Modular bioprinted multicellular scaffold and methods of making and using the same

Abstract

This invention relates to a modular bioprinted multicellular scaffold, its preparation method, and its applications. The modular bioprinted multicellular scaffold is constructed by assembling cell-carrying modular units using a dynamic dual-network splicable bio-ink equipped with cells via bio-3D printing technology. The modular bioprinted multicellular scaffold allows for flexible adjustment of its key spatiotemporal characteristics, including biophysical, biochemical, and biological properties, by modifying the bio-ink composition, cell type, and density of each cell-carrying modular unit. This invention provides a novel strategy for promoting complex tissue regeneration and organ reconstruction.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and relates to a modular bioprinted multicellular scaffold, its preparation method, and its application. Background Technology

[0002] The human body is an organism composed of various tissues and organs, which are formed by the orderly assembly of various tissue cells and extracellular matrix in a three-dimensional environment. The inherent biological (e.g., cell type, cell density), biochemical (e.g., protein content, inorganic minerals), and biophysical (e.g., matrix stiffness, orientation, porosity) characteristics of tissues / organs are crucial for maintaining tissue and organ homeostasis and normal physiological function. However, these complex and orderly physiological characteristics are often disrupted after tissue trauma or disease, making effective tissue regeneration a significant challenge. Tissue engineering aims to create in vitro biomimetic engineered artificial grafts to induce in vivo tissue regeneration, with 3D bioprinting emerging as a transformative method capable of precisely arranging cells, biomaterials, and growth factors in three-dimensional space. Currently, many complex biomimetic structures, such as artificial skin, artificial hearts, and artificial lungs, have been developed based on bioprinting technology, demonstrating promising potential for in vivo regeneration and repair. However, existing bioprinting strategies have inherent limitations, making it difficult to simultaneously achieve key physiological characteristics such as multicellular composition, hierarchical component distribution, and structural complexity. For example, extrusion bioprinting typically only produces simple structural scaffolds, such as mesh-like and layered structures, and struggles to fabricate complex gradient scaffolds. While light-based bioprinting strategies, such as photopolymerization, can construct more complex structures at higher resolutions, they struggle to integrate multiple tissue cells simultaneously, leading to significant compromises in mimicking certain physiological characteristics of natural tissues (such as loaded cell types or structural complexity). Therefore, there is an urgent need to develop a universal and simple engineering strategy to fabricate large-scale, highly biomimetic multicellular constructs for complex tissue regeneration. Summary of the Invention

[0003] To address the aforementioned problems, this invention aims to provide a modular bioprinted multicellular scaffold, its preparation method, and its applications. This invention proposes constructing a static-dynamic dual-network splicable bio-ink carrying cells, using bio-3D printing technology to prepare cell-carrying modular units, which are then rationally spliced ​​and assembled. Utilizing the inherent self-healing properties of the static-dynamic dual-network bio-ink, these modular units are assembled into a whole, and further photocrosslinked and cured to form a stable multicellular scaffold. This invention successfully prepares various multicellular scaffolds through a modular bioprinting splicing strategy and applies them to the regeneration of complex tissues such as bone and cartilage.

[0004] In a first aspect, the present invention provides a modular bioprinted multicellular scaffold. The modular bioprinted multicellular scaffold is a modular bioprinted multicellular scaffold obtained by assembling cell-carrying modular units prepared using bio-3D printing technology with intrinsically self-healing properties and carrying cells through a static-dynamic dual-network splicable bio-ink. Preferably, the splicing is a physical splicing.

[0005] In an optional embodiment, the static-dynamic dual-network splicable bio-ink comprises a static-dynamic dual-network hydrogel capable of forming a static cross-linked network and a dynamic cross-linked network, and cells uniformly dispersed in the dual-network hydrogel; wherein the dual-network hydrogel comprises a static cross-linked network gel matrix and a dynamic cross-linked network gel matrix.

[0006] In optional embodiments, the static crosslinking network is a three-dimensional network capable of forming at least one of irreversible chemical bonds or stable physical effects; preferably, the irreversible chemical bonds form stable chemical bonds under light irradiation; more preferably, the static crosslinking network gel matrix includes at least one of methacrylated gelatin, methacrylated hyaluronic acid, methacrylated silk fibroin, and methacrylated sodium alginate, preferably methacrylated gelatin; even more preferably, the mass-volume concentration of the static crosslinking network gel matrix is ​​1%~20% (W / V).

[0007] In optional embodiments, the dynamic cross-linked network is a three-dimensional network formed through reversible cross-linking, capable of dynamically breaking and forming cross-links, including at least one of metal coordination chelation, Schiff base reaction, hydrogen bonding, electrostatic interaction, and host-guest interaction, preferably metal coordination chelation; more preferably, the dynamic cross-linked network gel matrix is ​​composed of metal ions and polymers containing specific ligand groups capable of chelating with metal ions, wherein the metal ions are at least one of copper, zinc, magnesium, strontium, and silver ions, and the polymers containing specific ligand groups are polymers having at least one ligand group selected from bisphosphate, histidine, catechol, and thiol groups, particularly preferably thiol-modified gelatin; even more preferably, the concentration of the metal ions is 0.1~50 mM, and the mass-volume concentration of the polymers containing specific ligand groups is 0.5%~5% (W / V).

[0008] In an optional embodiment, the bio-3D printing technology is at least one of extrusion 3D printing, projection photopolymerization 3D printing, surface projection photopolymerization 3D printing, and inkjet bio-3D printing.

[0009] In an optional embodiment, the cells include at least one of neural stem cells, bone marrow mesenchymal stem cells, endothelial cells, Schwann cells, chondrocytes, and tendon stem cells; preferably, the cell density is 1×10⁻⁶.7 ~5×10 8 per mL.

[0010] In an optional implementation, the key biophysical, biochemical, and biological characteristics of modular bioprinted multicellular scaffolds can be precisely controlled by adjusting the composition of the static-dynamic dual-network splicable bio-ink carrying cells, as well as the type and density of the cells.

[0011] In an optional embodiment, the modular bioprinted multicellular scaffold is a modular bioprinted multicellular scaffold with a multi-level structure and multi-cell spatial distribution; preferably, the modular bioprinted multicellular scaffold is a biomimetic osteochondral multicellular construct, formed by stacking five cell-carrying module units layer by layer; the bio-ink of the first cell-carrying module unit from top to bottom (forming the cartilage layer) includes 3%~20% methacrylated gelatin, 0.5%~5% thiol-modified gelatin, 0.1~20 mM zinc ions, and 1×10 7 ~8×10 7 The second cell-carrying module unit contains 3%–20% methacrylated gelatin, 0.5%–5% thiol-modified gelatin, 0.1–10 mM zinc ions, and 1 × 10⁻⁶ chondrocytes / mL. 7 ~5×10 7 The cell-carrying module unit contains chondrocytes per mL. The third cell-carrying module unit's bio-ink (forming the interface layer) includes 5%–20% methacrylated gelatin and 1%–5% thiol-modified gelatin. The fourth cell-carrying module unit's bio-ink (forming the subchondral bone layer) includes 5%–20% methacrylated gelatin, 0.5%–5% thiol-modified gelatin, 0.1–10 mM copper ions, and 1×10⁻⁶ ions. 7 ~5×10 7 Bone marrow mesenchymal stem cells per mL, the fifth cell-carrying module unit's bio-ink (forming the subchondral bone layer) includes 5%–20% methacrylated gelatin, 0.5%–5% thiol-modified gelatin, 0.1–20 mM copper ions, and 1×10 7 ~8×10 7 Bone marrow mesenchymal stem cells per mL.

[0012] In an optional embodiment, the zinc ion content of the bio-ink in the second cell-carrying module unit is lower than that in the first cell-carrying module unit, and the chondrocyte content of the bio-ink in the second cell-carrying module unit is lower than that in the first cell-carrying module unit.

[0013] In an optional embodiment, the bio-ink of the third cell-carrying module unit has a higher content of methacrylated gelatin than the bio-ink of the second cell-carrying module unit, and the bio-ink of the third cell-carrying module unit has a higher content of methacrylated gelatin than the bio-ink of the first cell-carrying module unit.

[0014] In an optional embodiment, the copper ion content of the bio-ink in the fifth cell-carrying module unit is higher than that in the bio-ink in the fourth cell-carrying module unit, and the bone marrow mesenchymal stem cell content of the bio-ink in the fifth cell-carrying module unit is higher than that in the bio-ink in the fourth cell-carrying module unit.

[0015] In an optional embodiment, the methacrylated gelatin content of the bio-ink in the third cell-carrying module unit is lower than that in the fourth cell-carrying module unit, and the methacrylated gelatin content of the bio-ink in the third cell-carrying module unit is lower than that in the fifth cell-carrying module unit.

[0016] Secondly, the present invention provides a method for preparing the modular bioprinted multicellular scaffold. The method includes the following steps: uniformly mixing a static-dynamic dual-network hydrogel with cells to obtain a cell-carrying static-dynamic dual-network splicable bio-ink; using bio-3D printing technology to prepare cell-carrying module units from the cell-carrying static-dynamic dual-network splicable bio-ink; assembling the cell-carrying module units, and after self-healing fusion and covalent cross-linking curing of each cell-carrying module unit, obtaining a modular bioprinted multicellular scaffold with key biomimetic biophysical, biochemical, and biological characteristics.

