A large-sized biological tissue containing vascular structures and a construction method thereof

Abstract

The present invention relates to a large-sized biological tissue containing vascular structures and a construction method. In existing three-dimensional cell culture, there is a contradiction in the elastic modulus of the scaffold in ensuring structural stability and cell compatibility. Moreover, when preparing large-sized structures, vascular-like structures are required to provide nutrient exchange channels, and there is relatively little research on the regulation of the wall porosity of tubular structures that affects diffusivity. The present invention uses a hollow scaffold as a support scaffold, and pours a matrix material loaded with cells onto the support scaffold. By regulating the mechanical properties of the support scaffold, the stability of the overall scaffold structure can be ensured, effectively solving the contradictory requirements for the mechanical properties of the scaffold material in both ensuring structural stability and biocompatibility in traditional three-dimensional cell culture. By adding a temperature-sensitive material to the coaxial printing outer layer material, the porosity of the structure wall can be increased by removing the outer layer temperature-sensitive material, further improving the diffusivity of the hollow tube.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological tissues, and particularly relates to a large-sized biological tissue with a vascular structure and a construction method thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] In the three-dimensional culture of cells, to ensure the stability of the scaffold structure, the scaffold material needs to have a relatively high elastic modulus. During in vitro culture, the mechanical properties of the scaffold will affect the survival, proliferation, and differentiation of cells. Therefore, to ensure good biocompatibility of the scaffold, it is necessary to make the mechanical properties of the scaffold as close as possible to the mechanical properties of cells in the in vivo environment. However, the mechanical properties of different cells in the in vivo survival environment vary. For example, brain cells require a relatively low elastic modulus, while bone cells require a relatively high elastic modulus. Therefore, there is a contradiction in the mechanical properties of the scaffold in terms of ensuring structural stability and cell compatibility.

[0004] In addition, due to the limited diffusion distance of nutrients, when preparing large-sized tissue organs, to ensure the long-term viability and function of the model, it is necessary to introduce a vascular structure. The methods for preparing vascular structures in vitro mainly include direct preparation and indirect preparation of vascular structures. Direct preparation mainly uses coaxial extrusion to print hollow pipes. Some scholars have also studied that when using coaxial extrusion, sacrificial materials are first printed in the inner layer part, and then after the entire structure is prepared, the sacrificial materials are removed to prepare a hollow structure. Coaxial printing can directly print a hollow tubular structure. Indirect preparation of vascular structures is to first prepare a model using sacrificial materials, and then wrap it in a hydrogel material. After the hydrogel material is cured, the sacrificial materials are removed to prepare a vascular structure.

[0005] The inventor believes that the diffusivity of the existing in vitro prepared vascular structure is affected by the porosity of its tube wall, and currently, whether it is directly prepared or indirectly prepared vascular, the porosity of its tube wall depends on the nature of the material itself and has not been effectively adjusted. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the present invention provides a large-sized biological tissue with a vascular structure and a construction method of the biological tissue.

[0007] In the first aspect of the present invention, there is provided a large-sized biological tissue with a vascular structure, wherein the biological tissue includes a cell-laden hydrogel matrix and a support scaffold. The support scaffold is a hollow pipeline that is interspersed in the cell-laden hydrogel matrix; the support scaffold contains a thermosensitive material.

[0008] The design concept of the large-sized biological tissue provided by the above first aspect is as follows: The support scaffold is similar to the steel bar scaffold in building construction, playing the role of enhancing mechanical properties and ensuring structural stability, while the cell-loaded hydrogel matrix material is similar to the concrete material and is poured on the support scaffold. Since the support scaffold can ensure structural stability, when designing the cell-loaded hydrogel matrix material, only its biological properties need to be considered, so that the material properties are close to the in-vivo tissue where the cells are located, without the need to balance the mechanical properties like the materials used in general bio-3D printing technology to meet the inconsistent or even contradictory requirements of the support structure stability and biocompatibility for the mechanical properties of the materials.

