Vascularized bone organoid and construction method therefor

Abstract

Provided are a vascularized bone organoid and a construction method therefor. By combining a suspension-based three-dimensional (3D) bioprinting method, a cell-laden printing strategy, and a sacrificial bioink material, a vascularized bone organoid model containing a hollow vascular network is constructed. The vascularized bone organoid model has excellent structural stability, mechanical properties, growth and development capability, and osteogenic capability, involves simple selected materials and fabrication process, implements engineered stable production, allows for long-term co-culture of cells for 3D cultivation, and has high applicability and research potential.

Description

A vascularized bone organoid and its construction method Technical Field

[0001] The present invention relates to the technical field of organoids, and in particular to vascularized bone organoids and construction methods thereof. Background Art

[0002] Organoids are in vitro models that use stem cells or organ progenitor cells as raw materials and are constructed into three-dimensional culture systems by simulating organ structure and function in vitro. They show broad application prospects in disease model establishment, drug screening, tissue regeneration, and research on tissue repair and regeneration mechanisms. However, existing organoid systems often suffer from problems such as insufficient construction accuracy and uncontrollable culture systems, resulting in insufficient controllability of their culture morphology and self-organization. At the same time, organoids are limited in size by nutrient transport and are difficult to grow beyond the millimeter level. They lack the large-scale structural characteristics of corresponding organs in the body and cannot achieve higher-level organ functional responses. This limits the development of organoid technology in the fields of in vitro bionic models and regenerative medicine.

[0003] Bone organoids are an emerging strategy developed based on organoid technology. They utilize biomaterials to construct three-dimensional bone biomimetic models in vitro with complex biological functions, further recreating the bone microenvironment. The principle of bone organoid construction is to achieve three-dimensional, self-renewing, self-organizing microbone tissues and to guide cell differentiation. However, due to the complex mechanisms of bone itself, the poor reproducibility of the bone microenvironment in in vitro models, and the insufficient vascularization of existing bone organoids, bone organoid research remains a novel field and faces significant challenges.

[0004] The traditional organoid culture method is to culture stem cells in vitro into 3D cultures such as cell clusters and microspheres, and then transfer them into Matrigel matrix gel for further induction of differentiation. Akiva et al. cultured bone marrow mesenchymal stem cells (BMSC) in vitro and induced differentiation to construct a functional 3D self-organized co-culture composed of osteoblasts and osteocytes, forming woven bone organoids (Adv.Funct.Mater.2021,31,2010524). Papantoniou et al. constructed microspheres of callus organoids based on human periosteal stem cells (hPDCs), which can self-assemble into three-dimensional engineered tissues in vitro and can be implanted to treat critical-sized long bone defects in mice. The regenerated bone shows morphological characteristics similar to natural tibia (Advanced Science,7(2),1902295). Bone organoids constructed by this traditional method have certain bionic functions, but lack the ability to continuously supply nutrients. Their growth and development capabilities are greatly limited, and it is difficult to grow to above the millimeter level.

[0005] Bio-3D printing has also been widely used for tissue engineering scaffolds and organoid construction, particularly for organoid systems composed of multiple materials and cells. Through coaxial printing, suspension printing, or multi-nozzle co-extrusion, corresponding multicellular co-culture structures can be engineered to further mimic the in vivo bone microenvironment. In 2017, Ali Khademhosseini et al. constructed a three-dimensional bone tissue construct for repairing large bone defects using extrusion 3D printing technology. They used a low-concentration methacrylated gelatin (GelMA) bioink containing human umbilical vein endothelial cells (HUVECs) and bone marrow mesenchymal stem cells (hBMSCs) to print a central vascular fiber construct. A high-concentration GelMA ink containing silicate nanosheets was printed around the fibers to induce osteogenesis (Adv. Healthc. Mater. 6 (2017) 1–15). This bone tissue construct demonstrated microscopic endothelial cell vascularization, but the co-printing method of the two cells involved direct mixing, resulting in a macroscopic structure significantly different from an actual vascular network and the inability to grow into a true vascular network. Furthermore, while single-component GelMA hydrogels offer advantages such as UV cross-linking and high cell viability as matrix materials for organoid culture, their applicability is significantly limited by their mechanical properties. In the three-dimensional environment of organoid culture, low-concentration GelMA hydrogels suffer from weak mechanical properties, rapid degradation, and easy cell sedimentation. High-concentration GelMA hydrogels, however, significantly restrict the growth of encapsulated cells due to the dense distribution of polymer chains. This often prevents encapsulated cells from proliferating for more than seven days, preventing them from completing the entire osteogenic induction process and significantly limiting the culture cycle of bone organoids.

