Myocardial tissue construct with bionic structure and function as well as preparation method and application thereof

Abstract

The invention relates to a myocardial tissue construct with a bionic structure and a function as well as a preparation method and application of the myocardial tissue construct. The myocardial tissue construct with both the bionic structure and the function comprises the following components: a hydrogel matrix suitable for 3D printing rheological properties, a cross-linking agent dispersed in the hydrogel matrix, inorganic biological material particles with myocardial protection and vascularization biological activity promotion, myocardial cells and vascularization related cells, the myocardial cells and the vascularization-related cells are regularly arranged in a three-dimensional space by simulating a cell distribution mode in a natural myocardial tissue; preferably, the regularly arranged structure comprises at least one of spatial arrangement of myocardial cell-angiogenesis related cells in a left-right / up-down side-by-side mode and spatial arrangement of myocardial cell-angiogenesis related cells in a core-shell / shell-core coaxial mode.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a myocardial tissue construct with bionic structure and function, and a preparation method and application thereof. Background Art

[0002] Overwork, excitement, and cold stimulation may induce acute myocardial infarction, causing irreversible necrosis of myocardial tissue and thus inducing death. At present, patients with large-area myocardial infarction can only undergo heart transplantation, but the source and number of donors are extremely limited. Therefore, reconstructing artificial myocardial tissue constructs that match the structure and function of natural myocardial tissue is expected to alleviate the urgent need for heart transplantation in patients with myocardial infarction and heart failure.

[0003] Bio-3D printed myocardial tissue constructs that mimic myocardial tissue have opened up new avenues for cardiac repair and myocardial infarction treatment. However, how to induce the functionality of biomimetic myocardial tissue constructs remains a key challenge. To effectively treat myocardial infarction, biomimetic myocardial tissue constructs need to have the following key functions: 1) activate myocardial cell activity and reconstruct myocardial function; 2) activate vasculogenic activity, reconstruct the vascular system and restore blood perfusion; 3) induce myocardial-vascular interaction.

[0004] Therefore, it is urgent to develop bionic myocardial constructs with multi-cellular bionic arrangements and capable of achieving the above key functions of myocardial tissue. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a bionic myocardial tissue construct with both myocardial function and vascularization activity, as well as a preparation method and application thereof. The bionic myocardial tissue construct has the properties of simulating the spatial arrangement structure of cells of natural myocardial tissue, being formed by 3D printing and maintaining cell survival; on the other hand, it can regulate myocardial maturity, synchronize contraction function, activate the activity of vascularization-related gene proteins, and stimulate the crosstalk and interaction between myocardial cells and vascular endothelial cells through the active ion signals generated by the bio-ink containing inorganic bioactive materials, thereby promoting the regeneration of damaged myocardial tissue and enhancing cardiac function.

[0006] In a first aspect, the present invention provides a myocardial tissue construct having both bionic structure and function, wherein the components of the myocardial tissue construct include: a hydrogel matrix having rheological properties suitable for 3D printing, and a cross-linking agent dispersed in the hydrogel matrix, inorganic biomaterial particles having myocardial protection and angiogenesis-promoting biological activities, myocardial cells, and angiogenesis-related cells; The cardiomyocytes and angiogenesis-related cells are regularly arranged in three-dimensional space, imitating the cell distribution pattern in natural myocardial tissue; preferably, the regularly arranged structure includes at least one of a left-right / up-down side-by-side spatial arrangement of cardiomyocytes and angiogenesis-related cells, and a core-shell / shell-core coaxial spatial arrangement of cardiomyocytes and angiogenesis-related cells.

[0007] Preferably, the hydrogel matrix includes at least one of methacrylated gelatin GelMA, methacrylated hyaluronic acid HAMA, gellan gum, sodium alginate, and polyethylene glycol; preferably, the methacryloyl substitution degree of the GelMA is 50-80%, and the mass concentration is 4-6%.

[0008] Preferably, the cross-linking agent is one of I2959, dimethyl paraphenylenediamine dinitrate VA-086, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate LAP, preferably LAP; The content of the crosslinking agent in the myocardial tissue construct is controlled to be 0.25-2wt% of the hydrogel matrix.

[0009] Preferably, the inorganic biomaterial particles contain ≥3 elements and ≥2 elements beneficial to myocardial protection or vascularization, and the elements beneficial to myocardial protection or vascularization include silicon, calcium, strontium, magnesium, copper, and zinc; preferably, the inorganic biomaterial is a strontium silicate inorganic material SS containing silicon and strontium elements; The particle size of the inorganic biomaterial particles is 10 nm to 10 μm; The content of the inorganic biomaterial particles in the myocardial tissue construct is 2-5% of the hydrogel matrix.

[0010] Preferably, the cardiomyocytes are primary extracted cardiomyocytes or cardiomyocytes differentiated from human pluripotent stem cells, preferably primary cardiomyocytes; the ratio of the number of cells to the volume of the hydrogel matrix is ​​2×10 7 ~4×10 7 / mL, preferably 3×10 7 Pieces / mL.

[0011] Preferably, the angiogenesis-related cells are at least one of aortic endothelial cells, umbilical vein endothelial cells, and smooth muscle cells, preferably aortic endothelial cells; the ratio of the number of cells to the volume of the hydrogel matrix is ​​1×10 6 ~5×10 6 / mL, preferably 3×10 6 Pieces / mL.

[0012] In a second aspect, the present invention provides a method for preparing the myocardial tissue construct having both bionic structure and function, the method comprising the following steps: (1) mixing inorganic biomaterial particles, a crosslinking agent, and a hydrogel matrix to obtain a hydrogel ink material; (2) mixing myocardial cells and the hydrogel ink material to obtain bio-ink A, and mixing angiogenesis-related cells and the hydrogel ink material to obtain bio-ink B; (3) Modeling is performed based on the cell distribution pattern in natural myocardial tissue and dual-channel extrusion coaxial or parallel bio-3D printing is performed using bio-ink A and bio-ink B to obtain a blank, which is then cured by photo-crosslinking to obtain the myocardial tissue construct having both bionic structure and function.

[0013] Preferably, in step (3), the pressure range of the two extrusion channels of the dual-channel extrusion coaxial or parallel biological 3D printing is 30-150 KPa, the temperature of the extrusion needle is 18-22°C, and the temperature of the deposition table is 0-10°C.