[0009] In addition, to ensure that the cells inside the tissue can obtain sufficient oxygen and nutrients, the support scaffold is designed as a hollow pipe structure. During the tissue culture process, the hollow pipe can transport nutrients and oxygen to the cells inside the tissue, and at the same time output the waste products of cell metabolism, completing the physiological functions of the blood vessel structure in the biological tissue. To further improve the nutrient diffusion effect of the support structure, a thermosensitive material is incorporated during the preparation of the support scaffold. The thermosensitive material can undergo reversible sol-gel conversion with the change of temperature. By regulating the environmental temperature, the thermosensitive material in the support scaffold changes from the gel state to the liquid state and dissolves into the surrounding environment, thereby increasing the porosity of the wall of the support structure and improving the diffusion effect of nutrients in the blood vessel structure.

[0010] Furthermore, in the second aspect of the present invention, a method for constructing the above large-sized biological tissue with a blood vessel structure is also provided. The construction method includes the following steps: coaxially printing the inner layer and outer layer materials onto the receiving platform to form a scaffold structure, where the outer layer material is a composite material composed of a thermosensitive material and a crosslinking material, and the inner layer material is a thermosensitive material; after printing, curing the outer layer material in the scaffold structure to obtain a stable scaffold structure; incubating the stable scaffold structure at the melting temperature of the thermosensitive material to dissolve the thermosensitive materials in the inner and outer layers, obtaining a hollow support scaffold; pouring the cell-loaded hydrogel matrix into the support scaffold and curing it.

[0011] The beneficial effects of the above one or more technical solutions are as follows:

[0012] The present invention introduces a support scaffold to provide structural support for the prepared biological tissue, pours the matrix material loaded with cells on the support scaffold, and can ensure the stability of the overall scaffold structure by regulating the mechanical properties of the support scaffold, while ensuring an ideal living environment for the cells by regulating the mechanical properties of the matrix material. By using this method, the contradictory requirements of the mechanical properties of the scaffold material for ensuring structural stability and biocompatibility in traditional three-dimensional cell culture can be effectively solved.

[0013] The support scaffold is prepared into a hollow structure. During the tissue culture process, through the hollow vascular structure, nutrients and oxygen can be transported to the cells of the matrix material, and the metabolic wastes of the cells of the matrix material can be output, which is beneficial to solving the problem that when culturing cells in three dimensions in vitro, due to the too large tissue size, the internal cells cannot obtain sufficient nutrients and necrosis occurs. Therefore, large-sized tissue structures can be prepared by using this method.

[0014] The coaxial printing is used to prepare the hollow support scaffold structure. By adding a thermosensitive material to the outer layer material, after the overall structure of the scaffold is prepared, the thermosensitive material in the outer layer composite material is removed, and the porosity of the hollow tube wall can be regulated, thereby improving the diffusivity of the hollow tube. Brief Description of the Drawings

[0015] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0016] Figure 1 Schematic diagram for preparing a large-sized biological tissue with a vascular structure in Example 1;

[0017] Among them, Figure a is a schematic diagram of the support scaffold structure, and Figure b is a schematic diagram of the overall structure of the biological tissue.

[0018] Figure 2 It is the large-sized biological tissue with a vascular structure and its preparation process in Example 1;

[0019] Scale 1 cm. Among them, Figure a is the sterilized hollow support scaffold structure, Figure b is the hollow support scaffold structure soaked in the culture medium, and Figure c is the overall structure after pouring the cell-laden hydrogel matrix material into the hollow support scaffold.

[0020] Figure 3 It is the cell live / dead staining characterization;

[0021] Scale 200 μm. Among them, Figure a is the staining diagram of cell culture for 1 day, and Figure b is the staining diagram of cell culture for 7 days.

[0022] Figure 4 It is the cell survival and proliferation situation;

[0023] Among them, Figure a is the characterization of the cell survival rate, and Figure b is the cell proliferation situation.

[0024] Figure 5 It is the diffusion situation of small molecules in the hydrogel tube. Detailed Embodiments

[0025] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] As introduced in the background art, in the existing cell 3D culture engineering, there are contradictions in the mechanical properties of the scaffold in ensuring structural stability and cell compatibility, and there is a lack of a regulatory mechanism for the permeability of the vascular structure. To solve the above technical problems, the present invention proposes a large-sized biological tissue with a vascular structure and a construction method.