[0006] Currently, the biomimetic extracellular matrix required for organoids is typically made from natural polymer hydrogels, which are solidified through UV, enzymatic, ionic, or chemical crosslinking for long-term culture. Numerous studies have demonstrated that single natural polymer materials often have significant deficiencies in inductive capacity, mechanical properties, and structural stability. Therefore, it is necessary to modify or blend the hydrogel matrix to obtain multifunctional hydrogels with both high mechanical properties and high bioactivity to meet the needs of biomimetic and engineered production.

[0007] Research on bone organoids is still in its initial stages of exploration. Most existing bone organoids have the following shortcomings: (1) Lack of preparation standards, low construction stability and reproducibility, and difficulty in engineering production; (2) Lack of structural characteristics in cell arrangement, making it difficult to mimic the real bone microenvironment; (3) Lack of connection with blood vessels, nerves, and the immune system, making it difficult for bone organoids to grow and develop continuously; (4) The structural stability and mechanical properties of the hydrogel material used as a culture matrix are insufficient, making it difficult to use for long-term culture of bone organoids; (5) Cells are easily damaged by shear stress during extrusion printing, which affects their ability to proliferate in the constructed hydrogel print body and generally cannot exceed 7 days.

[0008] Summary of the Invention

[0009] To address the shortcomings of existing bone organoid preparations in terms of stability and biomimetic capabilities, this present invention aims to provide a method for preparing hydrogel materials for bone organoid culture that can be stably used in 3D bioprinting, as well as a strategy for constructing centimeter-scale vascularized bone organoid models. This approach aims to improve the manufacturing stability, osteogenic mineralization, and growth potential of bone organoids. This bone organoid preparation process is simple, easily scalable, and widely applicable.

[0010] This study combines a suspended 3D bioprinting method, a cell-loaded printing strategy, and sacrificial bio-ink materials to construct a vascularized bone organoid model containing a hollow vascular network. This vascularized bone organoid model exhibits excellent structural stability, mechanical properties, growth and development capabilities, and osteogenesis. The materials and manufacturing methods are simple, enabling stable engineering production and long-term co-culture of cells in three dimensions, demonstrating its high applicability and research potential.

[0011] One aspect of the present invention provides a suspension printing support bath for preparing vascularized bone organoids, wherein the support bath material comprises methacrylated gelatin, methacrylated hyaluronic acid, glycerol, a photoinitiator, bone marrow mesenchymal stem cells, and a first solution.

[0012] Furthermore, the photoinitiator is selected from 2,4,6-trimethylbenzoyl phosphate lithium salt (LAP).

[0013] Furthermore, the density of bone marrow mesenchymal stem cells was 4×10 7 cells / mL.

[0014] Furthermore, the first solution is a buffer solution, preferably a PBS buffer solution.

[0015] Furthermore, the composition of the components in the hydrogel material is as follows by mass:

[0016] Furthermore, the composition of the components in the hydrogel material is as follows by mass:

[0017] Furthermore, the sum of the components in the hydrogel material is 100%.

[0018] Furthermore, the suspension printing support bath preparation method includes the following steps:

[0019] S11) mixing methacrylated gelatin, methacrylated hyaluronic acid, glycerol, and the first solution in a certain mass ratio and adding the mixture to a container, heating and stirring until the mixture is completely dissolved to obtain a GHG hydrogel solution;

[0020] S12) sterilizing the GHG hydrogel solution;

[0021] S13) preparing a working solution with the photoinitiator and the first solution and sterilizing the solution;

[0022] S14) mixing the working solution and the GHG hydrogel solution uniformly;

[0023] S15) Digest and resuspend human bone marrow mesenchymal stem cells into a suspension, and seed them at 4×10 7 The density of cells / mL was fully mixed with the GHG hydrogel solution to obtain a suspended printing support bath.

[0024] Furthermore, in step S14), the mass concentration of the photoinitiator is 0.06%-0.1%, preferably 0.08%.

[0025] Furthermore, the mass of the methacrylated gelatin is 4.5%.

[0026] Furthermore, the amino substitution degree of the methacrylated gelatin is 30%-90%, preferably 60%.

[0027] Furthermore, the molecular weight of the methacrylated gelatin is 100-200 kDa, preferably 150 kDa.

[0028] Furthermore, the mass ratio of the methacryloyl hyaluronic acid is 0.5%.

