A modular helical vane-like three-dimensional stent and its preparation method and application

Abstract

The application discloses a kind of modularization imitates helical blade three-dimensional scaffold and its preparation method and application.The modularization imitates helical blade three-dimensional scaffold has customizable geometric topology structure, can realize accurate regulation to microflow field after assembly, constructs controllable three-dimensional physical microenvironment for the multiple cells loaded in it.This kind of modularization three-dimensional scaffold based on geometric topology customization to realize space flow field management has important application value for researching cell interaction and the interaction of cell and microenvironment, and promoting the development of complex tissue engineering.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a modular helical blade-like three-dimensional scaffold, its preparation method, and its application. Background Technology

[0002] In natural tissues, the fluid microenvironment plays a crucial role in maintaining tissue homeostasis and regulating cellular function. The flow of interstitial fluid, the shear forces generated by blood flow, and the resulting chemical gradients together constitute a dynamic physicochemical signaling network that profoundly influences cell proliferation, migration, differentiation, and metabolism. However, for current in vitro models, simultaneously reconstructing the three-dimensional spatial distribution of vascular networks and simulating spatial fluid stimulation remains challenging. In the field of bone tissue engineering, the spatiotemporal coupling between angiogenesis and osteogenics is crucial, but existing models generally lack synergistic simulation of the bone-specific fluid microenvironment and the dynamic process of vascular budding. This not only limits in-depth research on the mechanisms of intraosseous angiogenesis but also hinders the screening of better strategies to promote vascularized bone repair.

[0003] In recent years, advancements in advanced technologies such as bioprinting and microfluidic chips have made it possible to simulate fluid microenvironments and the spatial patterning of various cell types. However, existing models still have some shortcomings. First, regarding the fluid microenvironment, most platforms can only provide unidirectional constant flow, making it difficult to reproduce the transient rheological characteristics induced by pulsating blood flow, pressure fluctuations, and mechanical loads in vivo; simultaneously, maintaining the stability and sterility of the fluid during long-term culture is also challenging. Second, regarding cell spatial distribution, current printing resolutions still cannot accurately reproduce the true topological relationships of multiple cells in bone tissue and the interactions between cells and the extracellular matrix, and the dynamic spatiotemporal coupling between angiogenesis and bone formation lacks systematic simulation. Therefore, existing platforms still face certain challenges in the study of mechanisms promoting vascularized bone repair and in high-throughput screening applications. Summary of the Invention

[0004] To address the aforementioned problems, this invention aims to provide a modular, helical blade-like three-dimensional scaffold, its fabrication method, and its applications. The modular scaffold possesses a programmable geometric topology, enabling precise control of the microfluidic field after assembly, thus constructing a controllable three-dimensional physical microenvironment for the various cells it houses. This modular three-dimensional scaffold, based on geometric topology customization for spatial flow field management, is of significant importance for studying intercellular interactions and cell-microenvironment interactions, and for advancing the development of complex tissue engineering.

[0005] In a first aspect, the present invention provides a modular helical blade-like three-dimensional scaffold. The modular helical blade-like three-dimensional scaffold includes an overall scaffold frame with a three-dimensional helical structure formed by assembling multiple scaffold module units with blades, and (multiple) cells distributed in a layered manner within the three-dimensional space of the overall scaffold frame.

[0006] This invention assembles helical leaf-like scaffolds to regulate fluid flow within the overall defect space, enabling the long-distance directional transport of previously diffuse and disordered paracrine signals in an open, macroscopic space. This improves the efficiency of information transmission and spatially specific control. Simultaneously, the leaf shape modulates localized fluid stress, causing cells adhering to it to produce different mechanical responses under varying degrees of mechanical stimulation, thereby dynamically regulating osteogenic processes through cellular behavior. Therefore, this modular, helical leaf-like three-dimensional scaffold loaded with living cells has broad application prospects in fields such as intercellular communication, drug screening, and complex tissue regeneration.

[0007] Preferably, the support module unit with blades includes a central entity, blades disposed on the outer periphery of the central entity and evenly distributed around the axis of the central entity, and keys and keyways located on the upper and lower end faces of the central entity.

[0008] Preferably, the central entity is a central cylinder, and the shape of the blade is a planar sheet-like fan ring and / or a cylindrical sheet-like fan ring; preferably, the number of blades is n, and the central angle of the fan ring of the blade is 180° / n.

[0009] Preferably, the diameter of the central cylinder is 2-3 mm and the height is 1-2 mm; the width of the key is 0.8-1.1 mm, the length is 1-1.3 mm, and the height is 0.8-0.9 mm; the width of the keyway is 0.9-1.2 mm, the length is 1.2-1.5 mm, and the depth is 0.9-1 mm; both the key and the keyway have their major axes along their respective long sides, and the angle between the major axes of the key and the keyway is 0-180°; the central angle of the blade is 45°, the outer arc diameter is 6-8 mm, the thickness is 0.4-0.5 mm, and the curvature of the blade edge cylindrical surface in the thickness direction (axial direction of the central cylinder) is 0-0.67 mm. -1 .

[0010] Preferably, the bladed support module units are independently distributed in alternating and staggered layers, and the area overlap rate between adjacent layers of the blades in the axial direction in the assembled three-dimensional support is 0-100%.