[0028] In a first aspect of the present invention, there is provided a large-sized biological tissue with a vascular structure, wherein the biological tissue includes a cell-laden hydrogel matrix and a support scaffold. The support scaffold is a hollow pipeline that is interspersed in the cell-laden hydrogel matrix to simulate the vascular structure; the support scaffold contains a thermosensitive material.

[0029] In the large-sized biological tissue described in the above first aspect, the support scaffold can simulate the vascular structure in the biological tissue to provide nutrients for the culture of cells and tissues and discharge waste metabolites.

[0030] The support scaffold is one or more pipelines, and the multiple pipelines may be interconnected or non-interconnected with each other; in an embodiment provided by the present invention, the pipelines of the support scaffold have multiple layers, which are stacked and arranged in the cell-laden gel matrix, and the pipelines of adjacent layers are staggered or vertically arranged.

[0031] The size of the biological tissue can be designed and prepared according to specific application requirements. In an embodiment verified by the present invention, the size of the biological tissue can be adjusted within the range of 16×16 mm - 40×40 mm.

[0032] The diameter of the pipeline in the support scaffold is 0.31 - 1.28 mm.

[0033] In order to meet the needs of different biological tissue cultures and achieve adjustable vascular permeability, the permeability of the above support scaffold is adjustable, and the adjustment method is: changing the concentration of the cross-linking material in the coaxial printing outer layer material.

[0034] In the second aspect of the present invention, there is provided a method for constructing the large-sized biological tissue containing vascular structures. The construction method includes the following steps: coaxially printing the inner layer and outer layer materials onto a receiving platform to form a scaffold structure, where the outer layer material is a composite material composed of a thermosensitive material and a crosslinking material, and the inner layer material is a thermosensitive material; after printing, curing the outer layer material in the scaffold structure to obtain a stable scaffold structure; incubating the stable scaffold structure at the melting temperature of the thermosensitive material to dissolve the thermosensitive materials in the inner and outer layers and obtain a hollow support scaffold; pouring a cell-laden hydrogel matrix into the support scaffold and curing it.

[0035] Preferably, the specific steps of coaxially printing the inner layer and outer layer materials onto a receiving platform to form a scaffold structure are as follows:

[0036] Configure the inner layer and outer layer materials for coaxial printing. The inner layer material is a thermosensitive material, and the outer layer material is a composite material composed of a thermosensitive material and a crosslinking material. Load the liquid inner and outer layer materials into syringes respectively, and incubate them in a first temperature environment to form a gel state.

[0037] Load the syringes containing the gel-like inner and outer layer materials into an extrusion 3D printer. At the same time, connect the coaxial printing nozzle to the syringes, set the process parameters of the 3D printer, start the receiving platform of the 3D printer, and set the temperature of the receiving platform.

[0038] Start the 3D printer software, make the coaxial printing nozzle move along a predetermined trajectory, and extrude the gel materials in the syringes onto the receiving platform to form a scaffold structure. Then, process the scaffold to cure the crosslinking material in the outer composite material and obtain a stable scaffold structure.

[0039] Further, in the above printing method, the inner layer thermosensitive material is Pluronic F127, its derivatives or gelatin; further, the inner layer thermosensitive material is Pluronic F127.

[0040] Further, the crosslinking material is one or a mixture of polyethylene glycol diacrylate, methacrylated gelatin, and sodium alginate; specifically, the crosslinking material is polyethylene glycol diacrylate.

[0041] In one implementation of the above preferred technical solution, the outer layer gel material is a composite material of polyethylene glycol diacrylate and Pluronic F127.

[0042] In the printing steps of the above-mentioned stent structure, the inner layer material is configured as follows: Pluronic F127 is dissolved in a phosphate solution with a mass fraction of 10-30%; the outer layer material is configured as follows: a photoinitiator and polyethylene glycol diacrylate are fully dissolved in a phosphate solution, and then Pluronic F127 is added and allowed to stand and dissolve. In the outer layer material, the mass concentration of Pluronic F127 is 10-30%, and the mass concentration of polyethylene glycol diacrylate is 5-20%.