[0017] Thirdly, the present invention provides the application of the modular bioprinted multicellular scaffold in constructing biomimetic in vitro multicellular models and in vivo complex tissue regeneration and organ reconstruction. Beneficial effects

[0018] This invention combines modular assembly with bio-3D printing technology to fabricate a biomimetic multicellular scaffold, effectively simulating the key biophysical, biochemical, and biological characteristics of natural complex tissues simultaneously, providing a new strategy for complex tissue regeneration and organ reconstruction. This invention relates to a modular bioprinted multicellular scaffold, its fabrication method, and its applications. Attached Figure Description

[0019] Figure 1This document presents the design and characterization of a dynamic dual-network bio-ink based on GM, G-SH, and copper ions. A shows a schematic diagram of bio-ink combinations with different G-SH and copper ion concentrations. B shows strain scanning and creep experiments of bio-inks with 6% GM, 1% G-SH, and different copper ion concentrations. C shows strain scanning and creep experiments of bio-inks with 6% GM, 2% G-SH, and different copper ion concentrations. B shows strain scanning and creep experiments of bio-inks with 6% GM, 3% G-SH, and different copper ion concentrations. EF shows the modular unit assembly and pourable modular unit assembly of the bio-ink with components of 6% GM, 2% G-SH, and 10 mM copper ions. G shows the assembled images of bio-ink modules with gradient copper ion and gradient G-SH concentrations.

[0020] Figure 2 The printability and splicing characterization of dynamic dual-network bio-inks based on GM, G-SH and copper ions are shown in Figure A. Printed images of 12 bio-inks are shown in Figure B. Statistical analysis of printability is shown in Figure C. Unit diagram of splicing printing module is shown in Figure D. Diagram of bioprinting module, splicing, photocrosslinking and stability test are shown in Figure D.

[0021] Figure 3 The preparation and characterization of dynamic dual-network bio-inks based on GM, G-SH and different metal ions are shown in Figure A, which is a schematic diagram of the dynamic dual-network bio-ink; B and E are strain scanning and creep experiments of dynamic bio-inks based on zinc, silver, magnesium and strontium ions, respectively; FG is a modular unit splicing diagram and grayscale analysis based on five ions: zinc, copper, magnesium, silver and strontium.

[0022] Figure 4 This study focuses on the preparation and characterization of dynamic dual-network bio-inks based on silicate biomaterials. A shows the creep recovery experiment of bio-inks with compositions of GM, G-SH, and copper silicate, zinc silicate, strontium silicate, and magnesium silicate, respectively. B shows the preparation and splicing diagram of four bioprinting module units.

[0023] Figure 5 The preparation and characterization of a dynamic dual-network bio-ink composed of HAMA, G-SH, and copper ions are shown in Figure A. Strain scanning and creep recovery experiments of the bio-ink are shown in Figure B. The preparation, splicing, and stability test images of the module units are shown in Figure B.

[0024] Figure 6 The preparation and characterization of a dynamic dual-network bio-ink composed of SilMA, G-SH, and copper ions are shown in Figure A. Strain scanning and creep recovery experiments of the bio-ink are shown in Figure B. The preparation, splicing, and stability test images of the module units are shown in Figure B.

[0025] Figure 7The preparation and characterization of a dynamic dual-network bio-ink composed of GM, Gelatin-PBA, and PVA includes dynamic borate ester reaction to impart dynamism, creep experiments, and a photograph of the assembled scaffold.

[0026] Figure 8 The preparation and characterization of a dynamic dual-network bio-ink composed of HAMA, ODex, and CMCS includes dynamic Schiff base reaction to impart dynamism, creep experiments, and a photograph of the assembled scaffold.

[0027] Figure 9 Design and characterization of bioprinted spliced ​​neural bone multicellular scaffolds; A is the spliced ​​image; B is a spatial distribution image of neural stem cells and bone marrow mesenchymal stem cells, with red representing neural stem cells and green representing bone marrow mesenchymal stem cells; C is an image of live and dead cell staining of neural stem cells and bone marrow mesenchymal stem cells, with green representing live cells and red representing dead cells; D is an image of immunofluorescence protein staining of neural stem cells and bone marrow mesenchymal stem cells.

[0028] Figure 10 Design and characterization of bioprinted neuromuscular multicellular scaffolds; A is the assembled image; B is a spatial distribution image of neural stem cells and muscle cells, with magenta representing neural stem cells and indigo representing muscle cells; C is an image of live and dead cell staining of neural stem cells and muscle cells, with green representing live cells and red representing dead cells; D is an image of immunofluorescence protein staining of neural stem cells and muscle cells.

[0029] Figure 11 Design and characterization of a bioprinted multicellular scaffold for assembling a neurally innervated musculoskeletal system; A is the assembled image; B shows the survival rate of four cell types; C shows images of live and dead cells of the four cell types, with green representing live cells and red representing dead cells; D shows images of immunofluorescent protein staining of the four cell types.

[0030] Figure 12 The images show the design, fabrication, and characterization of a biomimetic osteochondral multicellular scaffold; AB shows fluorescence images and statistical results of Runx2 and OPN proteins, osteogenic markers of bone marrow mesenchymal stem cells, in scaffolds with different copper ion concentrations; CD shows physical images and statistical results of alkaline phosphatase staining and Alizarin Red staining in scaffolds with different copper ion concentrations; EF shows fluorescence images and statistical results of Aggrecan and COL-II proteins in chondrocytes in scaffolds with different zinc ion concentrations; G is a schematic diagram of the fabrication of the biomimetic osteochondral multicellular scaffold, including printing modular units, splicing assembly, and photocrosslinking.

[0031] Figure 13Biological characterization of bioprinted, assembled biomimetic osteochondral multicellular scaffolds; A is a fluorescence image of cell distribution in the multicellular scaffold, where red represents chondrocytes and green represents bone marrow mesenchymal stem cells; B is a live-dead cell staining image of chondrocytes and bone marrow mesenchymal stem cells in the multicellular scaffold, where green represents live cells and red represents dead cells; C is the cell viability of chondrocytes and bone marrow mesenchymal stem cells.

[0032] Figure 14 Characterization of in vivo repair of osteochondral defects using bioprinted spliced ​​biomimetic osteochondral multicellular scaffolds; A shows macroscopic images and Micro-CT reconstructions of osteochondral tissue at 6 and 12 weeks post-surgery; BC shows the volume fraction of newly formed bone and statistical data on bone mineral density; D shows the ICRS score.

[0033] Figure 15 Histological staining and mechanical characterization of bioprinted spliced ​​biomimetic osteochondral multicellular scaffolds for in vivo repair of osteochondral defects; A shows the histological section staining at 6 and 12 weeks post-operation, including H&E, Masson, and SF; BD shows the force-displacement curves and statistical results of modulus and hardness obtained using nanoindentation testing. Detailed Implementation

[0034] The present invention is further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0035] The modular bioprinted multicellular scaffold is a modular bioprinted multicellular scaffold obtained by assembling cell-carrying module units prepared by bio-3D printing technology using static-dynamic dual-network splicable bio-ink carrying cells.

[0036] The dynamic dual-network splicable bio-ink comprises a static-dynamic dual-network hydrogel capable of forming both a static and a dynamic cross-linking network, and cells uniformly dispersed within the static-dynamic dual-network hydrogel. The static-dynamic dual-network hydrogel includes a static cross-linking network gel matrix and a dynamic cross-linking network gel matrix.

[0037] In other words, the static-dynamic dual-network splicable bio-ink comprises a static-dynamic dual-network hydrogel and (tissue) cells mounted on the hydrogel. This static-dynamic dual-network splicable bio-ink can form a dual-network structure including a static cross-linking network and a dynamic cross-linking network, exhibiting excellent printability and self-healing properties. Both the static and dynamic cross-linking networks contribute to the excellent printability and self-healing properties of the dynamic dual-network splicable bio-ink. Precise control of the biophysical, biochemical, and biological characteristics of multicellular scaffolds can be achieved by adjusting the composition of the static-dynamic dual-network splicable bio-ink and the types and densities of the tissue cells.

[0038] The static cross-linked network is a three-dimensional network capable of forming at least one of irreversible chemical bonds or stable physical interactions. Preferably, the irreversible chemical bonds are stable chemical bonds formed under light irradiation. Preferably, the light irradiation is blue light. The static cross-linked network gel matrix (the polymeric material forming the static cross-linked network) includes, but is not limited to, at least one of methacrylated gelatin (GM), methacrylated hyaluronic acid (HAMA), methacrylated silk fibroin (SilMA), and methacrylated sodium alginate, preferably methacrylated gelatin (GM). In an optional embodiment, the static cross-linked network gel matrix accounts for 1% to 20% (wV, g / mL) of the bioink. In an optional embodiment, the degree of substitution of methacrylated gelatin is 30% to 90%, and the concentration is 1% to 20%.