[0011] Preferably, the material of the three-dimensional scaffold is bioactive ceramic, organic matter, bioinert ceramic, or a composite of organic matter and bioactive ceramic, preferably tricalcium phosphate in the β phase.

[0012] Preferably, the cells are bone marrow mesenchymal stem cells or endothelial cells; more preferably, the cells loaded on the modular spiral blade-like three-dimensional scaffold are endothelial cells and bone marrow mesenchymal stem cells, and the scaffold module units loaded with endothelial cells and bone marrow mesenchymal stem cells are arranged in alternating layers.

[0013] Preferably, by changing the fluid velocity and direction in the space where the scaffold is located, the overall spatial flow field microenvironment in which the cells are located is changed, thereby constructing a three-dimensional multi-cell co-culture system with controllable flow field, promoting mutual communication between different cells and the interaction between cells and the fluid microenvironment.

[0014] Secondly, the present invention provides a method for preparing the modular helical blade-like three-dimensional scaffold described above. The preparation method includes the following steps: thoroughly mixing the material of the three-dimensional scaffold with photosensitive resin to obtain a printing slurry; using photocuring to print a ceramic scaffold green body; sintering the printed ceramic scaffold green body to obtain ceramic scaffold module units; sterilizing the sintered ceramic scaffold module units and then using them to load cells; selecting scaffold module units of specific shapes as needed and seeding them with bone marrow mesenchymal stem cells or endothelial cells respectively; after cell adhesion, assembling all scaffold module units into a complete, designed-oriented modular helical blade-like three-dimensional scaffold loaded with living cells.

[0015] Thirdly, the present invention provides an application of the modular helical blade-like three-dimensional scaffold described above in the preparation of drugs or products for drug screening, multi-cell co-culture models, and complex tissue regeneration.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The modular helical blade-like three-dimensional scaffold described in this invention is characterized by its ability to regulate the flow field within the scaffold, simulating the anisotropic fluid distribution in natural bone tissue on a three-dimensional macroscopic scale. Natural bone contains interstitial fluid driven by pressure differences, which exhibits anisotropy within the defect space, determining the transport pathways and concentration gradient directions of various chemical signaling molecules. This anisotropy can provide navigational clues for angiogenesis, determining the extension direction of vascular sprouting cells and the chemotactic migration of mesenchymal stem cells, and can also coordinate the synergistic functions of multiple cells through spatial heterogeneity. The overall repair efficiency of bone defects is highly dependent on the matching of fluid direction with the vascular network topology. This invention assembles a spirally extended scaffold by using keyways in different directions (keys and keyways between adjacent scaffold module units), allowing the blades to have different orientations. This regulates the fluid flow within the overall defect space, enabling the originally diffuse and disordered paracrine signals to achieve long-distance directional transport in an open, macroscopic space, thereby improving the efficiency and spatial specificity of information transmission. Simultaneously, the shape of the blades can regulate the local stress of the fluid, causing cells adhering to them to produce different mechanical responses under varying degrees of mechanical stimulation, and thus dynamically regulating the osteogenic process through cell behavior.

[0017] (2) This invention is the first to successfully prepare a modular spiral blade-like three-dimensional scaffold loaded with living cells using the above method, and construct a multi-cell three-dimensional co-culture platform, realizing the regulation of the layered distribution of different cells in the three-dimensional space inside the ceramic scaffold. Through the modular assembly strategy, a multi-cell tissue engineering scaffold that simulates the physiological structure of vascularized bone tissue in vivo and also has the function of promoting tissue repair was successfully constructed.

[0018] (3) The helical blade-like three-dimensional scaffold can regulate the fluid distribution in three-dimensional space; based on modular assembly technology, the distribution of different cells in the three-dimensional space inside the scaffold can be precisely controlled; at the same time, the scaffold can also be disassembled into individual modules, which facilitates the study of the contribution of each cell in the whole system. Therefore, this modular helical blade-like three-dimensional scaffold has great application potential in studying the interaction between complex hydrodynamic microenvironment and biological tissue repair. Attached Figure Description

[0019] Figure 1 This document presents design drawings and schematic diagrams of support module units with different shapes in this invention. It includes a top view of the module support unit, a planar blade C0 module support with zero curvature, and a cylindrical surface with a thickness direction curvature of 0.40 mm. -1 The C40 module support and blade thickness direction cylindrical curvature is 0.67 mm. -1 C67 module bracket.

[0020] Figure 2 This is a schematic diagram of the design of the support module unit in this invention.

[0021] Figure 3 The images show the design model (A: a - d), general appearance (A: e - h), and scanning electron microscope images (A: i - l) of the bioceramic scaffold module with simulated spiral blades in this invention, as well as phase characterization (B) and elemental distribution (C).