[0043] Based on the above configuration method of the outer layer material, the outer layer material can be cured by ultraviolet light irradiation or ionic crosslinking curing. Considering the convenience of operation, in the preferred solution, ultraviolet light irradiation is used for curing.

[0044] Furthermore, the first temperature environment is 4-40°C, and the incubation time is 10-30 min; for specific examples, the first environmental temperature is set to 37°C, and the incubation time is 20 min.

[0045] Furthermore, the outer layer specification of the coaxial printing nozzle is 14-21G, and the inner layer specification is 18-30G; for specific examples, the outer layer specification of the coaxial printing nozzle is 17G, and the inner layer specification is 22G.

[0046] Furthermore, during 3D printing, the moving speed of the coaxial nozzle is 400-800 mm / min, and the extrusion speed ratio of the inner and outer layers is 1:4-1:1.

[0047] Furthermore, the receiving platform is selected as a high-temperature platform or a low-temperature platform, and the temperature is set to 4-50°C; in a specific implementation manner, the receiving platform is a high-temperature platform, and the temperature is set to 50°C.

[0048] During the construction of the hollow support stent described in the second aspect above, the stable stent structure is soaked and incubated in a phosphate buffer solution or ultrapure water. The melting temperature of the thermosensitive material is 4-40°C, and the incubation time is 1-5 days.

[0049] Furthermore, the stable stent is soaked in ultrapure water, the incubation temperature is 4°C, and the incubation time is 1-3 days.

[0050] Furthermore, after the construction of the hollow support stent is completed, it also includes a sterilization step. The sterilization method is as follows: the hollow support stent is soaked in a 75% ethanol solution for 1-5 h, and a specific example is 3 h. After the above sterilization step is completed, the hollow support stent needs to be washed to avoid the influence of residual ethanol. The washing can be carried out with a phosphate buffer solution, and then it is placed in a complete medium for standby.

[0051] In the construction method described in the second aspect above, the cell-loaded hydrogel matrix material is one or a combination of several of collagen, hyaluronic acid, sodium alginate, methacrylated gelatin, etc., with a mass concentration of 5-15%. Preferably, in some embodiments, the cell-loaded hydrogel matrix material is methacrylated gelatin with a mass concentration of 5%.

[0052] Furthermore, the curing method of the cell-loaded hydrogel matrix includes ionic crosslinking or photo-crosslinking. Preferably, in some embodiments, the curing method is photo-crosslinking.

[0053] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0054] Example 1

[0055] Take 20 g of phosphate buffer solution, place it in an environment at 4°C for 10 min, then add 5 g of Pluronic F127 to it, and shake for 1 min to disperse Pluronic F127 in the phosphate buffer solution. Finally, place it in an environment at 4°C for 3 days to fully dissolve Pluronic F127, and finally obtain a Pluronic F127 solution with a concentration of 20 wt%, which is used as the coaxial printing inner layer material.

[0056] Take 0.0625 g of photoinitiator LAP, pour it into a beaker, then add 16.1875 g of phosphate buffer solution and 3.75 g of polyethylene glycol diacrylate to the beaker, and magnetically stir in the dark at 40°C for 20 min at a rotation speed of 500 r / min to fully dissolve the photoinitiator LAP and polyethylene glycol diacrylate. Then transfer the mixed solution to a brown bottle, place it in an environment at 4°C for 20 min, then add 5 g of Pluronic F127, shake for 1 min, and then stand in an environment at 4°C for 3 days to fully dissolve Pluronic F127. The obtained solution is used as the coaxial printing outer layer material.