[0039] The dynamic cross-linked network is a three-dimensional network formed through reversible cross-linking, capable of dynamically breaking and forming cross-links, including at least one of metal coordination chelation, Schiff base reaction, hydrogen bonding, electrostatic interaction, and host-guest interaction, preferably metal coordination chelation. The metal coordination chelation consists of metal ions and a polymer containing specific ligand groups. The metal ions are at least one of copper, zinc, magnesium, strontium, and silver ions, preferably copper and zinc ions. In an optional embodiment, the concentration of metal ions in the bio-ink is 0.1~50 mM. The polymer containing specific ligand groups is a polymer material modified with at least one ligand group such as bisphosphate, histidine, catechol, or thiol groups, capable of chelating with metal ions. The mass-volume concentration of the polymer containing specific ligand groups in the bio-ink can be 0.5%~5% (WV, g / mL). The ligand group is preferably a thiol group. The polymer containing specific ligand groups is preferably thiol-modified gelatin (G-SH). More preferably, the concentration of thiol-modified gelatin (G-SH) is 0.5% to 5%.

[0040] The cells include at least one of neural stem cells, bone marrow mesenchymal stem cells, endothelial cells, Schwann cells, chondrocytes, and tendon stem cells, preferably bone marrow mesenchymal stem cells and chondrocytes. The cell density relative to the concentration of the bio-ink can be 1 × 10⁻⁶. 7 ~5×10 8 per mL.

[0041] The bio-3D printing technology is at least one of extrusion 3D printing, projection photopolymerization 3D printing, surface projection photopolymerization 3D printing, and inkjet bio-3D printing.

[0042] The modular bioprinted multicellular scaffold is composed of multiple bioprinted cell-carrying module units. The shape, size, structure, and composition of each unit can be flexibly designed according to actual needs, and they can be assembled into a personalized three-dimensional complex multicellular scaffold through various assembly methods such as layer stacking and side-by-side splicing.

[0043] The assembly process involves customizing and assembling each cell-carrying module according to specific requirements. Then, leveraging the intrinsic self-healing properties of the bio-ink, the interfaces between the modules fuse together to form a unified multi-cell scaffold. Precise control over the key biophysical, biochemical, and biological characteristics of the assembled multi-cell scaffold is achieved by adjusting the composition of the dynamic dual-network splicable bio-ink and the types and densities of tissue cells.

[0044] Therefore, this invention utilizes bio-3D printing technology to prepare cell-carrying modular units of different components, shapes, and sizes. These units are then rationally assembled to obtain multi-cell scaffolds with personalized multi-level structures and multi-cell spatial distributions. The modular bioprinted multi-cell scaffold can flexibly adjust its biophysical, biochemical, and biological spatiotemporal key characteristics by modifying the bio-ink composition, cell type, and density of each module. This modular bioprinted multi-cell scaffold provides a novel strategy for promoting complex tissue regeneration and organ reconstruction.

[0045] This invention also proposes a method for preparing modular bioprinted multi-cell scaffolds. The preparation method includes the following steps: Step (1) Mix the dual-network hydrogel with cells to obtain a static-dynamic dual-network splicable bio-ink carrying cells; Step (2) Bio-3D printing technology is used to deposit bio-ink layer by layer to obtain a three-dimensional modular unit scaffold; Step (3) Assemble each modular unit bracket in a reasonable manner by stacking layers and splicing side by side, and perform self-healing in a suitable environment; Step (4) After the self-healing fusion and covalent cross-linking curing of each module unit, a multi-cell scaffold with key biomimetic biophysical, biochemical and biological characteristics is obtained.

[0046] In an optional implementation, the bio-3D printing technology in step (2) is extrusion bio-3D printing, with a pressure range of 10~100 kPa, an extrusion needle temperature of 15~22 ℃, a deposition stage temperature of 0~4℃, and a needle inner diameter of 150~500 μm.

[0047] In an optional implementation, the self-healing temperature in step (3) is set to 10~25℃ and the self-healing time is 10~30min.

[0048] In an optional embodiment, the covalent crosslinking is photocrosslinking curing, preferably using blue light of a specific wavelength, and the crosslinking time is 10~60 s. This results in a photo-driven covalently crosslinked curing network after self-healing.

[0049] This invention also provides the application of the modular bioprinted multicellular scaffold in constructing biomimetic in vitro multicellular models and in vivo complex tissue regeneration and organ reconstruction. The modular bioprinted multicellular scaffold is particularly useful in the field of regenerative medicine, especially in the construction of multicellular co-culture models and the regeneration of complex tissues.

[0050] As one specific implementation, the modular bioprinted multicellular scaffold is a biomimetic osteochondral multicellular scaffold. The dynamic dual-network splicable bio-ink is composed of copper / zinc ions, GM, and G-SH. The cells are bone marrow mesenchymal stem cells and chondrocytes. The biomimetic osteochondral multicellular construct is formed by stacking five bioprinted modular units, wherein the top two layers are simulated cartilage layers, the middle layer is an interface layer, and the bottom two layers are simulated subchondral bone layers. Preferably, from top to bottom, the first layer is composed of 6% GM, 2% G-SH, 2.5 mM zinc ions, and 5×10⁻⁶ ions. 7 The first layer was prepared by printing with bio-ink composed of chondrocytes per mL; the second layer consisted of 6% GM, 2% G-SH, 1 mM zinc ions, and 2.5 × 10⁻⁶ ions. 7 The first layer was prepared by printing with bio-ink composed of chondrocytes per mL; the second layer was prepared by printing with ink composed of 7% GM and 2% G-SH; the third layer was prepared by printing with ink composed of 8% GM, 2% G-SH, 0.5 mM copper ions and 2.5 × 10⁻⁶ mol / mL. 7 The fifth layer was prepared by printing with bio-ink composed of bone marrow mesenchymal stem cells per mL; the fifth layer was composed of 8% GM, 2% G-SH, 1 mM copper ions and 5×10 7 The bio-ink composed of bone marrow mesenchymal stem cells per mL was used for printing. After all five module units were printed, they were stacked layer by layer and then subjected to self-healing and photocuring cross-linking to obtain a biomimetic osteochondral multicellular scaffold.

[0051] Natural osteochondrium is known to be an anisotropic, gradient, multi-layered structure, mainly composed of cartilage, subchondral bone, and the interfacial layer between them. Mechanically, the cartilage layer is primarily composed of extracellular matrix mainly composed of type II collagen fibers and chondrocytes, exhibiting relatively low mechanical strength. The subchondral bone layer, on the other hand, is composed of extracellular matrix mainly composed of hydroxyapatite and type I collagen fibers, along with osteocytes, with the content of inorganic minerals gradually increasing. The osteochondral interface is a cell-free transitional layer. In summary, natural osteochondrium tissue exhibits a gradient, multi-level structure in terms of mechanical strength, chemical composition, cell types, and distribution. The focus of this invention is to employ a modular bioprinting strategy to construct a biomimetic multicellular scaffold that mimics several key characteristics of natural osteochondrium tissue for complex in vivo integrated osteochondrium regeneration.

[0052] The biomimetic osteochondral multicellular scaffold has some significant features and advantages: (1) In terms of biological characteristics, chondrocytes and bone marrow mesenchymal stem cells are distributed in a three-dimensional gradient, which highly simulates the cell distribution characteristics of natural osteochondral tissue; (2) In terms of biophysical characteristics, the concentration of static cross-linked network GM in the bio-ink increases from top to bottom, and the mechanical properties also increase accordingly, simulating the physiological and mechanical properties of natural osteochondral tissue; (3) In terms of biochemical characteristics, zinc / copper ions not only chelate with G-SH to mediate the self-healing behavior between modules, but the layered gradient distribution of zinc / copper ions can also act as a bioactive factor to regulate cell behavior. Among them, the zinc ions in the upper layer can effectively promote the maturation of chondrocytes and the secretion of extracellular matrix, while the copper ions in the lower layer can induce osteogenic differentiation and calcium deposition of bone marrow mesenchymal stem cells. The biomimetic osteochondral multicellular scaffold described in this invention can simulate the key physiological characteristics of natural osteochondral tissue in three dimensions: biophysical, biochemical and biological, and is expected to significantly promote the repair and regeneration of osteochondral tissue.

[0053] The following exemplifies a method for fabricating the modular bioprinted multicellular scaffold. The fabrication method includes the following steps: (1) GM, G-SH, copper / zinc ions, photoinitiator, chondrocytes and bone marrow mesenchymal stem cells were mixed to obtain bio-ink material; bio-ink materials corresponding to the cartilage layer, subchondral bone layer and interface layer were prepared according to the concentration and type of GM, copper / zinc ions and cells respectively. (2) Five kinds of bio-inks were deposited sequentially using extrusion bioprinting technology to obtain five kinds of bioprinted cell-carrying modular units; (3) The five modular units are stacked layer by layer according to the structural characteristics of osteocartilage tissue, and the modules are self-healed in a suitable temperature environment. Then, they are further cured by photocrosslinking to obtain a biomimetic multicellular construct.