[0029] Furthermore, the molecular weight of the methacryloyl hyaluronic acid is 150-400 kDa, preferably 400 kDa.

[0030] Furthermore, the mass ratio of the glycerol is 5%.

[0031] Furthermore, the mass ratio of the photoinitiator is 0.08%.

[0032] Furthermore, the mass ratio of methacrylated gelatin, methacrylated hyaluronic acid and glycerol in the hydrogel material is 9:1:10.

[0033] Another aspect of the present invention provides a raw material for preparing a suspension printing support bath for vascularized bone organoids, comprising the above-mentioned hydrogel material and the bio-ink for suspension printing, which are placed separately.

[0034] The biological ink for suspension printing comprises umbilical vein endothelial cells, gelatin and a second solution.

[0035] Furthermore, the density of umbilical vein endothelial cells in the bio-ink for suspension printing is 1×10 7 cells / mL.

[0036] Furthermore, the concentration of gelatin in the bio-ink for suspension printing is 10%-15%, preferably 12%;

[0037] Furthermore, the second solution is a buffer solution, preferably a PBS buffer solution.

[0038] Furthermore, the method for preparing bio-ink for suspension printing comprises the following steps:

[0039] S21) preparing a gelatin solution with a mass ratio of 10% to 15% using gelatin and a second solution, heating and stirring until completely dissolved, and sterilizing;

[0040] S22) Umbilical vein endothelial cells were digested and resuspended into a suspension, and 1×10 7 The density of cells / mL was fully mixed with the gelatin solution to obtain the bio-ink for suspension printing.

[0041] Another aspect of the present invention provides use of the hydrogel material for preparing a suspension printing support bath for vascularized bone organoids as a suspension printing support bath for vascularized bone organoids.

[0042] Another aspect of the present invention provides a method for preparing vascularized bone organoids, the method comprising the following steps:

[0043] S1) preparing a hydrogel support bath as described above for use as a suspension 3D printing medium and vascularized bone organoid culture;

[0044] S2) preparing the bio-ink as described above;

[0045] S3) Suspension printing of vascularized bone organoids;

[0046] Wherein step S3) comprises the following steps:

[0047] S31) performing suspension printing with bio-ink in a support bath, constructing a suspended vascularized network in the support bath according to the model;

[0048] S32) After printing is completed, irradiating with an ultraviolet light source to complete the curing and cross-linking of the support bath to obtain a cross-linked and cured vascularized bone organoid model precursor;

[0049] S33) After the culture medium is added to the vascularized bone organoid model precursor, cell culture is performed. The bio-ink inside the vascularized bone organoid model precursor melts and flows out under the cell culture temperature conditions, and the endothelial cells in the bio-ink adhere to the hollow tubes left at the original bio-ink printing site and grow to form a vascular network structure; dead cells and residual gelatin in the tubes are removed by flushing, and fresh culture medium is replaced every 2-3 days for long-term culture.

[0050] Furthermore, the printing temperature in S31) is 22-26°C, preferably 25°C.

[0051] Furthermore, the printing extrusion pressure in S31) is 1.0-1.8 bar, preferably 1.5 bar.

[0052] Furthermore, the ultraviolet light source in S32) is an ultraviolet light source of 365-405 nm, preferably 365 nm.

[0053] Furthermore, the irradiation time of the ultraviolet light source in S32) is 10-20s, preferably 15s.

[0054] Furthermore, S1) comprises the following steps:

[0055] S11) mixing methacrylated gelatin, methacrylated hyaluronic acid, glycerol, and the first solution according to a mass ratio and adding the mixture to a centrifuge tube, heating and stirring until completely dissolved to obtain a GHG hydrogel solution;

[0056] S12) sterilizing the GHG hydrogel solution;

[0057] S13) preparing a working solution of the photoinitiator with the first solution and sterilizing the solution;

[0058] S14) mixing the photoinitiator working solution and the GHG hydrogel solution uniformly;

[0059] S15) Digest and resuspend human bone marrow mesenchymal stem cells into a suspension, and seed them at 4×10 7 cells / mL density was thoroughly mixed with the GHG hydrogel solution to obtain a suspended printing support bath.

[0060] Furthermore, S2) comprises the following steps:

[0061] S21) preparing a gelatin solution with a mass ratio of 10% to 15% using gelatin and a second solution, heating and stirring until completely dissolved, and sterilizing;

[0062] S22) Umbilical vein endothelial cells were digested and resuspended into a suspension, and 1×10 7 The density of cells / mL was fully mixed with the gelatin solution to obtain the bio-ink for suspension printing.