[0057] Coaxial printing of inner and outer layer materials was carried out, and they were respectively loaded into corresponding 5 mL syringes and left standing for 20 min in an environment of 37 °C. The parameters of the 3D printer were set as follows: the extrusion speed of the inner layer was 0.1 mm / min, the extrusion speed of the outer layer was 0.3 mm / min, the printing speed was 700 mm / min, the outer layer specification of the coaxial nozzle was 17G, the inner layer specification was 22G, the receiving platform was selected as a high-temperature platform with the temperature set at 50 °C. The layer height of the three-dimensional scaffold was 2 mm, the number of layers was 3, the number of repeated printing was 1, the spacing was 4 * 4 mm, the included angle was 90°, the length was 16 mm, the width was 16 mm, the Brim Width was 1 mm, and the Brim speed was 500 mm / min. After coaxial printing, the scaffold was irradiated with ultraviolet light (wavelength 405 nm, light intensity 25 mW / cm 2 ) for 10 min to cause photocrosslinking of the outer layer material, and finally a three-dimensional hydrogel scaffold with stable structure was formed. Then the hydrogel scaffold was transferred to ultrapure water and incubated in an environment of 4 °C for 3 days to remove the temperature-sensitive material Pluronic F127 in the inner and outer layers of the scaffold, and finally a hydrogel hollow support scaffold was obtained, as shown in Figure 2 (a).

[0058] The hydrogel hollow support scaffold was soaked in 75% ethanol for sterilization. After soaking in the ethanol solution for 3 h, it was washed with PBS and soaked for 12 h, then the PBS was changed and soaked for another 12 h to remove the residual ethanol in the scaffold. Then the scaffold was soaked in complete medium and placed in an incubator at 37 °C and 5% CO2 for 1 day to obtain the scaffold shown in Figure 2 (b).

[0059] To prepare the cell-loaded hydrogel matrix material, methacrylated gelatin was selected as the matrix material. First, 27.75 g of phosphate buffer solution was taken, 0.75 g of photoinitiator LAP was added, and then 1.5 g of GelMA material was added. It was stirred in a water bath at 40 °C for 30 min at a rotation speed of 500 r / min, and then filtered with a 0.45 μm filter for sterilization. The L929 fibroblasts were treated with trypsin. After centrifugation by rotation, the sterilized hydrogel material was added and pipetted to prepare the cell-loaded hydrogel matrix material with a cell density of 2×10 6 cells / mL.

[0060] The hollow support scaffold was placed in a culture dish, and then the culture dish was placed on ice. The cell-loaded hydrogel matrix material was poured onto the hollow support scaffold, 1.5 mL was poured for each scaffold, and then it was placed under ultraviolet light (wavelength 405 nm, light intensity 25 mW / cm 2 ) for light curing for 30 s to obtain a large-sized biological tissue with a vascular structure, as shown in Figure 2 (c).

[0061] In this example, the survival of cells in the biological tissue described in Example 1 was also investigated through performance tests:

[0062] Characterization of cell viability: Calcein-AM / PI was used to stain live / dead cells respectively, and then a fluorescence microscope was used to characterize the cell viability. Calcein-AM can stain live cells and emit green fluorescence, while PI can stain dead cells and emit red fluorescence. Figure 3 In the example, the live / dead cell staining images of the cells after 1 day and 7 days of cell culture are shown. Figure 4 (a) shows the survival rates of the cells after 1 day and 7 days of cell culture in Example 1. It can be seen that in the large-sized biological tissue with a vascular structure prepared in the example, the survival rate of the cells was higher than 95% throughout the culture period and exceeded 99% on the 7th day.

[0063] Characterization of cell proliferation: The CCK-8 assay was used to characterize the cell viability. The scaffolds cultured for 1, 3, 5, and 7 days were taken and placed in a new 6-well plate, and rinsed once with PBS. A working solution was prepared by mixing complete medium and CCK-8 solution at a volume ratio of 10:1. 5 ml of the working solution was added to each well, and then the 6-well plate was placed in an incubator at 37 °C and 5% CO2 for 3 hours. After incubation, the culture plate was taken out, and the incubation solution in the 6-well plate was transferred to a 96-well plate and injected into 5 wells, 100 μL into each well. The 96-well plate filled with the incubation solution was placed in a microplate reader, and the absorbance was measured at 450 nm. The proliferation of the cells at 1, 3, 5, and 7 days of cell culture is shown as Figure 4 (b). It can be seen that the cells can proliferate well in the large-sized biological tissue with a vascular structure.