[0054] In some embodiments, step (1) of the preparation of the bio-ink material includes the following process: lyophilized GM and a suitable amount of copper chloride are dissolved in a PBS solution containing the photoinitiator LAP, heated in a water bath to 50-60°C, and stirred for 1 hour until completely dissolved. The solution is then sterilized by passing it through a bacterial filter membrane (0.22 μm pore size), which is solution A. Lyophilized GM and a suitable amount of zinc chloride are dissolved in a PBS solution containing the photoinitiator LAP, heated in a water bath to 50-60°C, and stirred for 1 hour until completely dissolved. The solution is then sterilized by passing it through a bacterial filter membrane (0.22 μm pore size), which is solution B. Lyophilized GM is dissolved in a PBS solution containing the photoinitiator LAP, and heated in a water bath to 50-60°C, and stirred for 1 hour until completely dissolved. Solution C is prepared by heating to 50-60°C and stirring for 1 hour until completely dissolved, then sterilizing the solution by passing it through a bacterial filter membrane (0.22 μm pore size). Solution D is prepared by dissolving freeze-dried G-SH in PBS solution, heating in a water bath to 50-60°C, stirring for 1 hour until completely dissolved, then sterilizing the solution by irradiating it under a UV lamp (e.g., for 3 hours). Mixing solutions A and D with bone marrow mesenchymal stem cells yields osteogenic bioink composed of GM, G-SH, and copper ions. Mixing solutions B and D with chondrocytes yields chondrogenic bioink composed of GM, G-SH, and zinc ions. Mixing solutions C and D yields a cell-free interface bioink composed of GM and G-SH. These bioinks are stored at 37°C for later use.

[0055] In some embodiments, in step (2), the bio-3D printing is carried out by layer-by-layer deposition using an extrusion bioprinting device to prepare a three-dimensional modular unit scaffold. First, the ink is loaded into a barrel and pre-cooled in a 4°C refrigerator for pre-gelation. Then, it is loaded into the printer for printing. The extrusion pressure is set to 30~100 kPa, the barrel temperature to 15~25°C, the deposition stage temperature to 0~4°C, and the needle diameter to 200~500 μm. After each module is printed, it is transferred to a 4°C environment for short-term storage.

[0056] In some implementations, in step (3), the prepared modules are stacked layer by layer according to the physiological structural characteristics of natural osteochondrium using tweezers, and then placed in an environment of 10~15℃. The self-healing properties of the dynamic dual-network bio-ink are used to fuse the modules into a whole, and then blue light is used for further photocuring and crosslinking, with a crosslinking time of 10~60 s.

[0057] The biomimetic osteochondral multicellular construct was then placed in a 12-well plate, mixed cell culture medium (MEM-α and DMEM medium mixed at a 1:1 ratio) was added, and the plate was placed in a cell culture incubator for in vitro culture, with the medium changed every other day.

[0058] This invention is the first to combine modular assembly and splicing with bio-3D printing technology to construct a biomimetic multicellular construct, achieving a three-dimensional spatial arrangement of key biophysical (mechanical gradient caused by hydrogel concentration), biochemical (layered and gradient distribution of copper-zinc dual ions), and biological (layered and gradient distribution of chondrocytes and bone marrow mesenchymal stem cells). Furthermore, in the metal ion-based static-dynamic dual-network bio-ink designed in this invention, copper / zinc ions not only act as chelating agents, endowing the bio-ink with excellent self-healing properties, but also act as bioactive factors to regulate the bioactivity of chondrocytes and bone marrow mesenchymal stem cells, inducing chondrocyte maturation and extracellular matrix secretion, as well as the expression of osteogenic differentiation-related genes and proteins and calcium deposition in bone marrow mesenchymal stem cells.

[0059] In this invention, the modular bioprinted biomimetic multicellular scaffold possesses gradient biophysical, biochemical, and biological distribution characteristics, highly mimicking the physiological structure and composition of natural osteochondral tissue, and can simultaneously promote the regeneration of cartilage and subchondral bone. Compared to traditional biomimetic layered scaffolds that can only mimic some of the physical, chemical, or cellular distribution characteristics of natural osteochondral tissue, this invention has significant advantages.

[0060] The static-dynamic dual-network bio-ink designed in this invention possesses excellent printability, self-healing properties, and superior bioactivity. Specifically, the static network of methacrylamide gelatin (GM) provides structural stability and printability; while the dynamic chelating effect between thiol-modified gelatin (G-SH) and metal ions endows the bio-ink with excellent self-healing properties, allowing the printed modules to spontaneously fuse together to form a unified structure. Furthermore, the metal ions not only act as chelating agents but also as bioactive components, flexibly regulating cell behavior and endowing the bio-ink with excellent cell regulation functions. For example, zinc ions promote chondrocyte maturation and soft differentiation, while copper ions promote osteogenic differentiation and calcium deposition of bone marrow mesenchymal stem cells. Existing technologies use dual-channel printing technology to construct upper and lower layered biomimetic osteochondral scaffolds, exhibiting gradient layered structures and gradient mechanical properties. This is precisely what this invention refers to in the background section: existing printing technologies can only achieve simple layered structures, mimicking single physical, chemical, or biological characteristics of natural tissues. The modular splicing strategy described in this invention can simultaneously achieve integrated simulation of key biophysical, biochemical, and biological features, resulting in more complex biomimetic tissues and demonstrating significant innovation. Furthermore, traditional static network bio-inks, such as methacrylated gelatin, methacrylated hyaluronic acid, methacrylated silk fibroin, and methacrylated sodium alginate, possess good printability and shape fidelity, but lack self-healing or cell-regulating activity. While standalone dynamic networks may exhibit self-healing properties, they lack printability and shape fidelity, making them unsuitable for standalone bio-3D printing. The static-dynamic dual-network bio-ink described in this invention combines excellent printability, shape fidelity, self-healing properties, and bio-inducible activity. The static network GM exhibits printability and shape fidelity; the dynamic network of G-SH and metal ions possesses self-healing properties and bio-inducible activity. On one hand, the self-healing property effectively assembles individual modules into a whole; on the other hand, metal ions not only participate in dynamic self-healing as chelating agents but also regulate cell behavior as active components. For example, zinc ions induce chondrocyte maturation, i.e., chondrogenesis; copper ions induce osteogenic differentiation and calcium deposition in bone marrow mesenchymal stem cells.

[0061] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation 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; that is, those skilled in the art can make appropriate selections within the range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0062] Example 1

[0063] This embodiment provides the preparation of a static-dynamic dual-network-based bio-ink (cell-free) based on methacrylated gelatin (GM), thiol-modified gelatin (G-SH), and copper ions, comprising the following steps: (1) Weigh out GM dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55°C water bath for 1 hour until fully dissolved. Then weigh out copper chloride powder and dissolve it fully in the above solution. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). This is solution A. (2) Dissolve the freeze-dried G-SH in PBS solution, heat in a water bath to 60°C for 1 hour until completely dissolved, and then sterilize the solution by irradiating it under a UV lamp (e.g., for 3 hours) to obtain solution B. (3) Mix solutions A and B thoroughly at a volume ratio of 1:1 to obtain a static-dynamic dual-network bio-ink based on GM, G-SH, and copper ions. In the bio-ink, the concentration of copper ions is 1~10 mM, the mass-volume concentration of G-SH is 1%~3% (g / mL), the mass-volume concentration of GM gel is 6% (g / mL), and the mass-volume concentration of initiator is 0.25% (g / mL).

[0064] Modular scaffolds were fabricated using extrusion-based bio-3D printing, with the entire printing process conducted in a clean bench. First, ink was loaded into a barrel and pre-cooled at 4 °C for pre-gelation. Then, it was loaded into the printer for printing. The extrusion pressure was set to 30 kPa, the barrel temperature to 20 °C, the deposition stage temperature to 4 °C, and the needle diameter to 220 μm. After each module was printed, the scaffold was photocrosslinked by irradiating it with a 405 nm wavelength light source for 20 s.

[0065] For the assembly process of the modular units: after the modular units are printed, they are first stored at 4 ℃, and then the modular units are assembled together as needed using tweezers. They are then allowed to self-heal at 15 ℃ for 20 min, and finally cross-linked with blue light for 30 s to obtain the assembled complex structure scaffold.

[0066] Based on the concentrations of G-SH (1%, 2%, and 3%) and copper ions (1 mM, 2.5 mM, 5 mM, and 10 mM) in the dynamic dual-network bio-ink, 12 groups were designed, such as... Figure 1 As shown in A in the figure. The rheological and self-healing properties of 12 bio-inks were evaluated using a rotational rheometer, such as... Figure 1As shown in the BD diagram, strain scanning results indicate that at low strain, the storage modulus (G') of the bio-ink is greater than its loss modulus (G''), exhibiting a gel state that suggests suitability for extrusion bioprinting. Creep experiments show that at low strain, the storage modulus (G') of the 12 bio-inks is greater than its loss modulus (G''), while at high strain, the storage modulus (G') is less than its loss modulus (G''), indicating the self-healing properties of this dynamic dual-network bio-ink. Further characterization of its splicing and self-healing effects was conducted using bio-inks composed of 6% GM, 2% G-SH, and 10 mM copper ions. Figure 1 As shown in Figure E, multiple hydrogel modules can be easily assembled side-by-side into strip and rectangular structures, and after photocrosslinking, they form stable structures that can be picked up with tweezers. In addition, multiple hydrogel modules with hollow channel structures can be easily assembled into a Z-shape and achieve stable infusion of blue dye without leakage, further demonstrating the excellent self-healing effect of bio-ink. Furthermore, hydrogel modules with different copper ion concentrations and different G-SH concentrations were prepared and assembled into strips. After self-healing and photocrosslinking, the assembled hydrogels can be easily picked up with tweezers and gently shaken or pulled, exhibiting excellent structural stability.

