with a volume concentration of 2% 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.

(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 (the solvent was PBS buffer) at a ratio of 1 g:8 ml and loaded into a syringe. The mixture was pushed steadily at 32°C. The inner diameter of the syringe needle was 2 mm and the pushing speed was 10 mm/min. The continuous phase continuously applied shear force to the gel microspheres to cause them to deform in a directional manner, thereby obtaining filamentous microgels. The microfilaments were arranged in a directional manner in the extruded continuous phase material in a state similar to that of fiber bundles. The microfilaments were transferred to 4°C PBS buffer and gently washed to remove the continuous phase, thereby collecting the 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 seeps out of the screen as the centrifuge is centrifuged, and the microfilaments remain on the surface of the screen. After scraping and weighing, it can be used. After mixing with the liquid phase, it can be resuspended by blowing thoroughly.

FIG4 is a statistical diagram of the diameters of sacrificial phase filamentous microgels.

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

(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 cells/ml and the concentration of fibroblasts is 2.5*10 ⁶ cells/ml. Mix sterile gelatin microfilaments and the cell suspension at a ratio of 1 g:4 ml and blow evenly. Place in a metal bath at 20°C and incubate for 10 hours to achieve cell adhesion on the gelatin microfilaments.

FIG. 6 is an observation diagram of gelatin microfilaments adhering to cells.

(4) Preparation of capillary-like structures: Prepare a 5% GelMA solution with PBS buffer as the matrix phase, and mix it with 0.15% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator. Mix the cell-loaded gelatin microfilaments with the GelMA solution at a ratio of 1g:10ml and blow evenly. After casting in the template and fixing it, cross-link the GelMA with ultraviolet light at a wavelength of 405nm for 180 seconds. Then soak it in culture medium and place it in a 37°C incubator for culture. Change the culture medium every day. Gelatin microfilaments can achieve more than 80% dissolution after 24 hours of culture. Capillary-like structures can be observed on the third day of culture, and the structure remains stable on the fifth day of culture.

FIG. 7 is a staining image of capillary-like structures induced in a GelMA environment.

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

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

(1) Preparation of cell-loaded gelatin microfilaments: The same as steps (1) to (3) of Example 1 was used to prepare gelatin microfilaments with vascular endothelial cells and fibroblasts loaded on their surfaces.

(2) Preparation of bio-ink: Bio-ink was prepared by mixing cell-laden gelatin microfilaments with a GelMA solution with a mass concentration of 7.5% at a ratio of 1 g:10 ml. The mixture was mixed at 25 °C to prevent the gelatin microfilaments from dissolving.

(3) Extrusion bio-3D printing: The mixed bio-ink is transferred into a syringe and loaded onto an extrusion bio-3D printer. The sleeve temperature is controlled at 17°C and the base temperature is controlled at 10°C. After printing, the GelMA is solidified by UV cross-linking, immersed in culture medium and placed in a 37°C incubator for culture. The culture medium is changed every day and the porous hydrogel sample containing capillary-like structure is obtained after 3-7 days of culture.

(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, pre-cool it in a 10°C environment 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°C to remove the un-photocrosslinked materials and take out the printed structure. Use ultraviolet light to post-crosslink for 1 minute to fully solidify the structure, immerse it in culture medium and place it in a 37°C incubator for culture. Change the culture medium every day and culture it for 3-7 days to obtain a porous hydrogel sample containing a capillary-like structure.

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

(1) Preparation of bio-ink: Bio-ink A containing cell-laden gelatin microfilaments was prepared by the same steps (1)-(2) as in Example 2; and bio-ink B was prepared by mixing a gelatin solution with a mass concentration of 5% with vascular endothelial cells with a concentration of 1*10 ⁶ -1*10 ⁷ cells/ml.

(2) Biological 3D printing: Use two inks to alternately print and fill the gaps between each other to construct a complex three-dimensional structure. After printing, perform UV cross-linking, soak in culture medium and culture in a 37°C incubator for 3-7 days. The GelMA in ink A provides mechanical support for the overall structure after solidification, and the gelatin microfilaments inside are sacrificially dissolved to construct a vascular structure with a diameter of 15-20 microns in situ; the gelatin in ink B is sacrificially dissolved to construct a vascular structure with a diameter of 100-200 microns in situ, and the two inks are used to achieve cross-scale microvascular construction.