[0064] Example 2

[0065] To verify that the biological tissue provided by the present invention can adjust the porosity of the wall of the support scaffold structure by changing the concentration of the cross-linking material in the coaxial printing outer layer material, the adjustment method of the above porosity is described in this example, and the cross-linking material is polyethylene glycol diacrylate.

[0066] Take 20 g of phosphate buffer solution, place it in an environment at 4 °C for 10 min, then add 5 g of Pluronic F127, shake for 1 min to disperse Pluronic F127 in the phosphate buffer solution, and finally place it in an environment at 4 °C for 3 days to fully dissolve Pluronic F127, and finally obtain a Pluronic F127 solution with a concentration of 20 wt%, and use it as the coaxial printing inner layer material.

[0067] Take the photoinitiator LAP and pour it into a beaker. Then add phosphate buffer solution and polyethylene glycol diacrylate to the beaker. Stir magnetically in the dark at 40 °C for 20 min at a rotation speed of 500 r / min to fully dissolve the photoinitiator LAP and polyethylene glycol diacrylate. Then transfer the mixture to a brown bottle and store it in an environment at 4 °C for 20 min. After that, add Pluronic F127 and shake for 1 min, then let it stand in an environment at 4 °C for 3 days to fully dissolve Pluronic F127. Finally, obtain solutions with a Pluronic F127 concentration of 20 wt% and polyethylene glycol diacrylate concentrations of 10 wt%, 15 wt%, and 20 wt% respectively, which are labeled as P10, P15, and P20. Use the obtained solutions as the outer layer materials for coaxial printing.

[0068] Take the inner and outer layer materials for coaxial printing and load them into corresponding 5 mL syringes respectively. Let them stand in an environment at 37 °C for 20 min. Set the parameters of the 3D printer: the inner layer extrusion speed is 0.1 mm / min, the outer layer extrusion speed is 0.3 mm / min, the printing speed is 700 mm / min, the outer layer specification of the coaxial nozzle is 17G, and the inner layer specification is 22G. Select a high-temperature platform for the receiving platform and set the temperature to 50 °C. The layer height of the three-dimensional scaffold is 2 mm, the number of layers is 1 layer, the number of repeated printing is 1 time, the spacing is 4 * 4 mm, the angle is 90°, the length is 40 mm, the width is 25 mm, the Brim Width is 1 mm, and the Brim speed is 500 mm / min. After coaxial printing, irradiate the scaffold with ultraviolet light (wavelength is 405 nm, light intensity is 25 mW / cm 2 ) for 10 min to cause photo-crosslinking of the outer layer material and finally form a three-dimensional hydrogel scaffold with a stable structure. Then prepare the hydrogel scaffold into a U-shaped structure and transfer it to ultrapure water. Soak it in an environment at 4 °C for 1 and 3 days respectively to remove the temperature-sensitive material Pluronic F127 in the inner and outer layers of the scaffold.

[0069] After soaking, the scaffold prepared from the solution with a polyethylene glycol diacrylate concentration of 10 wt% cannot be formed. Therefore, select the solutions with concentrations of 15 wt% and 20 wt% for subsequent experiments.

[0070] The U-shaped structures soaked for 1 day and 3 days were taken out respectively. The U-shaped structures prepared from the solution with a polyethylene glycol diacrylate concentration of 15 wt% were labeled as P15-1 and P15-3 respectively, and the U-shaped structures prepared from the solution with a polyethylene glycol diacrylate concentration of 20 wt% were labeled as P20-1 and P20-3 respectively. Then, a diffusivity experiment was carried out. According to the diffusion of small molecules from inside the pipeline to outside the pipeline, the regulation effect of the concentration of the cross-linked material in the outer layer material on the wall porosity was evaluated. When conducting the diffusion experiment, 30 μL of 0.5 wt% small molecule eosin Y dye was injected into the U-shaped tube, which was placed in an EP tube. 800 μL of PBS was injected into the EP tube, and then it was placed in an environment at 37 °C. Samples were taken out at 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h respectively. The PBS in the EP tube was aspirated, and 300 μL was aspirated from each specimen and transferred to a 96-well plate. 100 μL was injected into each well, that is, the PBS aspirated from one specimen was injected into 3 wells. Then, the absorbance was measured under an enzyme-linked immunosorbent assay (ELISA) reader, and the test wavelength was 525 nm.