[0067] Figure 2 Figure A shows physical images of bioprinting scaffolds made from 12 different bio-inks. The mesh-like structures of each group are clearly visible. Only the bio-inks composed of 6% GM, 3% G-SH, and 10mM copper ions exhibit slightly poorer printability, with less clear structural outlines. Printability statistics show that all 12 bio-inks maintain good printability, with Pr values ​​close to 1 (…). Figure 2 (B) Further, a bio-ink composed of 6% GM, 2% G-SH, and 10mM copper ions was selected as an example for printing and assembling modular units. For example... Figure 2 As shown in Figure C, square and annular modular units were successfully fabricated and assembled in an orderly manner through layer-by-layer stacking and nesting. After self-healing and photocrosslinking curing, they formed a whole scaffold, maintaining structural integrity. To demonstrate the superiority of this modular splicing strategy, nine modular units were printed and then arranged side by side to form a large scaffold in a nine-square grid shape. After self-healing and photocrosslinking, a whole scaffold was obtained. It was found that the scaffold composed of nine modules could maintain its intact structure even after being immersed in PBS solution for 7 days.

[0068] Example 2

[0069] This embodiment provides the following steps for the preparation of a static-dynamic dual-network-based bio-ink (cell-free) based on GM, G-SH and other metal ions (Zn, Ag, Mg, Sr): (1) Weigh out GM dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55°C water bath for 1 hour until fully dissolved. Then weigh out zinc chloride powder and dissolve it fully in the above solution. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). This is solution A1. Weigh out GM dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55°C water bath for 1 hour until fully dissolved. Then weigh out silver chloride powder and dissolve it fully in the above solution. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). This is solution A2. Solution A3: Weigh out GM dry gel and LAP photoinitiator, dissolve them in PBS solution, and heat in a 55°C water bath for 1 hour until fully dissolved. Then weigh out magnesium chloride powder and dissolve it completely in the above solution. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). Solution A4: Weigh out GM dry gel and LAP photoinitiator, dissolve them in PBS solution, and heat in a 55°C water bath for 1 hour until fully dissolved. Then weigh out strontium chloride powder and dissolve it completely in the above solution. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). (2) Dissolve the freeze-dried G-SH in PBS solution, heat in a water bath to 60°C for 1 hour until completely dissolved, and then sterilize the solution by irradiating it under a UV lamp (e.g., for 3 hours) to obtain solution B. (3) By thoroughly mixing solutions A1 and B at a volume ratio of 1:1, a static-dynamic dual-network bio-ink based on GM, G-SH, and zinc ions can be obtained; by thoroughly mixing solutions A2 and B at a volume ratio of 1:1, a static-dynamic dual-network bio-ink based on GM, G-SH, and silver ions can be obtained; by thoroughly mixing solutions A3 and B at a volume ratio of 1:1, a static-dynamic dual-network bio-ink based on GM, G-SH, and magnesium ions can be obtained; by thoroughly mixing solutions A4 and B at a volume ratio of 1:1, a static-dynamic dual-network bio-ink based on GM, G-SH, and strontium ions can be obtained. In each group of bio-inks, the mass-volume concentration of GM dry gel is 6% (g / mL), the mass-volume concentration of LAP photoinitiator is 0.25% (g / mL), and the mass-volume concentration of G-SH is 2% (g / mL). Among them, the concentration of zinc ions in group A1 is 10 mM, the concentration of silver ions in group A2 is 10 mM, the concentration of magnesium ions in group A3 is 10 mM, and the concentration of strontium ions in group A4 is 10 mM.

[0070] Figure 3 Figure A shows a schematic diagram of a metal ion-based static-dynamic dual-network bio-ink design. The rheological and self-healing properties of four bio-inks were evaluated using a rotational rheometer. Figure 3As shown in the BE diagram, strain scanning results indicate that the storage modulus (G') of the bio-ink is greater than its loss modulus (G'') at low strain, exhibiting a gel state that suggests suitability for extrusion bioprinting. Creep experiments show that at low strain, the storage modulus (G') of the four bio-inks is greater than its loss modulus (G''), while at high strain, the storage modulus (G') is less than its loss modulus (G''), indicating the self-healing properties of this dynamic dual-network bio-ink. Furthermore, the four bio-inks of this embodiment and the bio-ink of Example 1 were combined to construct five hydrogel modules, and their splicability and self-healing effects were further characterized. Figure 3 As shown in F, multiple hydrogel modules can be easily assembled side-by-side into a long strip structure, and after photocrosslinking, a stable structure is formed that can be picked up with tweezers; linear grayscale analysis results show that the modules are well integrated. Figure 3 The above results indicate that dynamic dual-network bioinks based on metal chelation exhibit a certain degree of universality in component design, allowing for flexible adjustment of the concentration of G-SH, the type and concentration of metal ions, thereby enabling the preparation of various dynamic dual-network bioinks.

[0071] Example 3

[0072] This embodiment provides the preparation of a static-dynamic dual-network-based bio-ink (cell-free) based on GM, G-SH, and silicate bioceramics, including the following steps: (1) Weigh out GM dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55°C water bath for 1 hour until fully dissolved. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). Subsequently, weigh out sterile copper silicate powder and fully disperse it in the above solution. Set the solid content ratio of copper silicate to GM to be 2%, which is solution A1. Weigh out GM dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55°C water bath for 1 hour until fully dissolved. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). Subsequently, weigh out sterile zinc silicate powder and fully disperse it in the above solution. Set the solid content ratio of zinc silicate to GM to be 2%, which is solution A2. Weigh out GM dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55°C water bath for 1 hour until fully dissolved. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). Subsequently, weigh out sterile zinc silicate powder and fully disperse it in the above solution. Set the solid content ratio of zinc silicate to GM to be 2%, which is solution A2. Heat the solution in a sterilizing bath for 1 hour until fully dissolved. Then, sterilize the solution by passing it through a bacterial filter membrane (0.22 μm pore size). Subsequently, weigh out sterile strontium silicate powder and fully disperse it in the above solution. Set the solid content ratio of strontium silicate to GM to be 2%, which is solution A3. Weigh out GM dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat the solution in a 55°C water bath for 1 hour until fully dissolved. Then, sterilize the solution by passing it through a bacterial filter membrane (0.22 μm pore size). Subsequently, weigh out sterile magnesium silicate powder and fully disperse it in the above solution. Set the solid content ratio of magnesium silicate to GM to be 2%, which is solution A4. (2) Dissolve the freeze-dried G-SH in PBS solution to make the concentration of G-SH so that the solution is heated to 60°C in a water bath for 1 hour until it is completely dissolved. Then sterilize the solution by irradiating it under a UV lamp (e.g., for 3 hours). This solution is solution B. (3) By thoroughly mixing solutions A1 and B at a volume ratio of 1:1, a static-dynamic dual-network basic bio-ink based on GM, G-SH, and copper silicate can be obtained; by thoroughly mixing solutions A2 and B at a volume ratio of 1:1, a static-dynamic dual-network basic bio-ink based on GM, G-SH, and zinc silicate can be obtained; by thoroughly mixing solutions A3 and B at a volume ratio of 1:1, a static-dynamic dual-network basic bio-ink based on GM, G-SH, and strontium silicate can be obtained; by thoroughly mixing solutions A4 and B at a volume ratio of 1:1, a static-dynamic dual-network basic bio-ink based on GM, G-SH, and magnesium silicate can be obtained. In each group of bio-inks, the mass-volume concentration of GM dry gel is 6% (g / mL), the mass-volume concentration of LAP photoinitiator is 0.25% (g / mL), and the mass-volume concentration of G-SH is 1%~3% (g / mL).

[0073] The self-healing properties of four silicate-based static-dynamic dual-network bio-inks were evaluated using a rotational rheometer. Figure 4 As shown in A in the figure. Creep test results show that at low strain, the storage modulus (G') of the four bio-inks is greater than the loss modulus (G''), while at high strain, the storage modulus (G') is less than the loss modulus (G''), indicating the good self-healing properties of this dynamic dual-network bio-ink. Further characterization of the printability and self-healing properties of the four silicate bio-inks was performed using the printing parameters of Example 1 to fabricate modular scaffolds, such as... Figure 4 As shown in B, multiple printing modules have good three-dimensional structure and can be spliced ​​side by side into a square structure or stacked into a pyramid structure as needed, and form a stable structure after photocrosslinking.

[0074] Example 4

[0075] This embodiment provides the preparation of a static-dynamic dual-network-based bio-ink (cell-free) based on HAMA, G-SH, and copper ions, including the following steps: (1) Weigh HAMA dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55°C water bath for 1 hour until fully dissolved. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). Then weigh copper chloride powder and fully disperse it in the above solution. Set the copper ion concentration to 20 mM, which is solution A. (2) Dissolve the freeze-dried G-SH in PBS solution to make the concentration of G-SH so that the solution is heated to 60°C in a water bath for 1 hour until it is completely dissolved. Then sterilize the solution by irradiating it under a UV lamp (e.g., for 3 hours). This solution is solution B. (3) Mix solutions A and B thoroughly at a volume ratio of 1:1 to obtain the static-dynamic dual-network basic bio-ink. In each group of bio-inks, the mass-volume concentration of HAMA dry gel is 2.5% (g / mL), the mass-volume concentration of LAP photoinitiator is 0.25% (g / mL), the mass-volume concentration of G-SH is 2% (g / mL), and the copper ion concentration is 10 mM.