[0022] Figure 4 The images show the design rendering (A), the printed assembly (B), and the sintered assembly bracket (C) of the different orientation modules in this invention. C0-0 refers to a C0 three-dimensional bracket where the axial overlap rate of adjacent layers of the blades is 0, and the angle between the major axes of the key and keyway is 45º. C0-50 refers to a C0 three-dimensional bracket where the axial overlap rate of adjacent layers of the blades is 50%, and the angle between the major axes of the key and keyway is 22.5º. C0-100 refers to a C0 three-dimensional bracket where the axial overlap rate of adjacent layers of the blades is 100%, and the angle between the major axes of the key and keyway is 0º. C40-0 refers to a C40 three-dimensional bracket where the axial overlap rate of adjacent layers of the blades is 0, and the angle between the major axes of the key and keyway is 45º. C40-50 refers to a C40 three-dimensional bracket where the axial overlap rate of adjacent layers of the blades is 50%, and the angle between the major axes of the key and keyway is 22.5º. C40-100 refers to a C40 3D support where the blades have a 100% overlap in area between adjacent layers along the axial direction, and the major axis angle between the key and keyway is 0º. C67-0 refers to a C67 3D support where the blades have a 0% overlap in area between adjacent layers along the axial direction, and the major axis angle between the key and keyway is 45º. C67-50 refers to a C67 3D support where the blades have a 50% overlap in area between adjacent layers along the axial direction, and the major axis angle between the key and keyway is 22.5º. C67-100 refers to a C67 3D support where the blades have a 100% overlap in area between adjacent layers along the axial direction, and the major axis angle between the key and keyway is 0º.

[0023] Figure 5 This invention illustrates the effect of scaffold structural parameters on single cells. (A) Endothelial cell proliferation on scaffolds with different orientations; (B-E) Expression of angiogenesis-related genes in scaffolds with different orientations; (F, G) Levels of angiogenesis-related proteins in scaffolds with different orientations; (H, I) Adhesion of bone marrow mesenchymal stem cells on scaffolds of different shapes; (J-N) Expression of mechanotransduction genes in bone marrow mesenchymal stem cells on scaffolds of different shapes; (O-R) Proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells on scaffolds of different shapes and orientations.

[0024] Figure 6This diagram illustrates the use of a modular, spiral-shaped bioceramic scaffold to construct a multicellular three-dimensional platform for studying chemotaxis. (A-C) Activation of the SDF-1 / CXCR4 signaling axis in the endothelial cell-bone marrow mesenchymal stem cell (BMSC) configuration under different orientations. (D-F) Number density distribution of BMSCs at different spatial locations.

[0025] Figure 7 This diagram illustrates the use of a modular, spiral-blade-like bioceramic scaffold to construct a multicellular three-dimensional platform for studying the mutual migration of two cell types. (A) Endothelial cells and bone marrow mesenchymal stem cells attract each other under different orientation structures, with stem cells migrating upwards across the module and forming contact with endothelial cells. (B) Number density map of the migration distance of endothelial cells on the central cylindrical sidewall of the endothelial cell-loaded module. (C) Number density map of the migration distance of bone marrow mesenchymal stem cells on the central cylindrical sidewall of the endothelial cell-loaded module.

[0026] Figure 8 This diagram illustrates the use of a modular, spiral-blade-like bioceramic scaffold to construct a multicellular three-dimensional platform for studying the influence of interstitial flow on the relative migration of two cell types. (A) The relative migration and contact of the two cell types within the C40-50 scaffold space under flow field conditions. (B) The overlap distance between the migration fronts of the two cell types on the sidewall of the central cylinder of the endothelial cell-loaded module. (C) The number of cells in contact within the overlap region of the sidewall of the central cylinder of the endothelial cell-loaded module. (D, E) The effect of interstitial flow stimulation on the mechanotransduction protein levels of bone marrow mesenchymal stem cells. (F) Organelle communication between bone marrow mesenchymal stem cells and endothelial cells under interstitial flow stimulation. (G-L) The effect of interstitial flow stimulation on osteogenic / angiogenic differentiation of bone marrow mesenchymal stem cells and endothelial cells. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not intended to limit the present invention.

[0028] The modular helical blade-like three-dimensional scaffold of this invention includes an overall flow-guiding frame assembled from scaffold module units of various shapes and splicing directions, and various cells distributed in layers within the three-dimensional space of the overall scaffold frame. Since the modular helical blade-like three-dimensional scaffold is assembled from multiple layers of scaffold module units with flow-guiding blades of specific shapes, these scaffold module units can be designed with different materials, shapes, orientations, and size parameters, or can load different types of living cells, according to different needs and application scenarios.

[0029] In an optional embodiment, the modular helical blade-like three-dimensional scaffold includes an overall scaffold frame with a three-dimensional helical structure formed by assembling multiple scaffold module units with blades, and a variety of cells distributed in layers within the three-dimensional space of the overall scaffold frame.

[0030] As an example, the bladed support module unit includes a central body, blades disposed on the outer periphery of the central body and evenly distributed around the axis of the central body, and keys and keyways located on the upper and lower end faces of the central body. It should be understood that the keys and keyways are located on opposite end faces of the central body. This includes, but is not limited to, a key on the upper end face and a keyway on the lower end face of the central body, or a keyway on the upper end face and a key on the lower end face. In an optional embodiment, the lower end face of the blade and the lower end face of the central body are located on the same plane. The central body and the blades can be integrally formed. Adjacent support module units are assembled and spliced ​​using keys and keyways.

[0031] For example, the support module unit includes a central cylinder, blades located around the central cylinder, and keys and keyways located on the upper and lower end faces of the central cylinder respectively; the blades include, but are not limited to, fan-shaped planar plates and cylindrical plates.