[0071] Figure 5 As for the diffusion of small molecules in the hydrogel tube, it can be seen from the figure that for the two coaxial printing outer layer materials with a polyethylene glycol diacrylate concentration of 15 wt% and 20 wt%, there is no obvious difference in the diffusion behavior of the hydrogel tubes obtained after soaking for 1 day and 3 days, indicating that after soaking for 1 day, the thermosensitive sacrificial materials in both materials are basically removed. At all time points, the absorbance of the solution outside the hydrogel tube prepared from the material with a polyethylene glycol diacrylate concentration of 15 wt% is higher than that of the material with a polyethylene glycol diacrylate concentration of 20 wt%, indicating that the porosity of the hydrogel tube prepared from the material with a polyethylene glycol diacrylate concentration of 15 wt% is higher than that of the material with a polyethylene glycol diacrylate concentration of 20 wt%. Therefore, by changing the concentration of the cross-linked material in the coaxial printing outer layer material, the porosity of the wall of the support bracket structure can be adjusted.

[0072] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A large-sized biological tissue containing vascular structures, characterized in that, The biological tissue includes a cell-laden hydrogel matrix and a support scaffold. The support scaffold is a hollow tube inserted into the cell-laden hydrogel matrix to simulate a vascular structure. The support scaffold contains a temperature-sensitive material. The outer layer material of the support bracket is configured as follows: a photoinitiator and polyethylene glycol diacrylate are fully dissolved in a phosphate solution, and then Pluronic F127 is added and allowed to dissolve. In the outer layer material, the mass concentration of Pluronic F127 is 10-30%, and the mass concentration of polyethylene glycol diacrylate is 5-20%. The cell-laden hydrogel matrix material is poured into the hollow support scaffold.

2. The large-sized biological tissue containing blood vessel structures as described in claim 1, wherein The supporting bracket is one or more pipelines, and the multiple pipelines may be interconnected or not.

3. The large-sized biological tissue containing a vascular structure according to claim 1, wherein The tubes of the support stent have multiple layers, which are stacked and arranged in the cell-laden gel matrix, and the tubes of adjacent layers are staggered or arranged vertically.

4. The construction method of the large-sized biological tissue containing blood vessel structures according to any one of claims 1-3, characterized in that, The construction method comprises the following steps: coaxially printing inner and outer layer materials onto a receiving platform to form a stent structure, wherein the outer layer material is a composite material composed of a temperature-sensitive material and a cross-linked material, and the inner layer material is a temperature-sensitive material; After printing is completed, the outer layer material in the support structure is solidified to obtain a stable support structure; placing the stable support structure at the melting temperature of the temperature-sensitive material for incubation, so that the temperature-sensitive material in the inner and outer layers dissolves, thereby obtaining a hollow support support; The cell-laden hydrogel matrix was poured onto the support scaffold and solidified.

5. The construction method of the large-sized biological tissue containing blood vessel structures according to claim 4, wherein The specific steps of coaxially printing the inner and outer layer materials onto the receiving platform to form the support structure are as follows: The inner and outer layer materials are coaxially printed, wherein the inner layer material is a temperature-sensitive material and the outer layer material is a composite material composed of a temperature-sensitive material and a cross-linked material. The liquid inner and outer layer materials are respectively loaded into syringes and placed in a first temperature environment for incubation to form a gel; Load the syringe containing the gel-like inner and outer layer materials into the extrusion 3D printer, connect the coaxial print head to the syringe, set the 3D printer process parameters, start the 3D printer receiving platform, and set the receiving platform temperature; Start the 3D printer software, make the coaxial printing nozzle move along a predetermined trajectory, and extrude the gel material in the syringe onto the receiving platform to form a stent structure. Then, the stent is processed to solidify the cross-linking material in the outer layer composite material to obtain a stable stent structure.