[0076] First, the self-healing properties of the static-dynamic dual-network bio-ink based on HAMA / G-SH / Cu were evaluated using a rotational rheometer. Figure 5 As shown in Figure A. Strain scanning results showed that the storage modulus (G') of the bio-ink was greater than the loss modulus (G'') at low strain, exhibiting a gel state that indicated its suitability for extrusion bioprinting. Creep experiments showed that the storage modulus (G') of the bio-ink was greater than the loss modulus (G'') at low strain, while at high strain, the storage modulus (G') was less than the loss modulus (G''), indicating the good self-healing properties of this dynamic dual-network bio-ink. Five modular units were further prepared and then assembled into a pentagram-shaped scaffold. After self-healing and photocrosslinking, a complete scaffold was obtained, which remained structurally stable after immersion in PBS solution for 7 days.

[0077] Example 5

[0078] This embodiment provides the preparation of a static-dynamic dual-network-based bio-ink (cell-free) based on SilMA, G-SH, and copper ions, including the following steps: (1) Weigh out SilMA dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55°C water bath for 1 hour until fully dissolved. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). Then weigh out copper chloride powder and fully disperse it in the above solution to obtain solution A. (2) Dissolve the freeze-dried G-SH in PBS solution to make the concentration of G-SH so that the solution is heated to 60°C in a water bath for 1 hour until it is completely dissolved. Then sterilize the solution by irradiating it under a UV lamp (e.g., for 3 hours). This solution is solution B. (3) Mix solutions A and B thoroughly at a volume ratio of 1:1 to obtain a static-dynamic dual-network bio-ink based on a mass-volume concentration of 10% SilMA (g / mL), a mass-volume concentration of 0.25% LAP photoinitiator (g / mL), a mass-volume concentration of 2% G-SH (g / mL), and 10 mM copper ions.

[0079] First, the self-healing properties of the SilMA / G-SH / Cu static-dynamic dual-network bio-ink were evaluated using a rotational rheometer. Figure 6 As shown in Figure A. Strain scanning results showed that the storage modulus (G') of the bio-ink was greater than the loss modulus (G'') at low strain, exhibiting a gel state that indicated its suitability for extrusion bioprinting. Creep experiments showed that the storage modulus (G') of the bio-ink was greater than the loss modulus (G'') at low strain, while at high strain, the storage modulus (G') was less than the loss modulus (G''), indicating the good self-healing properties of this dynamic dual-network bio-ink. Four modular units were further prepared and then spliced ​​side-by-side into a long strip scaffold. After self-healing and photocrosslinking, a complete scaffold was obtained. It was found that the scaffold could be easily picked up with tweezers and maintained structural integrity even when shaken in the air. It also maintained structural stability after immersion in PBS solution for 7 days, demonstrating the superiority of this combined strategy.

[0080] Example 6

[0081] This embodiment provides the preparation of a static-dynamic dual-network-based bio-ink (cell-free) based on borate ester reaction, comprising the following steps: (1) Weigh out GM dry gel, polyvinyl alcohol PVA and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55℃ water bath for 1 hour until fully dissolved. Then sterilize the solution by passing it through a filter membrane (pore size of 0.22 μm). This is solution A. (2) The freeze-dried phenylboronic acid modified gelatin-PBA was dissolved in PBS solution and heated in a water bath to 60°C for 1 h until completely dissolved. The solution was then sterilized by passing it through a bacterial filter membrane (pore size of 0.22 μm) to obtain solution B. (3) Mix solutions A and B thoroughly at a volume ratio of 1:1 to obtain a static-dynamic dual-network basic bio-ink based on a mass-volume concentration of 5% GM (g / mL), a mass-volume concentration of 0.25% LAP photoinitiator (g / mL), a mass-volume concentration of 2% Gelatin-PBA (g / mL), and a mass-volume concentration of 1% PVA (g / mL).

[0082] To further expand the material system of dynamic dual-network bio-ink, a photocrosslinking-based GM hydrogel was designed as the static crosslinking network, and the dynamic borate ester reaction between Gelatin-PBA and PVA was designed as the dynamic network.

[0083] First, the self-healing properties of the static-dynamic dual-network bio-ink based on GM / Gelatin-PBA / PVA were evaluated using a rotational rheometer. Figure 7As shown in the figure. Strain scanning results show that the storage modulus (G') of the bio-ink is greater than the loss modulus (G'') at low strain, exhibiting a gel state that indicates its suitability for extrusion bioprinting. Creep experiments show that at low strain, the storage modulus (G') of the bio-ink is greater than the loss modulus (G''), while at high strain, the storage modulus (G') is less than the loss modulus (G''), indicating the good self-healing properties of this dynamic dual-network bio-ink. Four modular units were further fabricated using bioprinting and then spliced ​​side-by-side into a large square scaffold. After self-healing and photocrosslinking, a complete scaffold was obtained. It was found that the scaffold could be easily picked up with tweezers and maintained structural integrity even when shaken in the air. It also remained structurally stable after being soaked in PBS solution for one day.

[0084] Example 7

[0085] This embodiment provides the preparation of a static-dynamic dual-network-based bio-ink based on the Schiff base reaction, including the following steps: (1) Weigh out HAMA dry gel, carboxymethyl cellulose CMCS and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55℃ water bath for 1 hour until fully dissolved to obtain solution A. (2) Dissolve oxidized dextran ODex in PBS solution, heat in a water bath to 60°C, and heat for 1 hour until completely dissolved to obtain solution B; (3) Mix solutions A and B thoroughly at a volume ratio of 1:1 to obtain a static-dynamic dual-network basic bio-ink based on a mass-volume concentration of 2% HAMA (g / mL), a mass-volume concentration of 0.25% LAP photoinitiator (g / mL), a mass-volume concentration of 3% ODex (g / mL), and a mass-volume concentration of 3% CMCS (g / mL).

[0086] A photocrosslinked HAMA hydrogel was designed as a static crosslinking network, and a Schiff base reaction between ODex and CMCS was used as a dynamic crosslinking network.

[0087] First, the self-healing properties of the static-dynamic dual-network bio-ink based on HAMA / ODex / CMCS were evaluated using a rotational rheometer. Figure 8 As shown in the figure, creep experiments revealed that at low strain, the storage modulus (G') of the bio-ink is greater than its loss modulus (G''), while at high strain, the storage modulus (G') is less than its loss modulus (G''), indicating the excellent self-healing properties of this dynamic dual-network bio-ink. Furthermore, three modular units were fabricated using bioprinting and then spliced ​​side-by-side into a long strip-shaped scaffold. After self-healing and photocrosslinking, a complete scaffold was obtained, which could be easily picked up with tweezers, demonstrating structural stability.

[0088] Example 8

[0089] This embodiment provides the following steps for the preparation of a modular bioprinted multi-cell scaffold containing neural stem cells and bone marrow mesenchymal stem cells: (1) Weigh out GM dry gel and LAP photoinitiator and dissolve them in PBS solution. Heat in a 55°C water bath for 1 hour until fully dissolved. Then weigh out copper chloride powder and dissolve it in the above solution. Then sterilize the solution by passing it through a bacterial filter membrane (pore size 0.22 μm). This is solution A. Dissolve freeze-dried G-SH in PBS solution and heat in a water bath to 60°C for 1 hour until completely dissolved. Then sterilize the solution by irradiating it under a UV lamp (e.g., for 3 hours). This is solution B. Mix solutions A and B thoroughly at a volume ratio of 1:1 to obtain a static-dynamic dual-network basic bio-ink with the following components: 6% GM (g / mL), 0.25% LAP photoinitiator (g / mL), 2% G-SH (g / mL), and 1 mM copper ions.

[0090] (2) The basic ink was mixed with neural stem cells and bone marrow mesenchymal stem cells respectively to obtain a static-dynamic dual-network splicable bio-ink for carrying cells. The module units were printed, spliced ​​and photocrosslinked using the printing parameters described in Example 1 to obtain a neural-bone multicellular scaffold. Finally, the scaffold was placed in a well plate and cultured in a cell culture incubator with supplemented culture medium.

[0091] Figure 9 In the diagram, A represents the multicellular scaffold of the neural bone model prepared in Example 8. To facilitate observation of cell distribution, bone marrow mesenchymal stem cells were marked in green, and neural stem cells were marked in red, and observed under a confocal microscope. Figure 9 As shown in B, the two cell types exhibit a left-right distribution, and this distribution remains stable during culture. Further characterization of cell survival over 10 days of culture was performed using a dual-color fluorescent staining agent for live and dead cells. Figure 9 As shown in Figure C, live cells appear green, and dead cells appear red, indicating that both cell types survive well in the spliced ​​scaffold. Immunofluorescence staining was used to characterize the specific differentiation behavior of the two cell types. First, the cells in the scaffold were fixed by soaking the cell patch in 4% paraformaldehyde solution for 30 min. Then, the cells were infiltrated with 0.1% Triton-X 100 solution for 10 min, followed by blocking with 5% BSA solution for 30 min. Finally, the patch was incubated with primary antibody working solution overnight at 4 °C. After incubation, the primary antibody working solution was washed away with PBS, and the patch was incubated with secondary antibody working solution at 37 °C for 1 h. After incubation, the secondary antibody working solution was washed away with PBS, and the patch was incubated with DAPI for 10 min. After staining, images were taken using a laser confocal microscope. Figure 9 As shown in D, the neural stem cell scaffold partially expresses neuronal markers GFAP and Tuj1, while the bone marrow mesenchymal stem cell scaffold partially expresses osteogenic marker OCN, indicating that the two cell types maintain their respective differentiation activity and function.