[0032] In a specific embodiment, the diameter of the central cylinder is 2-3 mm and the height is 1-2 mm; the key width is 0.8-1.1 mm, the length is 1-1.3 mm, and the height is 0.8-0.9 mm; the keyway width is 0.9-1.2 mm, the length is 1.2-1.5 mm, and the depth is 0.9-1 mm; the direction of the longer side (excluding the height and depth directions) of the key and keyway is defined as their respective major axes, and the angle between the major axes of the key and keyway is 0-180°. Correspondingly, in the axial direction of the central cylinder, the area overlap rate between adjacent layers of the assembled blade is 0-100%.

[0033] In a specific implementation, based on the number of blades n, the central angle of the fan ring of the blade can be extended to 180° / n. For example, the central angle of the fan ring of the blade is 45°, and the four blades are evenly distributed. The diameter of the circumcircle of the fan ring of the blade is 6-8 mm, the blade thickness is 0.4-0.6 mm (e.g., 0.4-0.5 mm), and the curvature of the cylindrical surface where the blade is located in the thickness direction is 0-0.67 mm. -1 The blade's thickness direction is along the axis of the central cylinder. The surface containing the blade's edge in the thickness direction is a cylindrical surface. The curvature of this cylindrical surface is the same as the curvature of the cylindrical surface containing the blade in the thickness direction. The blade's top view is fan-shaped. The circumcircle is the outer circumference of the fan-shaped area.

[0034] The assembled scaffold is loaded with endothelial cells and bone marrow mesenchymal stem cells, which are arranged in alternating layers to simulate the physiological process of vascular sprouting to form a dendritic network and nourishing surrounding mesenchymal stem cells to promote osteogenic formation during real bone repair. In an optional embodiment, the three-dimensional scaffold mimicking a spiral blade includes a layered structure with a (AB)m cyclic arrangement. A is a scaffold module unit loaded with endothelial cells, and B is a scaffold module unit loaded with bone marrow mesenchymal stem cells.

[0035] The material of the three-dimensional scaffold of the simulated helical blade is bioactive ceramic, bioinert ceramic, organic matter, metal, biocompatible polymer, or a composite of organic matter and bioactive ceramic, preferably β-tricalcium phosphate bioceramic, which can also be called β-phase tricalcium phosphate (Ca3(PO4)2).

[0036] Bioceramics, metals, and other biocompatible polymers can effectively adsorb proteins on their surfaces, which is beneficial for cell adhesion and growth; the active products released by the degradation of materials can promote cell proliferation and differentiation; and the modular helical structure itself can regulate the information transmission pathways of the cell environment, which helps to intervene in and regulate the paracrine pathway to a certain extent.

[0037] Unlike conventional scaffolds that are fabricated as a single unit with a single configuration, this invention designs blades with specific shapes to guide flow. By utilizing the angled splicing of assembly points, the orientation of the blades between different modules can be easily controlled, thereby regulating the flow field within the entire scaffold space. Simultaneously, specific cells can be seeded onto the scaffold modules before assembly, allowing for precise control of the distribution of different cells within the scaffold's internal space. By changing the fluid velocity and direction within the scaffold space, the overall microenvironment of the flow field surrounding the cells can be altered, thus constructing a flow-controlled three-dimensional multi-cell co-culture system that promotes communication between different cells and the interaction between cells and the fluid microenvironment. Therefore, the cells within the scaffold exhibit varying degrees of proliferation, differentiation, and migration. The interaction between the loaded endothelial cells and bone marrow mesenchymal stem cells provides a foundation for achieving vascularized bone repair within the scaffold. Furthermore, after culture, the scaffold can be disassembled into individual scaffold modules for studying the performance of various cells under flow field conditions and their contribution to the co-culture system.

[0038] The modular helical blade-like three-dimensional scaffold is assembled from multiple modular units, each with a customizable blade shape, forming a three-dimensional helical structure after assembly. The shape, size, and material of each module can be designed according to actual needs, and the spatial distribution of various cells loaded within it can be precisely controlled by adjusting different cell-carrying modules. Each modular unit of the scaffold is fabricated using photopolymerization 3D printing.

[0039] This invention also provides a method for preparing the modular, spiral-blade-like three-dimensional scaffold as described above. A slurry containing different ceramic powders and photosensitive resin is thoroughly mixed using a planetary ball mill to form a printing slurry; then, a scaffold green is prepared using a photopolymerization 3D printer; and the shape and size parameters of the scaffold module are designed and adjusted using computer software to control various parameters of the final printed scaffold; the printed scaffold green is sintered in a high-temperature furnace to obtain the scaffold module.

[0040] Specifically, the preparation method of the cell-loaded modular helical leaf-like three-dimensional scaffold includes the following steps: Ceramic powder and photosensitive resin are stirred in a planetary ball mill until fully mixed, and the mixture is filtered to obtain a printing slurry. The shape and size parameters of the scaffold module units are designed and adjusted using 3D modeling software, and a 3D solid general file is exported. The 3D solid model is imported into slicing software for slicing, and then the sliced ​​model is imported into a photopolymerization printer for printing to prepare a scaffold green body. The printed ceramic scaffold green body is sintered in a high-temperature furnace to obtain ceramic scaffold module units. The sintered ceramic scaffold module units are placed in a high-temperature and high-pressure sterilizer for sterilization before being used for cell loading. Scaffold module units of specific shapes are selected as needed and seeded with bone marrow mesenchymal stem cells or endothelial cells, respectively. After cell adhesion, all scaffold module units are assembled into a complete, design-oriented modular helical leaf-like three-dimensional scaffold loaded with live cells.