[0014] Preferably, in step (3), the two channels of the coaxial biological 3D printing are connected by a coaxial needle; preferably, the inner diameter of the shell needle is 300-600 μm, and the inner diameter of the core needle is 150-250 μm.

[0015] In a third aspect, the present invention provides an application of the above-mentioned myocardial tissue construct having both bionic structure and function in the preparation of myocardial cell-vascular related cell interaction materials and myocardial repair related materials in an in vitro three-dimensional microenvironment.

[0016] Beneficial Effects (1) The myocardial tissue construct with biomimetic structure and function provided by the present invention is expected to be applied to the treatment of heart diseases, effectively promoting the repair of myocardial damage and the recovery of cardiac function in vivo, and has good biological activity and biosafety; (2) The cell structure of the bionic myocardial tissue construct provided by the present invention simulates the structural characteristics of the intermingled capillaries of myocardial cells in the natural myocardium, and is achieved by regularly arranging myocardial cells and angiogenesis-related cells through multi-channel 3D printing technology; the bio-ink used to prepare the construct is prepared by combining inorganic biomaterials with myocardial protection and angiogenesis activity and biocompatible hydrogels, as well as myocardial / vascular related cells, to prepare an inorganic material-organic material-myocardial / vascular cell composite bio-ink, which has the functions of promoting myocardial maturation and synchronous contraction, inducing angiogenesis-related gene expression, and activating myocardial-vascular interaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1-3This is a fluorescence microscopic image of the myocardial tissue construct SS-Core-Shell prepared in Example 1; Figure 4 This is a fluorescence microscopic image of the myocardial tissue construct SS-Parallel prepared in Example 2; Figure 5 This is a fluorescence microscopic image of the myocardial tissue construct SS-Shell-Core prepared in Example 3; Figure 6 Optical photos of cardiomyocyte scaffolds printed with bio-ink containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%, 10%), and fluorescence microscopic photos and cell survival rate statistics after 1 and 7 days of culture; Figure 7 The synchronous contraction function characterization diagram of the cardiomyocyte scaffold printed with bio-ink containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%, 10%) after 7 days of culture; Figure 8 A characterization diagram of the maturation of cardiomyocyte scaffolds printed with bio-ink containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%, 10%) after 7 days of culture; Fig. 9 Characterization of myocardial-specific proteins and vascularization-related proteins in Core-Shell constructs printed with bio-inks containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%) after 7 days of culture; Fig.10 Characterization of myocardial maturation-related genes in Core-Shell constructs printed with bio-inks containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%) after 7 days of culture; Fig.11 Characterization of genes related to vascularization activity in Core-Shell constructs printed with bio-inks containing different concentrations of strontium silicate inorganic particles (0%, 2%, and 5%) after 7 days of culture; Fig.12 Fluorescence microscopy images of biomimetic myocardial tissue constructs constructed based on strontium silicate bio-ink with cardiomyocytes-aortic endothelial cells spatially arranged in three different patterns; Fig.13 The transient change maps of calcium ions and the corresponding peak times and peak time statistics of bionic myocardial tissue constructs with three different spatial arrangements of cardiomyocytes and aortic endothelial cells based on strontium silicate bio-ink; Fig.14 Characterization of the expression of myocardial maturation-related functional genes in biomimetic myocardial tissue constructs based on strontium silicate bio-ink with cardiomyocytes and aortic endothelial cells spatially arranged in three different patterns; Fig.15Characterization of vascularization-related gene expression in biomimetic myocardial tissue constructs based on strontium silicate bio-ink with cardiomyocytes-aortic endothelial cells spatially arranged in three different patterns; Fig.16 To characterize the promotion of cardiac function recovery in vivo by constructing biomimetic myocardial tissue constructs with three different spatial arrangements of cardiomyocytes and aortic endothelial cells based on strontium silicate bio-ink: (a) echocardiogram 4 weeks after surgery; (be) statistics of ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at diastole (LVIDd) and left ventricular internal diameter at systole (LVIDs) after 4 weeks; (fi) changes in EF, FS, LVIDd and LVIDs from 1 week to 4 weeks; Fig.17 Masson staining histological characterization of biomimetic myocardial tissue constructs based on strontium silicate bio-ink with three different spatial arrangements of cardiomyocytes and aortic endothelial cells to promote cardiac function recovery in vivo, and the corresponding statistical results of ventricular wall thickness and infarction ratio; Fig.18 To construct bionic myocardial tissue constructs with cardiomyocytes and aortic endothelial cells spatially arranged in three different patterns based on strontium silicate bio-ink, CD31 / α-SMA immunofluorescence staining was used to promote angiogenesis in vivo, and the corresponding statistical results of arterial vascular density and total vascular density were obtained. DETAILED DESCRIPTION

[0018] The present invention is further described below by the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0019] First, the present invention provides a myocardial tissue construct with both bionic structure and function. The components of the myocardial tissue construct may include: a hydrogel matrix with rheological properties suitable for 3D printing, a cross-linking agent dispersed in the hydrogel matrix, inorganic biomaterial particles with myocardial protection and angiogenesis-promoting biological activities, myocardial cells, and angiogenesis-related cells; The cardiomyocytes and angiogenesis-related cells are regularly arranged in three-dimensional space, imitating the cell distribution pattern in natural myocardial tissue; preferably, the structural characteristics of the regular arrangement can include at least one of the following: the cardiomyocytes-angiogenesis-related cells are spatially arranged in a left-right / up-down side-by-side pattern, and the cardiomyocytes-angiogenesis-related cells are spatially arranged in a core-shell / shell-core coaxial pattern.

[0020] In some embodiments, the hydrogel matrix can be a hydrogel material that has a gel state at room temperature, has a rheological property that the viscosity decreases with shear force, and can be formed by photocrosslinking or ionic crosslinking; preferably, the hydrogel matrix can include at least one of methacrylated gelatin GelMA, methacrylated hyaluronic acid HAMA, gellan gum, sodium alginate, and polyethylene glycol; more preferably, the methacryloyl substitution degree of the GelMA can be 50-80%, and the mass concentration can be 4-6% (e.g., 5%).