[0092] Example 9 This embodiment provides the following steps for the fabrication of a modular bioprinted multicellular scaffold containing neural stem cells and muscle cells: (1) The static-dynamic dual-network bio-based ink with components of 6% GM (g / mL), 2% G-SH (g / mL) and 1mM copper ions was prepared in the same manner as in Example 8; (2) The ink was mixed with neural stem cells and muscle cells respectively to obtain a static-dynamic dual-network splicable bio-ink for carrying cells. The module units were printed, spliced ​​and photocrosslinked using the printing parameters described in Example 1 to obtain a neural-muscle multicellular scaffold. Finally, the scaffold was placed in a well plate and cultured in a cell culture incubator with supplemented culture medium.

[0093] Figure 10 In the diagram, A represents the multicellular scaffold of the neuromuscular model prepared in Example 9. To facilitate observation of cell distribution, muscle cells were labeled indigo, and neural stem cells were labeled magenta, and observed under a confocal microscope. Figure 10 As shown in B, the two cell types exhibit a left-right distribution, and this distribution remains stable during culture. Further characterization of cell survival over 10 days of culture was performed using a live / dead cell dual-color fluorescent staining agent. Figure 10 As shown in C, live cells appear green and dead cells appear red, indicating that both cell types survive well in the spliced ​​scaffold. Immunofluorescence staining was used to characterize the specific differentiation behavior of the two cell types. The immunofluorescence protein staining procedure was the same as in Example 8. Figure 9 As shown in D, the neural stem cell scaffold partially expresses neuronal markers GFAP and Tuj1, while the muscle cell scaffold partially expresses myoblastic differentiation marker MHC, indicating that the two cell types maintain their respective differentiation activity and function.

[0094] Example 10 This embodiment provides the following steps for the preparation of a modular bioprinted multicellular scaffold containing neural stem cells, bone marrow mesenchymal stem cells, tendon stem cells, and muscle cells: (1) The dynamic dual-network bio-based ink with components of 6% GM (g / mL), 2% G-SH (g / mL) and 1mM copper ions was prepared in the same manner as in Example 8;

[0095] (2) The ink was mixed with neural stem cells, muscle cells, bone marrow mesenchymal stem cells, and tendon stem cells respectively to obtain a static-dynamic dual-network splicable bio-ink for carrying cells. The printing parameters described in Example 1 were used to print, splice, and photocrosslink the module units to obtain a multicellular scaffold for the neural innervation skeletal muscle system model. Finally, the scaffold was placed in a well plate and cultured in a cell culture incubator with supplemented culture medium.

[0096] Figure 11 In this context, A represents the multicellular scaffold for the neural innervation skeletal muscle system model prepared in Example 10. Cell survival was further characterized using a dual-color fluorescent staining agent for live and dead cells over 10 days of culture. Figure 11 As shown in Figure BC, live cells appear green, and dead cells appear red, indicating that all four cell types survived well in the spliced ​​scaffold. Immunofluorescence staining was used to characterize the specific differentiation behavior of the two cell types. The immunofluorescence protein staining procedure was the same as in Example 9. Figure 11 As shown in Figure D, the neural stem cell scaffold partially expresses neuronal markers GFAP and Tuj1, the muscle cell scaffold partially expresses myogenic differentiation marker MHC, the bone marrow mesenchymal stem cells express osteogenic differentiation marker OCN, and the tendon stem cells express tendonogenic differentiation marker TNMD, indicating that the four cell types maintain their respective differentiation activities and functions.

[0097] Example 11 This embodiment provides the following steps for the fabrication of a modular bioprinted biomimetic osteochondral multicellular scaffold containing bone marrow mesenchymal stem cells and chondrocytes: (1) Bio-inks based on mass-volume concentrations of 8% GM (g / mL), 2% G-SH (g / mL), and different copper ion concentrations (0, 0.5, 1, 2.5, 5 mM) were prepared and labeled as GG, GG-0.5Cu, GG-1Cu, GG-2.5Cu, and GG-5Cu, respectively. They were then mixed with bone marrow mesenchymal stem cells to obtain static-dynamic dual-network splicable bio-inks for carrying cells. Cell-carrying scaffolds were prepared and placed in an incubator for culture. The optimal copper ion concentration was screened by protein expression. (2) Bio-inks based on mass-volume concentrations of 6% GM (g / mL), 2% G-SH (g / mL), and different zinc ion concentrations (0, 0.5, 1, 2.5, 5 mM) were prepared and labeled as GG, GG-0.5Zn, GG-1Zn, GG-2.5Zn, and GG-5Zn, respectively. These bio-inks were mixed with chondrocytes to obtain static-dynamic dual-network splicable bio-inks for carrying cells. Cell-carrying scaffolds were prepared and placed in an incubator for culture. The optimal zinc ion concentration was screened by protein expression. (3) Five types of bioinks were prepared, and the specific preparation methods are the same as in Example 8. The main difference is that Bioink I consists of 6% GM, 2% G-SH, 2.5 mM zinc ions and 5×10 7 10c / mL chondrocytes; Bioink II components consist of 6% GM, 2% G-SH, 1 mM zinc ions, and 2.5 × 10c 7 Bioink III contained 7% GM and 2% G-SH, and was free of metal ions and cells; Bioink IV contained 8% GM, 2% G-SH, 0.5 mM zinc ions, and 2.5 × 10⁻⁶ cells / mL chondrocytes. 7 / mL bone marrow mesenchymal stem cells; Bioink V components are 8% GM, 2% G-SH, 1 mM zinc ions and 5×10 7 Bone marrow mesenchymal stem cells per mL; (4) Subsequently, five types of module units were prepared using extrusion bioprinting technology, and spliced ​​and photocrosslinked. The steps were the same as in Example 8. After preparation, they were placed in an incubator for co-culture.

[0098] Furthermore, the regulatory effect of bioinks with different copper ion concentrations on osteogenic differentiation of bone marrow mesenchymal stem cells was investigated. Immunofluorescence staining experiments were performed following the steps outlined in Example 8. Figure 12 As shown in Figure A, cells in all groups survived well, with the GG-1Cu group exhibiting the highest expression of osteogenic differentiation-specific proteins Runx2 and OPN. Statistical results also showed that GG-1Cu had the highest expression of Runx2 and OPN proteins, followed by GG-0.5Cu. Further alkaline phosphatase (ALP) and Alizarin Red (ARS) staining was performed on the scaffolds to assess alkaline phosphatase expression and calcium deposition. Figure 12 As shown in CD, GG-1Cu exhibits the best osteogenic activity, demonstrating the highest ALP activity and calcium deposition characteristics. Therefore, both GG-1Cu and GG-0.5Cu groups are considered for subsequent modular printing.

[0099] Similarly, the regulatory effects of bio-inks with different zinc ion concentrations on chondrocyte maturation were investigated. Immunofluorescence staining experiments were performed following the steps outlined in Example 8. Figure 12 As shown in the EF diagram, cells in all groups survived well, with the GG-2.5Zn group exhibiting the highest expression of chondrocyte differentiation-specific proteins Aggrecan and COL-II. Statistical results also showed that GG-2.5Zn had the highest expression of Aggrecan and COL-II proteins, followed by GG-1Zn.

[0100] like Figure 12As shown in G, after selecting the optimal copper-zinc ion concentration, five modules were further prepared, assembled, and cross-linked. To characterize the spatial distribution of chondrocytes and bone marrow mesenchymal stem cells in the assembled biomimetic osteochondral multicellular scaffold, chondrocytes were labeled with red fluorescence, and bone marrow mesenchymal stem cells were labeled with green fluorescence. Figure 13 As shown in Figure A, the red and green fluorescence indicates the distribution of upper and lower layers, with a non-fluorescent interface layer in the middle. This layered structure was well maintained even after 7 days of culture, indicating the structural integrity of the assembled scaffold. Furthermore, live / dead cell staining was used to characterize the cell viability of chondrocytes and bone marrow mesenchymal stem cells in different layers of the assembled multicellular scaffold, as shown in Figure A. Figure 13 As shown in Figure B, live cells emit green fluorescence, while dead cells emit red fluorescence, indicating that the chondrocytes in layers 1 and 2 and the bone marrow mesenchymal stem cells in layers 4 and 5 are all surviving well. Statistical results also show that both cell types maintained a survival rate of over 85% during co-culture. Figure 13 (C in the middle).

[0101] Example 12 This embodiment provides a modular bioprinted biomimetic osteochondral multicellular scaffold containing bone marrow mesenchymal stem cells and chondrocytes for integrated osteochondral repair.