[0041] Thirdly, this invention also provides the application of the modular helical leaf-like three-dimensional scaffold loaded with living cells, as described above, in the study of various intercellular communication and vascularized bone regeneration. In particular, the modular helical leaf-like three-dimensional scaffold is particularly useful in regenerative medicine, especially in drug screening, multi-cell co-culture models, and complex tissue regeneration.

[0042] like Figure 1 As shown, the overall structure of the C0 support module (planar blade) consists of a central cylinder, four planar, sheet-like fan-shaped blades evenly distributed on the outer circumference with a central angle of 45°, and keys and keyways on the upper and lower end faces. The central cylinder has a diameter of 2-3 mm and a height of 1-2 mm. The blades have a central angle of 45°, an outer circumference diameter of 6-8 mm, a thickness of 0.4-0.5 mm, and a cylindrical curvature of 0 mm in the thickness direction. -1 (i.e., a completely planar sheet shape, without curvature). The key width is 0.8-1.1 mm, the length is 1-1.3 mm, and the height is 0.8-0.9 mm. The keyway width is 0.9-1.2 mm, the length is 1.2-1.5 mm, and the depth is 0.9-1 mm. C40 support module (cylindrical blade, curvature 0.40 mm) -1The overall structure consists of a central cylinder, four cylindrical fan-shaped blades evenly distributed on the outer circumference with a central angle of 45°, and keys and keyways on the upper and lower end faces. The central cylinder has a diameter of 2-3 mm and a height of 1-2 mm. The blades have a central angle of 45°, an outer circumference diameter of 6-8 mm, a thickness of 0.4-0.5 mm, and a cylindrical curvature of 0.40 mm in the thickness direction. -1 (The blade is cylindrically curved in the thickness direction). The key width is 0.8-1.1 mm, length is 1-1.3 mm, and height is 0.8-0.9 mm. The keyway width is 0.9-1.2 mm, length is 1.2-1.5 mm, and depth is 0.9-1 mm. C67 support module (cylindrical blade, curvature 0.67 mm) -1 The overall structure consists of a central cylinder, four cylindrical fan-shaped blades evenly distributed on the outer circumference with a central angle of 45°, and keys and keyways on the upper and lower end faces. The central cylinder has a diameter of 2-3 mm and a height of 1-2 mm. The blades have a central angle of 45°, an outer circumference diameter of 6-8 mm, a thickness of 0.4-0.5 mm, and a cylindrical curvature of 0.67 mm in the thickness direction. -1 (The blade exhibits a more pronounced cylindrical curvature in the thickness direction). The key width is 0.8-1.1 mm, the length is 1-1.3 mm, and the height is 0.8-0.9 mm. The keyway width is 0.9-1.2 mm, the length is 1.2-1.5 mm, and the depth is 0.9-1 mm.

[0043] like Figure 2 As shown, the support module of this invention has four core components: a central cylinder, blades, bonds, and keyways. The bonds and keyways between two support modules can be connected and assembled accordingly, ensuring that the entire support module will not detach during biological experiments. The central cylinder has a diameter of approximately 2.4 mm and serves to support and fix the blades; the central cylinder can also be designed in other shapes, and the shape and size of the bonds and keyways can be adjusted according to requirements. The blade shape varies from planar thin sheets to cylindrical plates, used for localized adjustment of flow field direction and stress distribution, and can also be adjusted in other shapes and sizes as needed.

[0044] like Figure 3 As shown, the modular scaffold in this invention can have its physicochemical properties matched to design requirements by adjusting its dimensional parameters. The material of the modular three-dimensional scaffold in this invention is not particularly limited; for example, it can be bioceramics such as tricalcium phosphate or hydroxyapatite, or it can be metal, organic material, or composite material.

[0045] like Figure 4As shown, the modular support in this invention can be assembled from pre-prepared modules. The outermost contour of the support is a circumscribed circle, which can be changed to other shapes according to actual needs. The angle between the major axes of the key and the keyway ranges from 0 to 180°. The helical supports obtained by assembling different modules with keys have different blade extension directions, and the area overlap rate between blade layers along the central cylinder axis can be flexibly adjusted within the range of 0-100%, thereby presenting different helical angles in space and achieving a certain degree of flow field control. By combining the shape changes of the blades, helical blade three-dimensional supports with various structural parameters can be prepared, and all can achieve the desired effect through program design. For example, a modular three-dimensional support with the same height, diameter, and porosity as the traditional staggered structure can be designed and prepared.

[0046] The following is an exemplary description of the fabrication method of the modular helical leaf-like three-dimensional scaffold loaded with living cells: (1) Preparation of slurry: Add photosensitive resin and raw material powder to the ball mill jar. The mass ratio of each component in the slurry is raw material powder: photosensitive resin = 1: (0.7 - 2.2). Place the slurry in a planetary ball mill and grind it thoroughly. After filtration, collect the slurry.

[0047] (2) 3D printing: The prepared slurry is poured into the printer's feed tank, the pre-written printing file is loaded into the printer system, and the support module is printed. 3D printing is a conventional technology in this field.