[0063] Furthermore, in S11), the mass proportions of methacryloylated gelatin, methacryloylated hyaluronic acid, glycerol and the first solution are 4.5%, 0.5%, 5% and 90% respectively.

[0064] Another aspect of the present invention provides a vascularized bone organoid prepared by the above preparation method.

[0065] Another aspect of the present invention provides use of the vascularized bone organoids in preparing implantable medical devices for bone repair. Beneficial effects

[0066] (1) The invention constructs a bone organoid hydrogel material with flexible mechanical properties and viscosity adjustments, which can ensure good cell proliferation while ensuring that the constructed bone organoid model has excellent structural stability and mechanical properties, so as to maintain the spatiotemporal distribution characteristics of multiple cells for a long time during the three-dimensional culture of bone organoids. At the same time, the hydrogel can be used as a supporting bath medium for suspended biological 3D printing. Among them, the present invention specifically adds HAMA and glycerol, and the blending of the two with GelMA improves the toughness and viscosity of the hydrogel respectively, while retaining the advantages of one-step UV cross-linking and curing, and can maintain structural stability without degradation during long-term three-dimensional cell culture, so that the culture process of bone organoids can achieve a balance between matrix structural stability and cell growth and development.

[0067] (2) A vascularized bone organoid model containing multiple cells was printed using suspended 3D printing technology. Compared with traditional bone organoids, the macroscopic structure of the vascular network embedded in the biomimetic extracellular matrix was reproduced. Compared with other co-culture systems in which endothelial cells and stem cells are directly mixed, it is more in line with the real bone physiological environment. Cell proliferation experiments have shown that mesenchymal stem cells in the vascularized bone organoid model under three-dimensional culture conditions can continue to proliferate and maintain high cell activity within 14 days after support bath curing, solving the problem of long-term cell culture and induction in hydrogel-based three-dimensional cell culture.

[0068] (3) The osteogenesis capacity of the vascularized bone organoids constructed by the present invention was verified. High expression of osteogenesis-related genes and mineralized nodules were observed within 14 days of induction, indicating the maturity of osteogenesis. The role of vascular endothelial cells in promoting osteogenesis was explored. Compared with traditional single-cell bone organoids, they have better growth and development potential, culture stability, and osteogenesis differentiation ability. They are more similar to the bone microenvironment in the human body and significantly shorten the culture cycle of bone organoids, which is conducive to engineering production and culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG1 is a rheological characterization diagram of the bio-ink and the supporting bath hydrogel in the embodiment.

[0070] FIG2 is a diagram of a method for constructing a vascularized bone organoid print in an embodiment.

[0071] FIG3 is a diagram showing the structure and characterization of the hollow vascular network structure in the embodiment.

[0072] FIG4 is a cell live-dead staining diagram of the embodiment.

[0073] FIG5 is a diagram of a cell proliferation experiment in an embodiment.

[0074] FIG6 is an immunofluorescence experiment diagram of an embodiment.

[0075] FIG7 is an Alizarin Red staining analysis diagram of the embodiment. DETAILED DESCRIPTION

[0076] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0077] This article focuses on the application of bone organoid technology and provides a method for constructing vascularized bone organoids through suspended 3D bioprinting.

[0078] The present invention will be further described below with reference to the embodiments.

[0079] (1) An embodiment of the present invention provides a hydrogel material for use as a suspension 3D printing medium and for culturing vascularized bone organoids. The hydrogel comprises the following raw material components in the following mass percentages:

[0080] Specifically, the mass ratio of the GelMA is 4%-6%, preferably 4.5%, the amino substitution degree is 30%-90%, preferably 60%, and the molecular weight is 100-200 kDa, preferably 150 kDa.

[0081] The mass ratio of the HAMA is 0.5%-1%, preferably 0.5%, and the molecular weight is 150-400 kDa, preferably 400 kDa.

[0082] The mass ratio of the glycerol is 3%-20%, preferably 5%.

[0083] The mass ratio of the LAP is 0.04%-0.12%, preferably 0.08%.

[0084] Preferably, the mass ratio of GelMA, HAMA and glycerol in the hydrogel material is 9:1:10.

[0085] (2) In addition to providing the hydrogel material, the present invention further provides a method for preparing the hydrogel material. In one embodiment, the preparation method comprises at least the following steps:

[0086] S11) GelMA, HAMA, glycerol, and PBS buffer were mixed in a proportion and added to a centrifuge tube. The mixture was incubated in a water bath at 60° C. for 4 h until all materials in the centrifuge tube were completely dissolved to obtain a GHG hydrogel solution.