[0021] Among them, the hydrogel matrix used in the present invention is a hydrogel material having a gel state at room temperature, a rheological property that the viscosity decreases with shear force, and can be formed by photocrosslinking or ion crosslinking. If these properties are not possessed, it is impossible to prepare and form by the 3D printing method described later. At the same time, if the methacryloyl substitution degree of GelMA used in the present invention is too low, the GelMA ink cannot be formed by the photocrosslinking method described later, and is thus not suitable for the preparation and forming method of 3D printing; if the substitution degree is too high, the hydrogel network will be too tightly crosslinked, which is not conducive to the material exchange and migration of cells. In addition, if the mass concentration of the GelMA hydrogel is too high, the hydrogel will be too hard and the polymer network will be too dense, which is not suitable for cell survival; if the concentration is too low, the polymer network will be too sparse, and it will be difficult to tightly crosslink to provide sufficient mechanical support for cells.

[0022] In some embodiments, the crosslinking agent can be one of I2959, dimethyl paraphenylenediamine dinitrate VA-086, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate LAP, preferably LAP; the content of the crosslinking agent in the myocardial tissue construct can be controlled to be 0.25-2wt% of the hydrogel matrix.

[0023] In some embodiments, the inorganic biomaterial particles may contain ≥3 constituent elements and ≥2 elements beneficial to myocardial protection or vascularization, and the elements beneficial to myocardial protection or vascularization may include silicon, calcium, strontium, magnesium, copper, and zinc; preferably, the inorganic biomaterial is a strontium silicate inorganic material SS containing silicon and strontium elements.

[0024] Wherein, the particle size of the inorganic biomaterial particles can be 10 nm to 10 μm. If the particle size is too large, the structure of the hydrogel matrix in the bio-ink will be destroyed; if the particle size is too small, it is easy to agglomerate, which is not conducive to the uniform dispersion of the inorganic material particles in the hydrogel matrix.

[0025] Wherein, in the myocardial tissue construct, the content of inorganic biomaterial particles can be 2-5% of the hydrogel matrix (such as GelMA). If the content is too low, the concentration of released ions is insufficient to produce the activity that promotes myocardial-vascular activity and its interaction; if the content is too high, excessive inorganic ions will be released, which will have a negative induction effect on cells.

[0026] In some embodiments, the cardiomyocytes may be primary extracted cardiomyocytes or cardiomyocytes differentiated from human pluripotent stem cells, preferably primary cardiomyocytes; the ratio of the number of cells to the volume of the hydrogel matrix may be 2×10 7 ~4×10 7 / mL, preferably 3×10 7 / mL. Controlling the cell concentration within this range can better maintain the survival of cardiomyocytes and induce their maturation. If the cell concentration is too high, it is difficult to obtain sufficient adhesion space and sufficient nutrients inside the hydrogel matrix of the bio-ink, making it difficult to maintain survival for a long time; if the cell concentration is too low, it will be difficult to maintain its survival due to low cell density and lack of cell-to-cell contact and interaction.

[0027] In some embodiments, the angiogenesis-related cells may be at least one of aortic endothelial cells, umbilical vein endothelial cells, and smooth muscle cells, preferably aortic endothelial cells; the ratio of the number of cells to the volume of the hydrogel matrix may be 1×10 6 ~5×10 6 / mL, preferably 3×10 6 The cell concentration is controlled within this range to better maintain the activity of cells participating in vascularization. If the cell concentration is too high, it will be difficult to obtain sufficient angiogenic space on the surface or inside of the stent, making it difficult to maintain its vascularization activity for a long time; if the cell concentration is too low, it will be difficult to maintain survival due to low cell density.

[0028] The myocardial cells and angiogenesis-related cells contained in the myocardial tissue construct with both bionic structure and function provided by the present invention can mature and differentiate under the induction of inorganic biomaterials, and at the same time induce the repair of myocardial tissue through intercellular communication and restore cardiac function. At the same time, the hydrogel component can provide mechanical structure and support for the cells therein, which is convenient for the cells to absorb nutrients; the inorganic biomaterial can release bioactive ions such as silicon, calcium, strontium, magnesium, copper, zinc, etc. that are beneficial in myocardial protection or vascularization, promote the maturation of myocardial cells and realize synchronous contraction and other functions, while activating vascularization activity and myocardial-vascular interaction, and efficiently promote the repair of myocardial tissue and the recovery of cardiac function.

[0029] It should also be noted that: (1) angiogenic cells and strontium silicate bio-ink have a synergistic effect. Angiogenic cells can directly participate in the angiogenesis process, while strontium silicate bio-ink has the activity of promoting and accelerating angiogenesis. Therefore, the two complement each other and synergistically promote angiogenesis; (2) angiogenic cells and cardiomyocytes play a synergistic role through cell communication modes such as paracrine. On the basis of participating in angiogenesis, angiogenic cells can also release specific factors to promote cardiomyocyte activity and reduce cardiomyocyte apoptosis under hypoxic conditions.

[0030] The following is an exemplary description of the preparation method of the myocardial tissue construct with both bionic structure and function provided by the present invention. The preparation method may include the following steps: (1) mixing inorganic biomaterial particles, a crosslinking agent, and a hydrogel matrix to obtain a hydrogel ink material; (2) mixing myocardial cells and the hydrogel ink material to obtain bio-ink A, and mixing angiogenesis-related cells and the hydrogel ink material to obtain bio-ink B; (3) Modeling is performed based on the cell distribution pattern in natural myocardial tissue and dual-channel extrusion coaxial or parallel bio-3D printing is performed using bio-ink A and bio-ink B to obtain a blank, which is then cured by photo-crosslinking to obtain the myocardial tissue construct having both bionic structure and function.

[0031] In some embodiments, in step (1), the inorganic biomaterial particles may be sterilized at high temperature and high pressure before mixing; the mixing method may be to add the hydrogel matrix solution into the container containing the inorganic biomaterial particles and mix them. Otherwise, the inorganic biomaterial particles may not be evenly dispersed in the hydrogel matrix solution.