[0102] Referring to step (3) of Example 11, modular units without copper / zinc ions, chondrocytes, and bone marrow mesenchymal stem cells were prepared and assembled into scaffolds (GG hydrogel, G2); modular units containing copper / zinc ions but without chondrocytes and bone marrow mesenchymal stem cells were prepared and assembled into scaffolds (GG-Zn / Cu ions, G3); several modular units without copper / zinc ions but containing chondrocytes and bone marrow mesenchymal stem cells were prepared and assembled into scaffolds (GG-RCs / BMSCs, G4); and the biomimetic osteochondral multicellular scaffold containing copper-zinc ions, chondrocytes, and bone marrow mesenchymal stem cells described in Example 11 (G5) was prepared.

[0103] A model of osteochondral defect was established using 2.5 kg New Zealand white rabbits. Rabbits were first anesthetized with sodium pentobarbital. Then, the fur on the rabbit's legs was removed using a shaving tool to expose the skin, which was then disinfected. The skin was incised to expose the femur, and a 5 mm diameter, 6 mm deep osteochondral defect was created at the articular cartilage using an electric drill. Each group of scaffolds was implanted into the injury site, with the blank group (G1) receiving no implantation. The wound was then sutured. At 6 and 12 weeks post-implantation, the rabbits were euthanized, and the osteochondral tissue was harvested for imaging and histological analysis.

[0104] Figure 14Figure A shows a macroscopic image of the osteochondral tissue and a Micro-CT 3D reconstruction model, where green represents newly formed bone tissue and red circles represent the damaged areas. It can be seen that at 6 weeks post-operation, all groups showed a small amount of new bone regeneration, with the control group (G1) having the least new bone area. At 12 weeks post-operation, new bone tissue further increased, with the biomimetic osteochondral multicellular scaffold group (G5) showing the highest new bone formation, while the control group (G1) still exhibited significant cavities, indicating ineffective self-healing. Statistical results showed that at both 6 and 12 weeks, the biomimetic osteochondral multicellular scaffold group (G5) had the highest bone volume fraction (BV / TV) and bone mineral density (BMD). Figure 14 (BC in the text). Furthermore, the status of osteochondral regeneration is scored based on the International Cartilage Repair Society Grading System (ICRS), such as... Figure 14 As shown in D, the biomimetic osteochondral multicellular scaffold group (G5) has the highest score, indicating that it has the best osteochondral repair capacity.

[0105] The osteochondral tissue was further decalcified by immersing it in EDTA decalcification solution, followed by paraffin embedding and H&E, Masson's Law, and Safranin-Fix Green SF staining. Figure 15 As shown in Figure A, at 6 weeks post-surgery, the defect was filled with fibrous tissue, with minimal new cartilage and bone formation. At 12 weeks post-surgery, all groups showed some degree of bone and cartilage regeneration, with the biomimetic osteochondral multicellular scaffold group (G5) exhibiting the most beneficial subchondral bone and cartilage regeneration, forming a continuous layer of cartilage tissue (red) on the defect surface. Based on this, nanoindentation testing was used to analyze the mechanical properties of the newly formed cartilage, such as... Figure 15 As shown in BD, the force-displacement curves and corresponding modulus and hardness indices show that the biomimetic osteochondral multicellular scaffold group (G5) has the highest values, which are much greater than other groups, indicating the effective recovery of cartilage mechanical properties.

[0106] The above results indicate that the biomimetic osteochondral multicellular scaffold based on the modular bioprinting strategy can simultaneously simulate the biophysical (mechanical gradient distribution), biochemical (copper-zinc ion stratification and gradient distribution), and biological (stratification and gradient distribution of chondrocytes and bone marrow mesenchymal stem cells) characteristics of natural osteochondral tissue, thereby promoting the integrated regeneration of cartilage and subchondral bone. It has great application potential in the treatment of osteochondral injury, osteoarthritis, and other degenerative diseases.

Claims

1. A modular bioprinted multi-cell scaffold, characterized in that, The modular bioprinted multicellular scaffold is a modular bioprinted multicellular scaffold obtained by assembling cell-carrying module units prepared by bio-3D printing technology using static-dynamic dual-network splicable bio-ink with self-healing properties and carrying cells.

2. The modular bioprinted multi-cell scaffold according to claim 1, characterized in that, The static-dynamic dual-network splicable bio-ink comprises a dual-network hydrogel capable of forming a static cross-linked network and a dynamic cross-linked network, and cells uniformly dispersed in the dual-network hydrogel; wherein the dual-network hydrogel includes a static cross-linked network gel matrix and a dynamic cross-linked network gel matrix.

3. The modular bioprinted multi-cell scaffold according to claim 2, characterized in that, The static cross-linked network is a three-dimensional network capable of forming at least one of irreversible chemical bonds or stable physical interactions; preferably, the irreversible chemical bonds form stable chemical bonds under light irradiation; more preferably, the static cross-linked network gel matrix is ​​selected from at least one of methacrylated gelatin, methacrylated hyaluronic acid, methacrylated silk fibroin, and methacrylated sodium alginate, preferably methacrylated gelatin; even more preferably, the mass-volume concentration of the static cross-linked network gel matrix is ​​1%~20% (W / V).

4. The modular bioprinted multicellular scaffold according to claim 2 or 3, characterized in that, The dynamic cross-linked network is a three-dimensional network formed through reversible cross-linking, capable of dynamically breaking and forming cross-links, including at least one of metal coordination chelation, Schiff base reaction, hydrogen bonding, electrostatic interaction, and host-guest interaction, preferably metal coordination chelation; more preferably, the dynamic cross-linked network gel matrix is ​​composed of metal ions and polymers containing specific ligand groups capable of chelating with metal ions, wherein the metal ions are at least one of copper, zinc, magnesium, strontium, and silver ions, and the polymers containing specific ligand groups are polymers having at least one ligand group selected from bisphosphate, histidine, catechol, and thiol groups, particularly preferably thiol-modified gelatin; even more preferably, the concentration of the metal ions is 0.1~50 mM, and the mass-volume concentration of the polymers containing specific ligand groups is 0.5%~5% (W / V).

5. The modular bioprinted multicellular scaffold according to any one of claims 1 to 4, characterized in that, The bio-3D printing technology is at least one of extrusion 3D printing, projection photopolymerization 3D printing, surface projection photopolymerization 3D printing, and inkjet bio-3D printing.

6. The modular bioprinted multicellular scaffold according to any one of claims 1 to 5, characterized in that, The cells include at least one of neural stem cells, bone marrow mesenchymal stem cells, endothelial cells, Schwann cells, chondrocytes, and tendon stem cells; preferably, the cell density is 1×10⁻⁶. 7 ~5×10 8 per mL.

7. The modular bioprinted multicellular scaffold according to any one of claims 1 to 6, characterized in that, By adjusting the composition of the static-dynamic dual-network splicable bio-ink carrying cells, as well as the type and density of cells, precise control over the key biophysical, biochemical, and biological characteristics of modular bioprinted multicellular scaffolds can be achieved.

8. The modular bioprinted multicellular scaffold according to any one of claims 1 to 7, characterized in that, The modular bioprinted multicellular scaffold is a modular bioprinted multicellular scaffold with a multi-level structure and multi-cell spatial distribution; preferably, the modular bioprinted multicellular scaffold is a biomimetic osteochondral multicellular construct, formed by stacking five cell-carrying module units layer by layer; the bio-ink of the first cell-carrying module unit from top to bottom includes 3%~20% methacrylated gelatin, 0.5%~5% thiol-modified gelatin, 0.1~20 mM zinc ions, and 1×10 7 ~8×10 7 The second cell-carrying module unit contains 3%–20% methacrylated gelatin, 0.5%–5% thiol-modified gelatin, 0.1–10 mM zinc ions, and 1×10⁻⁶ ions per mL of chondrocytes. 7 ~5×10 7 The cell-carrying module unit contains chondrocytes per mL. The bio-ink in the third cell-carrying module unit comprises 5%–20% methacrylated gelatin and 1%–5% thiol-modified gelatin. The bio-ink in the fourth cell-carrying module unit comprises 5%–20% methacrylated gelatin, 0.5%–5% thiol-modified gelatin, 0.1–10 mM copper ions, and 1×10⁻⁶ ions. 7 ~5×10 7 The fifth cell-carrying module unit contains bone marrow mesenchymal stem cells at a density of 10 cells / mL. The bio-ink comprises 5%–20% methacrylated gelatin, 0.5%–5% thiol-modified gelatin, 0.1–20 mM copper ions, and 1×10⁻⁶ ions. 7 ~8×10 7 Bone marrow mesenchymal stem cells per mL.

9. The method for preparing a modular bioprinted multicellular scaffold according to any one of claims 1 to 8, characterized in that, The preparation method includes the following steps: (1) The static-dynamic dual-network hydrogel was mixed evenly with cells to obtain a static-dynamic dual-network splicable bio-ink carrying cells; (2) Cell-carrying module units were prepared by using bio-3D printing technology to fabricate static-dynamic dual-network splicable bio-ink carrying cells; (3) The cell-carrying module units are spliced ​​and assembled. After the self-healing fusion and covalent cross-linking of the cell-carrying module units, a modular bioprinted multi-cell scaffold with key bio-physical, biochemical and biological characteristics is obtained.

10. The application of modular bioprinted multicellular scaffolds according to any one of claims 1 to 8 in constructing biomimetic in vitro multicellular models and in vivo complex tissue regeneration and organ reconstruction.

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