[0048] (3) Post-processing: The printed support module is removed from the printing table, cleaned and dried to obtain a ceramic green body, which is then placed in a crucible and sintered at high temperature and cooled to obtain a ceramic support module. The sintering process needs to be adjusted according to the type of ceramic and the properties of the photosensitive resin. For example, the sintering temperature of the β-tricalcium phosphate support module is 1150℃, and the sintering temperature of the zirconia support module is 1600℃. For photosensitive resin systems with different ratios, the heating rate and holding time need to be adjusted to ensure sufficient resin removal and prevent cracks and holes in the support.

[0049] (4) Cell seeding: The sintered scaffold modules are cleaned and sterilized, and bone marrow mesenchymal stem cells or endothelial cells are seeded in a clean bench. They are then incubated in a cell culture incubator for 4 hours. Once the cells have fully adhered, scaffold modules loaded with different cells are obtained.

[0050] (5) Scaffold assembly: In the clean bench, the scaffold modules loaded with different cells are spliced ​​together by the buttons and keyways. Pure bone marrow mesenchymal stem cell three-dimensional scaffolds, pure endothelial cell three-dimensional scaffolds, multi-cell three-dimensional platforms with stem cells and endothelial cells distributed alternately can be assembled, or three-dimensional scaffolds loaded with other cells can be designed and assembled according to the needs.

[0051] The following specific embodiments further illustrate the detailed fabrication method of the modular helical leaf-like three-dimensional scaffold loaded with living cells according to the present invention. It should be understood that the following embodiments are only for further illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range, and those skilled in the art can select within a suitable range based on the description herein, without being limited to the specific values ​​in the examples below.

[0052] Example 1

[0053] Fabrication of a modular, helical-blade-like three-dimensional scaffold for β-tricalcium phosphate bioceramic: (1) Weigh 45 g of β-phase tricalcium phosphate ceramic powder and 55 g of photosensitive resin and add them to a ball mill jar. Stir for 5 hours using a planetary ball mill and filter to obtain a uniform 3D printing slurry.

[0054] (2) Pour the slurry into the feed tank of the photopolymer 3D printer, load the designed slicing program, print out β-tricalcium phosphate ceramic green body, wash the slurry residue on the surface of the support module with ultrapure water and let it dry.

[0055] (3) The green support blank is placed in a crucible and sintered in a high-temperature furnace. The sintering temperature of the β-tricalcium phosphate support module is 1150℃ and the sintering time is 3 hours, resulting in ceramic support modules of different shapes (such as...). Figure 3 ).

[0056] (4) By assembling the sintered support modules, different three-dimensional supports for simulated helical blades can be obtained (such as...). Figure 4 ).

[0057] Example 2

[0058] A modular, spiral-blade-like three-dimensional scaffold fabricated from β-tricalcium phosphate bioceramics was used to investigate the interaction between cells and the extracellular matrix.

[0059] The sintered helical blade three-dimensional support module was obtained by the method in Example 1, and then dried after high-temperature sterilization for later use.

[0060] The sterilized scaffold modules were washed three times with sterile PBS in a laminar flow hood and then placed in 48-well cell culture plates, one scaffold module per well. Bone marrow mesenchymal stem cells were uniformly dispersed in α-MEM complete medium at a cell density of 5000 cells / mL to obtain a bone marrow mesenchymal stem cell suspension; cultured endothelial cells were uniformly dispersed in endothelial cell complete medium at a cell density of 15000 cells / mL to obtain an endothelial cell suspension. 1 mL of each of the two cell suspensions was added to each well of the plate containing the scaffold module, and the 48-well plate was then incubated at 37°C.

[0061] After incubating in an incubator for 4 hours, remove the 48-well plate. Figure 4 As shown, modules loaded with bone marrow mesenchymal stem cells were sequentially assembled to form a three-dimensional culture platform for bone marrow mesenchymal stem cells, and modules loaded with endothelial cells were sequentially assembled to form a three-dimensional culture platform for endothelial cells. The assembled modular three-dimensional scaffolds were placed in new 48-well culture plates, with 1 mL of the corresponding culture medium added to each well, and then placed in an incubator for continued culture, with the medium changed every other day.

[0062] The interaction between cells and the extracellular matrix on a three-dimensional endothelial cell culture platform was observed: the proliferative activity of cells on the scaffold on days 1, 3, and 7 was detected using a CCK-8 assay kit (e.g., Figure 5 (A) The angiogenic differentiation of endothelial cells on the scaffold was characterized by RT-qPCR experiments (e.g., Figure 5 The leaf orientation (B-E) was used to observe the angiogenic differentiation and adhesion of endothelial cells, combined with immunofluorescence staining (F-G in Figure 5). The results showed that, under the condition of the same scaffold shape, the orientation of the leaves significantly affected the proliferation and functional differentiation of endothelial cells, with leaves with 50% orientation having the most significant effect on promoting endothelial cell proliferation and enhancing their angiogenic differentiation.