[0032] In some embodiments, in step (1), the method for preparing the hydrogel matrix may include the following process: dissolving a dry gel (such as GelMA dry gel) in a culture medium (such as DMEM high-glucose basal culture medium), heating (such as 60° C.) and stirring (such as 30 minutes) until completely dissolved; filtering and sterilizing the obtained uniform liquid through a filter (such as a 0.22 μm filter), and keeping the sterile hydrogel matrix solution warm for standby use (such as 37° C.).

[0033] In some embodiments, in step (3), the pressure range of the two extrusion channels of the dual-channel extrusion coaxial or parallel biological 3D printing can be 30-150 KPa, the temperature of the extrusion needle can be 18-22°C, and the temperature of the deposition table can be 0-10°C.

[0034] In some embodiments, in step (3), the two channels of the coaxial biological 3D printing are connected by a coaxial needle; preferably, the inner diameter of the shell needle is 300-600 μm, and the inner diameter of the core needle is 150-250 μm.

[0035] In some embodiments, in step (3), the photo-crosslinking curing time may be 15 to 30 seconds.

[0036] The present invention uses the above composition and method for the first time to prepare a bionic myocardial tissue construct, and realizes the spatial structural arrangement of myocardial and vascular-related cells in the construct to simulate the regularity of natural myocardial tissue. At the same time, based on the bio-ink material mentioned in the present disclosure, it can support the long-term survival and physiological functions of cells in the construct, can induce physiological functions such as maturation and synchronous contraction of myocardial cells, and can effectively promote the expression of vascularization-related gene proteins in vascular cells.

[0037] In the present disclosure, the cardiomyocytes and vascular-related cells in the construct can maintain a high survival rate during the culture process. The combination of inorganic materials / hydrogels / myocardial cells and vascular cells in a specific structural pattern in three-dimensional space enables the construct to have the characteristics of simultaneously simulating the structure of myocardial tissue and exerting its physiological functions, and has the potential to effectively treat heart-related diseases, repair the myocardium, and restore heart function.

[0038] The 3D printed bionic myocardial tissue construct obtained by the preparation method provided by the present invention has a bionic myocardial tissue cell structure and myocardial tissue function, and can be used to prepare myocardial cell-vascular related cell interaction materials under an in vitro three-dimensional microenvironment and to treat heart-related diseases and promote myocardial repair.

[0039] The following further examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all belong to the scope of protection of the present invention. The specific process parameters of the following examples are also only examples within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description of this article, and are not limited to the specific values ​​​​exemplified below. If not specifically stated, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0040] Example 1

[0041] The method for preparing the strontium silicate-cardiac cell-aortic endothelial cell shell-core three-dimensional spatial arrangement bionic myocardial tissue construct with both bionic structure and function provided in this embodiment comprises the following steps: (1) Preparation of hydrogel ink materials: a. Preparation of strontium silicate inorganic biomaterial particles using a solvothermal method: Weigh 1.058 g of Sr(NO 3 ) 2 and 1.42 g of Na 2 SiO 3 , respectively, were dissolved in 25 mL of deionized water; then, Sr(NO 3 ) 2 The solution was slowly dripped into Na 2 SiO 3 The solution was added dropwise while stirring until 25 mL of Sr(NO 3 ) 2 After the solution was added dropwise, the cup was sealed and gently stirred for 10 minutes using a magnetic stirrer. Then, 50 mL of anhydrous ethanol was measured and poured into the solution, and stirring was continued for 20 minutes. The stirred mixed solution was transferred to a 50 mL hydrothermal autoclave liner and reacted at 160°C for 8 hours. After the reaction was completed, it was naturally cooled. After cooling to room temperature, the supernatant in the hydrothermal autoclave liner was poured out, and the white precipitate at the bottom was transferred to a 50 mL centrifuge tube. Deionized water was added to the centrifuge tube containing the white precipitate for ultrasonic cleaning for 15 minutes, and then centrifuged at 8000 rpm for 10 minutes. After repeating the ultrasonic cleaning with deionized water and centrifugation for 3 times, the deionized water was changed to anhydrous ethanol and the cleaning was continued for 3 times. Finally, it was placed in a vacuum drying oven and dried at 60°C for 24 hours. b. Preparation of strontium silicate composite methacryloyl gelatin (GelMA) ink material: 10 g of gelatin particles were dissolved in 100 mL of deionized water at 50°C; 5 mL of methacrylic anhydride solution was gradually added to the gelatin solution at 50°C and stirred for 1.5 hours; after the reaction, the mixed solution was centrifuged at 3500 rpm for 3 min, the supernatant obtained by centrifugation was collected and added to about 3 times the volume of deionized water; dialyzed at 40°C using a 12-14 kDa dialysis bag for 7 days, during which the water was changed 3 times a day; finally, the dialyzed solution was freeze-dried to obtain GelMA seawater. sponge; then, GelMA (6wt.%) and 0.3wt.% of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were dissolved in DMEM basal medium (high glucose) at 60°C; then, the obtained GelMA solution was sterilized by passing through a 0.22μm filter; thereafter, strontium silicate particles sterilized by ultraviolet irradiation were added to the GelMA solution to obtain a hydrogel ink material SS-GelMA (the amount of strontium silicate particles added accounted for 2% and 5% of the mass of the GelMA sponge, forming 2SS-GelMA and 5SS-GelMA composite bio-inks); (2) Preparation of bio-inks A and B: Rat primary cardiomyocytes (RCMs) were extracted from rat pups aged 24-48 hours after birth, and rat aortic endothelial cells (RAECs) were extracted from SD rats (150±20 g, female). The extracted primary RCMs and RAECs were cultured in high-glucose DMEM medium (DMEM, Gibco, USA) supplemented with 15% v / v fetal bovine serum (FBS), 1% v / v endothelial cell growth factor / heparin (ECGS / H) and 1% penicillin-streptomycin (P / S). RCMs were directly dispersed in the bio-inks of each component after extraction, and RAECs were collected and dispersed in the bio-inks of each component after amplification culture to 5-6 generations for 3D printing. 300 μL of 3×10 7 The cell suspension of 10 RCMs was added into 3 mL of SS-GelMA bio-ink and mixed evenly to form cell-carrying bio-ink A. At the same time, 300 μL of 3×10 6 The cell suspension of RAECs was added into 3 mL of SS-GelMA bio-ink and mixed evenly to form cell-laden bio-ink B; (3) 3D printing to prepare myocardial tissue constructs with both bionic structure and function: The entire 3D printing process was carried out in an ultra-clean bench; ink A was transferred to the shell barrel corresponding to the core-shell printing channel on the printer and placed at 4°C to cool for 20 minutes; ink B was transferred to the core barrel corresponding to the core-shell printing channel on the printer and placed at 4°C to cool for 20 minutes; then a core-shell extrusion needle with an inner diameter of 260 / 510 μm was installed on the two barrels and placed on the biological 3D printer for printing; the temperature of the barrel was set to 18°C, the extrusion pressure range was between 30kPa-60kPa, and the temperature of the printer receiving platform was set to 4°C; after printing, it was placed under 405nm light for crosslinking for 15s; finally, the construct was placed in a culture well plate and supplemented with culture medium at 37°C, 5% CO 2 The myocardial tissue construct of cardiomyocytes-aortic endothelial cells with a shell-core spatial distribution structure was named SS-Core-Shell construct.