Industrial Applicability

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 (10)

A method for preparing a bio-ink that promotes microvessel formation comprises the following steps: 1) Prepare filamentous microgels with a diameter of less than 20 μm from sacrificial phase material A as pore-forming templates; 2) planting vascular endothelial cells and perivascular cells on the surface of the filamentous microgel to obtain hydrogel microfilaments loaded with cells on the surface; 3) The surface-loaded cell hydrogel microfilaments are mixed with a solution of matrix phase material B to obtain a bio-ink that promotes microvessel formation. The method according to claim 1, characterized in that: in step 1), the sacrificial phase material A has suitable temperature-sensitive properties, maintains a gel state at a lower temperature and can be sacrificially dissolved at a higher temperature, while supporting the adhesion of vascular endothelial cells and perivascular cells; In step 1), the operation of preparing filamentous microgels with a diameter of less than 20 microns from sacrificial phase material A is as follows: preparing granular microgels of sacrificial phase material A; preparing a hydrogel structure by in situ shearing of microgels in a continuous phase, in which the microfilaments are directionally arranged in the continuous phase material in a state similar to fiber bundles; removing the continuous phase material while keeping the microfilaments in a gel state to obtain a filamentous microgel. The method according to claim 2, characterized in that the diameter of the obtained granular microgel is 10-5000 microns; The operation of preparing the hydrogel structure by the method of in-situ shearing of microgels in the continuous phase is as follows: the granular microgel of the sacrificial phase material A is mixed with the continuous phase material and loaded into a syringe, and under the condition of being greater than the sol temperature of the material A, the continuous phase continuously applies shear force to the granular microgel during the pushing process to cause it to undergo directional deformation, thereby obtaining a filamentous microgel, and the microfilaments are directional arranged in a state similar to a fiber bundle in the extruded continuous phase material; The continuous phase material maintains extrudability within the operating temperature range and has poor miscibility with the sacrificial phase material A. The method according to claim 1 is characterized in that: in step 2), the operation of planting vascular endothelial cells and perivascular cells on the surface of the filamentous microgel comprises: preparing a cell suspension containing vascular endothelial cells and perivascular cells, transferring the filamentous microgel into the cell suspension, and incubating. The incubation condition is to incubate at a temperature lower than the sol temperature of material A for 3-15 hours. The method according to claim 1, characterized in that: in step 3), the matrix phase material B has good biocompatibility, can be stably cross-linked and cured, and has certain mechanical properties; The matrix phase material B is selected from at least one of methacrylated gelatin, alginate, hyaluronic acid, Matrigel, and fibrinogen; If the matrix phase material B is a photo-crosslinkable polymer, the solution also contains a photoinitiator. 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 loaded with cells on the surface to the solution of the matrix phase material B is: 1g:2ml to 1g:20ml. A bio-ink that promotes microvessel formation prepared by the method described in any one of claims 1 to 6. The use of the biological ink described in claim 7 in the preparation of capillary-like structures and the construction of cross-scale microvessels. A capillary-like structure is prepared by a method comprising the following steps: shaping and solidifying the matrix phase in the biological ink of claim 8, dissolving and flowing out the sacrificial phase material A therein, forming tubular pores while achieving in situ delivery of cells, thereby obtaining a capillary-like structure; or The filamentous microgel is mixed with the matrix phase material B that embeds the vascular endothelial cells and perivascular cells. After being shaped and solidified, the sacrificial phase material A therein is dissolved and flows out to form tubular pores. During the subsequent culture process, the cells migrate into the pores to form a capillary-like structure. A method for constructing cross-scale microvessels, comprising the following steps: 1) preparing two or more bio-inks respectively by the method according to any one of claims 1 to 6, wherein the diameters of the hydrogel microfilaments contained in the two or more bio-inks are different; 2) Bio-3D printing: Use two or more bio-inks to print alternately and fill the gaps between them to build complex three-dimensional structures. After shaping and curing, the sacrificial phase material A is dissolved and flows out to achieve cross-scale microvascular construction.