[0063] The interaction between cells and the extracellular matrix on a three-dimensional culture platform for bone marrow mesenchymal stem cells was observed: RT-qPCR experiments were used to characterize the mechanosensory function of bone marrow mesenchymal stem cells on the scaffold (e.g., Figure 5 J-N), and combined with immunofluorescence staining to observe the adhesion of stem cells (e.g. Figure 5 The results showed that the shape of the scaffold could affect cell mechanosensory and influence cell adhesion through mechanotransduction; among them, the C40 curvature leaflets had the most significant effect on activating stem cell mechanotransduction and enhancing cell adhesion. Cell proliferation activity on the scaffold at days 1, 3, and 7 was detected using a CCK-8 assay kit. Figure 5 The osteogenic differentiation of stem cells on the scaffold was characterized using RT-qPCR experiments (e.g., O). Figure 5The P), and combined with immunofluorescence staining to observe the osteogenic differentiation of stem cells (e.g. Figure 5 The results showed that, among the effects of scaffold structural parameters on stem cell proliferation and osteogenic differentiation, C40 curvature and 50% orientation had the best promoting effect, indicating that this structural parameter can significantly promote stem cell proliferation and osteogenic differentiation by activating the mechanotransduction of stem cells.

[0064] Example 3

[0065] A modular, spiral-shaped three-dimensional scaffold fabricated from β-tricalcium phosphate bioceramics was used to investigate the chemotactic migration of bone marrow mesenchymal stem cells and endothelial cells.

[0066] Using the method described in Example 2, a C40 curvature was optimized to obtain modular bioactive scaffolds loaded with stem cells and modular bioactive scaffolds loaded with endothelial cells. These scaffolds were assembled in a bottom-to-top sequence of "stem cells-endothelial cells-stem cells-endothelial cells" to form multicellular three-dimensional platforms with different orientations. Each scaffold group was placed in a 48-well plate, and sufficient mixed culture medium was added at an ECM:α-MEM ratio of 1:1. The plates were then placed in a cell culture incubator for in vitro culture, with the medium changed every other day.

[0067] The paracrine signaling between bone marrow mesenchymal stem cells and endothelial cells was observed. For example... Figure 6 As shown in Figure C, ELISA results indicated that after 6 hours of co-culture, the levels of the chemokine SDF-1 secreted by endothelial cells on scaffolds with different orientations differed; after 7 days of culture, the levels of the CXCR4 receptor protein on the surface of stem cells also showed significant differences (e.g., Figure 6 (A and B) This indicates that different scaffold orientations, in addition to affecting the functional differentiation of individual cells, also influence paracrine signaling between different cell types. The distribution of stem cell numbers at different locations after 48 hours of culture (e.g., A and B) was observed. Figure 6 The D-F analysis revealed that the stem cell density was relatively uniform in the center of the leaf blades of different orientations, while a significant density difference was observed at the leaf edges. Simultaneously, the migration distance of cells at the leaf lateral margins also differed significantly, with the migration of stem cells on the 50% orientation scaffold surface being relatively more pronounced. This indicates that different scaffold orientations have varying degrees of influence on intercellular paracrine signaling pathways, with the 50% orientation promoting chemotactic distribution of stem cells by activating the SDF-1 / CXCR4 signaling axis between endothelial cells and stem cells.

[0068] Further observation of cell chemotaxis and migration over a longer period (e.g.) Figure 7The study (A-C) found that endothelial cells grew and migrated relatively slowly, while stem cells were able to grow upwards across modules to reach the endothelial cell module, which corresponds to the delayed vascularization that may occur during bone repair. The migration of stem cells also differed under different spatial orientations: in the 50% orientation, significantly more stem cells migrated towards endothelial cells over a significantly longer distance, exhibiting vigorous migration activity and dense contact. These results indicate that the spatial structure constructed with a 50% orientation can better ensure the diffusion and reception of chemokines, providing the best paracrine stimulation effect for cell chemotactic migration and promoting stem cell homing.

[0069] The above results indicate that the modular helical blade-like three-dimensional scaffold can be used to study the interaction between bone marrow mesenchymal stem cells and endothelial cells. Different orientation spaces have different paracrine stimulation effects on the two types of cells, resulting in significant differences in their chemotactic migration, which in turn leads to different vascularization and osteogenic microenvironments and may affect the final vascularization and osteogenic effect.

[0070] Example 4

[0071] A modular, spiral-shaped three-dimensional scaffold fabricated from β-tricalcium phosphate bioceramics was used to investigate the interaction between bone marrow mesenchymal stem cells and endothelial cells under interstitial flow stimulation.

[0072] A three-dimensional spiral-shaped scaffold loaded with bone marrow mesenchymal stem cells and endothelial cells was obtained using the method described in Example 3. The preferred curvature and orientation were C40-50. A C40-50 multi-cell three-dimensional co-culture platform was constructed by alternating layers of stem cells and endothelial cells to investigate the effect of mesenchymal flow on the interaction between the two cell types in the three-dimensional space constructed by this scaffold. The constructed three-dimensional scaffold platform was placed in a 48-well plate, and sufficient mixed culture medium was added. The static group was incubated statically, while the dynamic group was placed on a shaker with slow shaking. Both were placed in a cell culture incubator for in vitro culture, with the medium changed every other day.