[0042] Figure 1-3 This is a fluorescent microscopic image (2SS-GelMA) of the myocardial tissue construct SS-Core-Shell prepared in Example 1. As can be seen from the figure, the myocardial cells-aortic endothelial cells in the construct are spatially arranged in a regular shell-core pattern.

[0043] Example 2

[0044] The preparation method of the strontium silicate-cardiac cells-aortic endothelial cells parallel arranged in three-dimensional space bionic myocardial tissue construct with both bionic structure and function provided in this embodiment refers to that in Example 1, the main difference is that: In step (3), ink A is transferred to the barrel corresponding to channel 1 on the printer and placed at 4°C for cooling for 20 minutes; ink B is transferred to the barrel corresponding to channel 2 on the printer and placed at 4°C for cooling for 20 minutes; then, an inner diameter of 210 μm is installed on the two barrels and placed on the biological 3D printer for printing; the temperature of the barrel is set to 18°C, the extrusion pressure range is between 30kPa-60kPa, and the temperature of the printer receiving platform is set to 4°C; the 3D printing program is set to 1, 2 two channels and discharge the filaments to obtain a construct with two cells arranged side by side; after the two barrels are printed, they are placed under 405nm light for crosslinking for 15s; finally, the construct is placed in a culture well plate and supplemented with culture medium at 37°C and 5% CO 2 The cells were cultured in a humidified incubator; the myocardial tissue construct of cardiomyocytes-aortic endothelial cells with a shell-core spatial distribution structure was named SS-Parallel construct.

[0045] Figure 4 This is a fluorescent microscopic image of the myocardial tissue construct SS-Parallel (2SS-GelMA) prepared in Example 2. As can be seen from the figure, the two types of cells in the construct are spatially arranged in a regular left-right parallel pattern.

[0046] Example 3

[0047] The preparation method of the strontium silicate-cardiac cell-aortic endothelial cell core-shell three-dimensional spatial arrangement bionic myocardial tissue construct with both bionic structure and function provided in this embodiment refers to Example 1, with the main difference being: In step (3), ink A is transferred to the core barrel corresponding to the core-shell printing channel on the printer, and ink B is transferred to the shell barrel corresponding to the core-shell printing channel on the printer; the dual-cell construct with this distribution pattern is named SS-Shell-Core construct.

[0048] Figure 5 This is a fluorescent microscopic image (2SS-GelMA) of the myocardial tissue construct SS-Shell-Core prepared in Example 3. As can be seen from the figure, the myocardial cells-aortic endothelial cells in the construct are spatially arranged in a regular core-shell pattern.

[0049] Below, we explore the application of strontium silicate bio-ink materials in regulating myocardial function: Referring to the method of step (1) in Example 1, SS-free GelMA bio-ink, 2SS-GelMA, 5SS-GelMA, and 10SS-GelMA composite bio-ink were prepared respectively, and cardiomyocytes were loaded for 3D printing. First, live / dead cell staining was used to evaluate the survival of Rat-CMs in 3D printed cardiomyocyte scaffolds based on strontium silicate bio-ink (GelMA, 2SS-GelMA, 5SS-GelMA, 10SS-GelMA): the scaffolds were incubated with live / dead cell staining reagents for 30 minutes on the 1st and 7th days of scaffold culture, and then the scaffolds were photographed using a fluorescent inverted microscope. Secondly, after 4 and 7 days of culture, the cardiomyocyte scaffolds were treated with a calcium ion probe kit (Fluo-4, AM, Invitrogen), and then the calcium transient signals of cardiomyocytes in the constructs were observed and recorded under 488nm excitation light. In addition, real-time fluorescence quantitative polymerase chain reaction qPCR was used to characterize the expression of cardiomyocyte maturation-related genes in the cardiomyocyte scaffolds printed with the above four bio-inks. The expressions of related genes such as calcium voltage-gated channel protein (CACNA1A), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), and cardiac troponin 2 (TNNT2) were analyzed.

[0050] Figure 6 The optical photos of the cardiomyocyte scaffolds printed with bio-ink containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%, 10%), the fluorescence micrographs after 1 and 7 days of culture, and the statistical chart of cell survival rate. It can be seen from the figure that the cells are evenly distributed on the scaffolds, tightly attached to the scaffolds, and the cell survival rate is always greater than 90%, indicating that the cells are in good survival state.

[0051] Figure 7 The figure shows the synchronous contraction function of cardiomyocyte scaffolds printed with bio-inks containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%, 10%) after 7 days of culture. As can be seen from the figure, the cardiomyocyte scaffolds printed with 2SS-GelMA and 5SS-GelMA inks show regular and consistent calcium transients and synchronous contraction signals, while the signals of GelMA and 10SS-GelMA are more irregular and have a lower beating frequency, indicating that 2SS-GelMA and 5SS-GelMA bio-inks have a beneficial effect on promoting the synchronous contraction function of cardiomyocytes.

[0052] Figure 8The figure shows the maturation of cardiomyocyte scaffolds printed with bio-inks containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%, 10%) after 7 days of culture. As can be seen from the figure, 2SS-GelMA and 5SS-GelMA inks significantly promoted the expression of genes related to cardiomyocyte maturation, indicating that 2SS-GelMA and 5SS-GelMA bio-inks have a beneficial effect on promoting the phenotype of cardiomyocyte maturation.