6. The construction method of the large-sized biological tissue containing blood vessels according to claim 5, characterized in that In the printing method, the inner layer temperature-sensitive material is Pluronic F127, its derivatives or gelatin; or the cross-linking material is one of polyethylene glycol diacrylate, methacrylated gelatin, sodium alginate or a mixture thereof.

7. The construction method of the large-sized biological tissue containing vascular structures according to claim 5, characterized in that The inner layer temperature-sensitive material is Pluronic F127; Or, the cross-linking material is polyethylene glycol diacrylate; The outer layer gel material is a composite material of polyethylene glycol diacrylate and Pluronic F127.

8. The construction method of the large-sized biological tissue containing blood vessel structures according to claim 4, characterized in that, The inner layer material is configured as follows: Pluronic F127 is dissolved in a phosphate solution with a mass fraction of 10-30%; The outer layer material is configured as follows: The photoinitiator and polyethylene glycol diacrylate are fully dissolved in a phosphate solution, and then Pluronic F127 is added and allowed to dissolve statically. In the outer layer material, the mass concentration of Pluronic F127 is 10 - 30%, and the mass concentration of polyethylene glycol diacrylate is 5 - 20%.

9. The method for constructing a large - sized biological tissue with a vascular structure according to claim 4, wherein The outer layer material is cured by ultraviolet light irradiation or ionic cross - linking curing.

10. The construction method of the large-sized biological tissue containing blood vessel structures according to claim 9, characterized in that, The outer layer material is cured by ultraviolet light irradiation.

11. The construction method of the large-sized biological tissue containing blood vessel structures according to claim 5, characterized in that, The first temperature environment is 4 - 40 °C, and the incubation time is 10 - 30 min; Or, the outer layer specification of the coaxial printing nozzle is 14 - 21G, and the inner layer specification is 18 - 30G; Or, during 3D printing, the moving speed of the coaxial nozzle is 400 - 800 mm / min, and the extrusion speed ratio of the inner and outer layers is 1:4 - 1:1; Or, the receiving platform is selected as a high - temperature platform or a low - temperature platform, and the temperature is set to 4 - 50 °C.

12. The construction method of the large-sized biological tissue containing blood vessel structures according to claim 5, wherein The first temperature environment is set to 37 °C, and the incubation time is 20 min; Or, the outer layer specification of the coaxial printing nozzle is 17G, and the inner layer specification is 22G; Or, the receiving platform is a high - temperature platform, and the temperature is set to 50 °C.

13. The construction method of a large-sized biological tissue containing a vascular structure according to claim 4, characterized in that, During the construction of the hollow support scaffold, the stable scaffold structure is soaked and incubated in a phosphate buffer solution or ultrapure water. The melting temperature of the thermosensitive material is 4 - 40 °C, and the incubation time is 1 - 5 days.

14. The method for constructing a large-sized biological tissue containing vascular structures according to claim 13, characterized in that, The stable scaffold is soaked in ultrapure water, the incubation temperature is 4 °C, and the incubation time is 1 - 3 days.

15. The construction method of the large-sized biological tissue containing blood vessel structures as described in claim 13, characterized in that, After the construction of the hollow support scaffold is completed, a sterilization step is also included. The sterilization method is as follows: The hollow support scaffold is soaked in a 75% ethanol solution for 1 - 5 h.

16. The construction method of the large-sized biological tissue containing blood vessel structures according to claim 13, wherein, The soaking time is 3 h.

17. The construction method of the large-sized biological tissue containing blood vessel structures according to claim 4, characterized in that, The cell - loaded hydrogel matrix material is one or a combination of collagen, hyaluronic acid, sodium alginate, methacrylated gelatin, etc., and the mass concentration is 5 - 15%.

18. The method for constructing a large - sized biological tissue with a vascular structure according to claim 4, wherein The cell - loaded hydrogel matrix material is methacrylated gelatin, and the mass concentration is 5%.

19. The method for constructing a large - sized biological tissue with a vascular structure according to claim 4, wherein 20. The method for constructing a large-sized biological tissue containing vascular structures according to claim 19, wherein, The curing method of the cell - loaded hydrogel matrix includes ionic cross - linking or photo - cross - linking. The curing method is photo - cross - linking.

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