[0073] First, we observed the migration of stem cells across modules to endothelial cells and their subsequent contact with each other (e.g. Figure 8 (A-C). The results showed that under mesenchymal flow stimulation, the rate of migration of stem cells and endothelial cells towards each other was significantly faster than in the static group, and the number of cells that made direct contact with each other was also greater. Under mesenchymal flow stimulation, the Piezo 1 protein level in bone marrow mesenchymal stem cells was significantly increased (e.g., A-C). Figure 8 (D-E), and stem cells can establish normal contact communication with endothelial cells earlier, such as mitochondrial transfer (e.g., D-E), and stem cells can establish normal contact communication with endothelial cells earlier (e.g., D-E). Figure 8 The F1 cells provide energy to endothelial cells, supporting their normal life activities. With enhanced contact communication, the osteogenic differentiation capacity of bone marrow mesenchymal stem cells (e.g., [missing information]) is stimulated by mesenchymal flow. Figure 8G, I-J) and the angiogenic differentiation capacity of endothelial cells (e.g. Figure 8 Both H and K-L were improved to some extent.

[0074] The above results collectively indicate that the C40-50 helical blade platform regulates the flow of interstitial fluid within its internal flow field through spatial management. Interstitial fluid stimulation can activate the mechanosensory perception of stem cells in their surrounding hydrodynamic microenvironment, enhance the expression level of mechanotransduction proteins, and accelerate the migration of stem cells to endothelial cells. Furthermore, through synergistic paracrine spatial chemotaxis, it enables the two cell types to establish multiple communications more quickly, thereby effectively promoting information exchange and enhancing osteogenic differentiation of bone marrow mesenchymal stem cells and angiogenic differentiation of endothelial cells.

Claims

1. A modular three-dimensional support for a helical blade, characterized in that, The modular helical blade-like three-dimensional scaffold includes an overall scaffold frame with a three-dimensional helical structure formed by assembling multiple scaffold module units with blades, and cells distributed in layers within the three-dimensional space of the overall scaffold frame.

2. The modular three-dimensional support for a helical blade as described in claim 1, characterized in that, The bladed support module unit includes a central entity, blades disposed on the outer periphery of the central entity and evenly distributed around the axis of the central entity, and keys and keyways located on the upper and lower end faces of the central entity.

3. The modular helical blade-like three-dimensional support according to claim 1 or 2, characterized in that, The central entity is a central cylinder, and the blades are planar sheet-like fan rings and / or cylindrical sheet-like fan rings; preferably, the number of blades is n, and the central angle of the fan ring of the blades is 180° / n.

4. The modular helical blade-like three-dimensional support according to any one of claims 1 to 3, characterized in that, The central cylinder has a diameter of 2-3 mm and a height of 1-2 mm; the key has a width of 0.8-1.1 mm, a length of 1-1.3 mm, and a height of 0.8-0.9 mm; the keyway has a width of 0.9-1.2 mm, a length of 1.2-1.5 mm, and a depth of 0.9-1 mm; both the key and the keyway have their major axes along their respective long sides, and the angle between the major axes of the key and the keyway is 0-180°; the blade has a central angle of 45°, an outer arc diameter of 6-8 mm, a thickness of 0.4-0.5 mm, and a blade edge cylindrical surface curvature of 0-0.67 mm in the thickness direction. -1 .

5. The modular helical blade-like three-dimensional support according to any one of claims 1 to 4, characterized in that, The bladed support module units are independently distributed in alternating and staggered layers, and the area overlap rate between adjacent layers of the blades in the axial direction of the assembled three-dimensional support is 0-100%.

6. The three-dimensional support for the modular helical blade according to any one of claims 1 to 5, characterized in that, The three-dimensional scaffold is made of bioactive ceramics, organic matter, bioinert ceramics, or a composite of organic matter and bioactive ceramics, preferably tricalcium phosphate in the β phase.

7. The three-dimensional support for the modular helical blade according to any one of claims 1 to 6, characterized in that, The cells are bone marrow mesenchymal stem cells or endothelial cells; preferably, the cells loaded on the modular spiral blade-like three-dimensional scaffold are endothelial cells and bone marrow mesenchymal stem cells, and the scaffold module units loaded with endothelial cells and bone marrow mesenchymal stem cells are arranged in alternating layers.

8. The modular helical blade-like three-dimensional support according to any one of claims 1 to 7, characterized in that, By changing the fluid velocity and direction in the space where the scaffold is located, the overall spatial flow field microenvironment of the cells is altered, thereby constructing a three-dimensional multi-cell co-culture system with controllable flow field, promoting communication between different cells and the interaction between cells and the fluid microenvironment.

9. A method for preparing a three-dimensional support for a modular helical blade according to any one of claims 1 to 8, characterized in that, The preparation method includes the following steps: thoroughly mixing the material of the three-dimensional scaffold with photosensitive resin to obtain a printing slurry; using photocuring to print a ceramic scaffold green body; sintering the printed ceramic scaffold green body to obtain a ceramic scaffold module unit; sterilizing the sintered ceramic scaffold module unit and then using it to load cells; selecting scaffold module units of specific shapes as needed and seeding them with bone marrow mesenchymal stem cells or endothelial cells respectively; after the cells adhere, assembling all the scaffold module units into a complete, modular, spiral-shaped three-dimensional scaffold with a design orientation and loaded with living cells.

10. The use of a modular helical blade-like three-dimensional scaffold according to any one of claims 1 to 8 in the preparation of drugs or products for drug screening, multi-cell co-culture models and complex tissue regeneration.

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