[0053] The following is an exploration of the application of strontium silicate bio-ink in regulating the myocardial function and vascularization activity of biomimetic myocardial tissue constructs with myocardial cells and aortic endothelial cells arranged in shell-core three-dimensional space: As described above, constructs were prepared using GelMA ink without strontium silicate, 2SS-GelMA ink, and 5SS-GelMA ink, respectively, and named Co-GelMA, Co-2SS-GelMA, and Co-5SS-GelMA. First, the expression of myocardial maturation / vascularization-related proteins was characterized: after 7 days of culture, each group of constructs was fixed with 4% paraformaldehyde solution for more than 30 minutes and washed with PBS three times; then, the scaffolds were permeated with 0.5% Triton X-100 solution and blocked with 5% BSA solution at room temperature for 30 minutes; then, the constructs were incubated with the primary antibody solution of CX43 and α-actinin (1:500) at 4°C overnight; after PBS washing, they were continued to be incubated with the corresponding secondary antibody solution and diamidinophenylindole (DAPI) solution for 45 minutes, and washed with PBS solution three times; finally, they were observed and photographed under a laser confocal microscope. Secondly, the related physiological activities of the constructs under the influence of strontium silicate bio-ink were evaluated. First, the expression of genes related to cardiomyocyte maturation and vascularization activity in the constructs was detected and analyzed by qPCR. The expression of myocardial maturation-related genes: calcium voltage-gated channel protein (CACNA1A), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), cardiac troponin 2 (TNNT2) and other related genes were analyzed. On the other hand, the expression of vascularization-related genes: vascular endothelial growth factor (VEGF), vascular endothelial cadherin (VE-cad), endothelial nitric oxide synthase (eNOs) related genes were analyzed.

[0054] Fig. 9The Core-Shell constructs printed with bio-inks containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%) are characterized by myocardial specific proteins and vascularization-related proteins after 7 days of culture. As can be seen from the figure, both specific proteins are significantly expressed and evenly distributed, proving that in the Core-Shell construct, myocardial cells and aortic endothelial cells coexist and stably express corresponding functional proteins; in addition, the semi-quantitative statistics of CD31 expression indicate that 2% and 5% strontium silicate bio-inks can significantly promote vascularization activity.

[0055] Fig.10 The core-shell constructs printed with bio-inks containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%) are characterized by myocardial maturation-related genes after 7 days of culture. As can be seen from the figure, the expression of myocardial cell maturation-related genes in both the Co-2SS-GelMA and Co-5SS-GelMA groups was significantly increased, proving that under the condition of dual cell coexistence, 2SS-GelMA and 5SS-GelMA bio-inks can also significantly promote the expression of myocardial cell maturation-related genes, among which the promotion effect of 2SS-GelMA ink is more significant.

[0056] Fig.11 The figure shows the characterization of vascularization activity-related genes in the Core-Shell construct printed with bio-ink containing different concentrations of strontium silicate inorganic particles (0%, 2%, 5%) after 7 days of culture. As can be seen from the figure, the expression of vascularization-related genes in both the Co-2SS-GelMA and Co-5SS-GelMA groups was significantly increased, proving that under the condition of dual cell coexistence, 2SS-GelMA and 5SS-GelMA bio-inks can significantly promote the expression of vascularization-related genes and enhance the vascularization activity of the construct.

[0057] Below, the application of the above three bionic myocardial tissue constructs in regulating myocardial function and vascularization activity is explored: In order to explore whether the above bionic myocardial tissue constructs can realize the functions of bionic myocardial tissue, as well as the laws of their regulation of myocardial function and vascularization activity, bionic myocardial tissue constructs with three different cell spatial arrangement patterns as mentioned above were prepared, and their cell activity, synchronous contraction function and vascularization activity were explored. (1) To investigate whether the cell distribution in the three different distribution pattern constructs changes during the culture process, the three multicellular constructs were taken on the day after printing and after 7 days of culture, and one of the diameters was observed respectively. Fig.12Fluorescence microscopic images of biomimetic myocardial tissue constructs with three different spatial arrangements of cardiomyocytes and aortic endothelial cells based on strontium silicate bio-ink. As can be seen from the figure, the SS-Core-Shell construct with a core-shell distribution of aortic endothelial cells in the inner diameter and cardiomyocytes in the outer diameter basically maintains the original core-shell distribution pattern after 7 days, and the density of endothelial cells in the middle decreases, which may be due to the fact that the cell viability has decreased to a certain extent due to being wrapped in the bio-ink; the SS-Parallel construct with endothelial cells and cardiomyocytes distributed in a side-by-side mode also basically maintains the original left-right distribution after 7 days, and the two cells overlap to a certain extent in the middle part, which is caused by the proliferation and surface migration of the two cells during the culture process. In the SS-Shell-Core construct with a shell-core distribution in which cardiomyocytes are located at the outer diameter and endothelial cells are located at the inner diameter, almost no cells were observed at the middle core position after 7 days, while a small number of green cardiomyocytes were observed at the outer diameter position. This may be because cardiomyocytes have a high demand for oxygen and nutrients. If they are wrapped in hydrogels, their survival and growth may be severely hindered. Only a small number of cells obtain nutrients by migrating to the outer diameter. (2) Study the effects and rules of the 3D spatial distribution of the two cells on the activity of the two cells. First, the activity of the three constructs' synchronous contraction was characterized using a calcium ion probe. Fig.13 The transient change graphs of calcium ions and the corresponding statistical results of peak times and peak times for biomimetic myocardial tissue constructs with three different spatial arrangements of cardiomyocytes and aortic endothelial cells based on strontium silicate bio-ink. As can be seen from the figure, the SS-Core-Shell construct and the SS-Parallel construct show synchronous pulsation with consistent rhythm, while the SS-Shell-Core construct only shows irregular calcium ion signals in individual positions; through statistical analysis of the calcium signal graph, it can be seen that the construct with the Core-Shell distribution pattern has the most peak times and the shortest peak time, indicating that the construct with this distribution pattern has the best synchronous contraction activity, which may be due to the fact that the cardiomyocytes are distributed on the outside of the entire construct, which can better obtain and exchange oxygen and nutrients, and thus show the best functionality of cardiomyocyte contraction. This result shows that the cell arrangement in the biomimetic myocardial tissue construct has a regulatory effect on myocardial function. (3) Further analyze the expression of genes related to myocardial function and vascularization in three multicellular constructs with different distribution patterns. Fig.14Characterization of the expression of myocardial maturation-related functional genes in biomimetic myocardial tissue constructs with three different spatial arrangements of cardiomyocytes and aortic endothelial cells based on strontium silicate bio-ink. As can be seen from the figure, the expression of Cacna1a and TNNT2 genes in the SS-Core-Shell group was significantly higher than that in the SS-Shell-Core group. Fig.15 Characterization of the expression of vascularization-related genes in biomimetic myocardial tissue constructs with three different spatial arrangements of cardiomyocytes and aortic endothelial cells based on strontium silicate bio-ink. As can be seen from the figure, the expression of vascularization-related genes in the SS-Shell-Core group is much higher than that in the remaining two groups. Through the above characterization analysis, it can be concluded that the construct with the SS-Core-Shell distribution pattern has the best myocardial activity and function, while the construct with the SS-Shell-Core distribution pattern has the best vascularization activity; and the SS-Parallel distribution pattern has intermediate myocardial activity and angiogenic activity, which further proves that the cell arrangement in the biomimetic myocardial tissue construct has a regulatory effect on myocardial function and vascularization activity.

[0058] Below, we explore the application of the above three bionic myocardial tissue constructs in the treatment of myocardial infarction: Myocardial infarction (MI) model was established using male SD rats weighing 225-250 g and randomly divided into 6 groups: sham group (Sham, n = 3), myocardial infarction group (MI, n = 5), GelMA-Core-Shell group (n = 5) in which aortic endothelial cells-cardiomyocytes were distributed in core-shell space printed with GelMA ink without strontium silicate, SS-Core-Shell group (n = 5) printed with strontium silicate ink, SS-Parallel group (n = 5) and SS-Shell-Core group (n = 5). Myocardial infarction model was induced by ligating the left anterior descending coronary artery (LAD), and all constructs (diameter: 15 mm, height: 1 mm) were cultured for 7 days before implantation. For the sham group, rats only underwent thoracotomy, while rats in the other groups underwent LAD ligation after thoracotomy; for the GelMA-Core-Shell, SS-Core-Shell, SS-Parallel, and SS-Shell-Core groups, the printed constructs were implanted into the infarct site 15 minutes after ligation and fixed to the heart using fibrin glue. Cardiac function was monitored by B-ultrasound 1 week and 4 weeks after surgery. Four weeks after surgery, the rats were killed and their hearts were collected. The hearts were embedded and sliced, and the repair effect was evaluated by Masson trichrome staining and immunofluorescence staining.

[0059] Fig.16To characterize the promotion of cardiac function recovery in vivo by constructing biomimetic myocardial tissue constructs with three different spatial arrangements of cardiomyocytes and aortic endothelial cells based on strontium silicate bio-ink: (a) is the echocardiogram at 4 weeks after surgery; (be) is the statistics of ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at diastole (LVIDd) and left ventricular internal diameter at systole (LVIDs) after 4 weeks; (fi) is the changes of EF, FS, LVIDd and LVIDs from 1 week to 4 weeks. It can be seen from the echocardiogram and its statistical results in the figure that after 4 weeks of implantation, certain contraction waves appeared in the left ventricular wall of each group of implanted constructs. According to the echocardiogram, several important parameters reflecting cardiac function were analyzed: ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at diastole (LVIDd) and left ventricular internal diameter at systole (LVIDs). First, the SS-Core-Shell group had the best cardiac function performance after 4 weeks, followed by the SS-Parallel group. By comparing the GelMA-Core-Shell and SS-Core-Shell groups, it can be seen that SS bio-ink significantly enhanced the repair function of the construct, significantly improved the EF and FS of the rat heart, and inhibited left ventricular dilatation. By comparing the SS-Core-Shell, SS-Parallel, and SS-Shell-Core groups, it can be seen that the spatial arrangement in the construct significantly affects the repair effect of the construct. Among them, both the SS-Core-Shell and SS-Parallel groups had significant improvements in cardiac function, and the SS-Core-Shell construct had the best effect. However, SS-Shell-Core did not show significant improvement in cardiac function.

[0060] Four weeks after implantation, the rat hearts were harvested and their myocardial tissue repair was analyzed by histology. Fig.17 The Masson staining histological characterization and the corresponding statistical results of ventricular wall thickness and infarction ratio of biomimetic myocardial tissue constructs with three different spatial arrangements of cardiomyocytes and aortic endothelial cells constructed based on strontium silicate bio-ink to promote cardiac function recovery in vivo were performed. Fig.17Masson's Trichrome staining characterized the histological morphology and fibrosis state of rat myocardial infarction sections. It can be seen that the left ventricular wall of the MI group was significantly fibrotic and necrotic, and the ventricular dilatation was obvious. The other groups implanted with constructs more or less inhibited the fibrosis and necrosis of the left ventricle and ventricular dilatation. By comparing the GelMA-Core-Shell and SS-Core-Shell groups, it can be seen that the SS bio-ink significantly enhanced the repair function of the construct and inhibited the degree of fibrosis and thinning of the left ventricle. By comparing the SS-Core-Shell, SS-Parallel, and SS-Shell-Core groups, it can be seen that the spatial arrangement in the construct also significantly affected the repair effect of the construct. Among them, both the SS-Core-Shell and SS-Parallel groups had a significant inhibitory effect on the infarct size and ventricular wall thinning. Among them, the SS-Core-Shell construct had the best effect, effectively inhibiting the adverse remodeling of the ventricular wall and promoting its repair within 4 weeks. The SS-Shell-Core had the worst repair effect, and obvious large-area fibrotic tissue could be observed in the left ventricle.

[0061] In addition, the effect of the constructs in promoting angiogenesis was analyzed by CD31 and α-smooth muscle actin (α-SMA) immunofluorescence staining. Fig.18CD31 / α-SMA immunofluorescence staining characterization of biomimetic myocardial tissue constructs with three different spatial arrangements of cardiomyocytes and aortic endothelial cells based on strontium silicate bio-ink to promote angiogenesis in vivo and the corresponding statistical results of arterial vascular density and total vascular density. As can be seen from the figure, compared with MI, each patch group promoted angiogenesis. By comparing the GelMA-Core-Shell and SS-Core-Shell groups, it can be seen that SS bio-ink significantly enhanced angiogenesis, both microvessels (CD31 only) and arterial vessels (CD31 and α-SMA co-labeled) increased significantly under the stimulation of SS. By comparing the SS-Core-Shell, SS-Parallel, and SS-Shell-Core groups, it can be seen that the spatial arrangement of cells in the construct also significantly affected its angiogenesis effect. Among them, the SS-Core-Shell and SS-Parallel groups had similar angiogenesis-promoting effects, and SS-Shell-Core was slightly worse. This may be due to the poor myocardial repair effect of the SS-Shell-Core construct. Due to the complementary and mutually reinforcing effect between the myocardium and blood vessels, the SS-Shell-Core construct cannot promote the formation of more blood vessels in the harsh fibrotic myocardial environment. The vascularization effect of the SS-Parallel group was slightly better than that of the SS-Core-Shell group. This may be due to the better activity of aortic endothelial cells in SS-Parallel, which promoted the formation of blood vessels in the myocardium, especially arterial blood vessels.

[0062] Through the above analysis of the characterization results of in vivo myocardial infarction repair, we can conclude that the biological ink containing strontium silicate significantly promotes the in vivo repair effect of the construct. At the same time, the spatial arrangement of cells in the construct also has a significant regulatory and promoting effect on its in vivo repair effect. In summary, the myocardial tissue construct with both bionic structure and function prepared by the present invention effectively treats myocardial infarction, promotes the repair of myocardial tissue and the reconstruction of cardiac function.

[0063] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A myocardial tissue construct with both bionic structure and function, characterized in that: The components of the myocardial tissue construct include: a hydrogel matrix with rheological properties suitable for 3D printing, a cross-linking agent dispersed in the hydrogel matrix, inorganic biomaterial particles with myocardial protection and angiogenesis-promoting biological activities, myocardial cells, and angiogenesis-related cells; The cardiomyocytes and angiogenesis-related cells are regularly arranged in three-dimensional space, imitating the cell distribution pattern in natural myocardial tissue; preferably, the regularly arranged structure includes at least one of a left-right / up-down side-by-side spatial arrangement of cardiomyocytes and angiogenesis-related cells, and a core-shell / shell-core coaxial spatial arrangement of cardiomyocytes and angiogenesis-related cells.

2. The myocardial tissue construct with bionic structure and function according to claim 1, characterized in that: The hydrogel matrix includes at least one of methacrylated gelatin GelMA, methacrylated hyaluronic acid HAMA, gellan gum, sodium alginate, and polyethylene glycol; preferably, the methacryloyl substitution degree of the GelMA is 50-80%, and the mass concentration is 4-6%.

3. The myocardial tissue construct with bionic structure and function according to claim 1 or 2, characterized in that: The cross-linking agent is one of I2959, dimethyl paraphenylenediamine dinitrate VA-086, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate LAP, preferably LAP; The content of the crosslinking agent in the myocardial tissue construct is controlled to be 0.25-2wt% of the hydrogel matrix.

4. The myocardial tissue construct with bionic structure and function according to any one of claims 1 to 3, characterized in that: The inorganic biomaterial particles contain ≥3 elements and ≥2 elements beneficial to myocardial protection or vascularization, wherein the elements beneficial to myocardial protection or vascularization include silicon, calcium, strontium, magnesium, copper, and zinc; preferably, the inorganic biomaterial is a strontium silicate inorganic material SS containing silicon and strontium elements; The particle size of the inorganic biomaterial particles is 10 nm to 10 μm; The content of the inorganic biomaterial particles in the myocardial tissue construct is 2-5% of the hydrogel matrix.

5. The myocardial tissue construct with both bionic structure and function according to any one of claims 1 to 4, characterized in that: The cardiomyocytes are primary extracted cardiomyocytes and cardiomyocytes differentiated from human pluripotent stem cells, preferably primary cardiomyocytes; the ratio of the number of cells to the volume of the hydrogel matrix is ​​2×10 7 ~4×10 7 / mL, preferably 3×10 7 Pieces / mL.

6. The myocardial tissue construct with bionic structure and function according to any one of claims 1 to 5, characterized in that: The angiogenesis-related cells are at least one of aortic endothelial cells, umbilical vein endothelial cells, and smooth muscle cells, preferably aortic endothelial cells; the ratio of the number of cells to the volume of the hydrogel matrix is ​​1×10 6 ~5×10 6 / mL, preferably 3×10 6 Pieces / mL.

7. A method for preparing a myocardial tissue construct having both bionic structure and function according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: (1) mixing inorganic biomaterial particles, a crosslinking agent, and a hydrogel matrix to obtain a hydrogel ink material; (2) mixing myocardial cells and the hydrogel ink material to obtain bio-ink A, and mixing angiogenesis-related cells and the hydrogel ink material to obtain bio-ink B; (3) Modeling is performed based on the cell distribution pattern in natural myocardial tissue and dual-channel extrusion coaxial or parallel bio-3D printing is performed using bio-ink A and bio-ink B to obtain a blank, which is then cured by photo-crosslinking to obtain the myocardial tissue construct having both bionic structure and function.

8. The preparation method according to claim 7, characterized in that: In step (3), the pressure range of the two extrusion channels of the dual-channel extrusion coaxial or parallel biological 3D printing is 30-150 KPa, the temperature of the extrusion needle is 18-22°C, and the temperature of the deposition table is 0-10°C.

9. The preparation method according to claim 7 or 8, characterized in that: In step (3), the two channels of the coaxial biological 3D printing are connected by a coaxial needle; preferably, the inner diameter of the shell needle is 300-600 μm, and the inner diameter of the core needle is 150-250 μm.

10. Use of the myocardial tissue construct with bionic structure and function as claimed in any one of claims 1 to 6 in the preparation of myocardial cell-vascular related cell interaction materials and myocardial repair related materials in an in vitro three-dimensional microenvironment.

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