[0087] S12) sterilizing the GHG hydrogel solution by pasteurization and keeping it warm at 37° C.

[0088] S13) preparing a working solution of LAP photoinitiator and PBS solvent at a concentration of 40 mg / mL, incubating the solution in a water bath at 50°C for 15 minutes with several shakes, sterilizing the solution through a 0.22 μm filter, and storing in the dark.

[0089] S14) Before using the hydrogel material, add the LAP working solution to the GHG hydrogel solution in a clean bench according to the proportion, mix thoroughly and evenly, and then use it for cell culture. The GHG hydrogel solution can be stored at 4°C in the dark for several months.

[0090] (3) In addition to providing the hydrogel material, the present invention also provides a method for preparing a printing support bath for vascularized bone organoid suspension 3D printing using the hydrogel material, comprising at least the following steps:

[0091] S11) GelMA, HAMA, glycerol, and PBS buffer were mixed in a proportion and added to a centrifuge tube. The mixture was incubated in a water bath at 60° C. for 4 h until all materials in the centrifuge tube were completely dissolved to obtain a GHG hydrogel solution.

[0092] S12) sterilizing the GHG hydrogel solution by pasteurization and keeping it warm at 37° C.

[0093] S13) preparing a working solution of LAP photoinitiator and PBS solvent at a concentration of 40 mg / mL, incubating the solution in a water bath at 50°C for 15 minutes with several shakes, sterilizing the solution through a 0.22 μm filter, and storing in the dark.

[0094] S14) Before using the hydrogel material, add the LAP working solution to the GHG hydrogel solution in a clean bench according to the proportion and mix thoroughly;

[0095] S15) Digesting and resuspending human bone marrow mesenchymal stem cells, thoroughly mixing with the GHG hydrogel solution containing the LAP photoinitiator, and adding the required amount to the well plate to obtain a support bath for suspension 3D printing.

[0096] (IV) The present invention also provides a method for preparing a bio-ink for suspension printing, comprising the following steps:

[0097] S21) preparing a gelatin solution with a mass ratio of 10% to 15% (preferably 12%) using PBS buffer as a solvent, incubating in a water bath at 60° C. until completely dissolved, sterilizing the gelatin solution by pasteurization, and keeping it warm at 37° C.;

[0098] S22) Digesting and resuspending human umbilical vein endothelial cells into a small amount of suspension, and thoroughly mixing with the sterilized gelatin solution to obtain a bio-ink.

[0099] (V) The present invention also provides a method for preparing vascularized bone organoids:

[0100] S1) preparing a hydrogel support bath as described above for use as a suspension 3D printing medium and vascularized bone organoid culture;

[0101] S2) preparing the bio-ink as described above;

[0102] S3) Suspension printing of vascularized bone organoids;

[0103] Wherein step S3) comprises the following steps:

[0104] S31) Loading the bio-ink into the printing barrel, setting the print head height, and then performing suspension printing in the support bath. The printing temperature is 22-26°C, preferably 25°C, and the printing extrusion pressure is 1.0-1.8 bar, preferably 1.5 bar. A vascular network structure is printed in the support bath according to the model. Upon completion of printing, a vascularized bone organoid is obtained.

[0105] S32) After printing is completed, irradiate the orifice plate with an ultraviolet light source with a wavelength of 365-405 nm (preferably 365 nm) for 10-30 seconds (preferably 20 seconds) to solidify the support bath;

[0106] After adding culture medium, the cells are placed in a cell culture incubator. The endothelial cells carried by the melted gelatin bio-ink adhere to the hollow tubes left behind and form a vascularized co-culture structure. Dead cells and residual gelatin are removed by flushing with PBS. Long-term culture is achieved by replacing the culture medium with fresh medium every 2-3 days.

[0107] In this patent application document, the limitations on the molecular weight of the selected polymer raw materials are all limitations on the average molecular weight, not the molecular weight of each polymer molecule.

[0108] In the bone organoid hydrogel preparation method of the present invention, the raw materials for GelMA and HAMA, in the examples, have molecular weights of 150 kDa and 400 kDa, respectively; the amino substitution degree of GelMA used is 60%. In the hydrogel material, the mass ratios of the three main components, GelMA, HAMA, and glycerol, are 4.5%, 0.5%, and 5%, respectively. The blending of HAMA and glycerol improves the hydrogel's toughness and viscosity, while retaining the advantages of rapid UV crosslinking and curing. This allows for structural stability and non-degradation during long-term 3D cell culture, enabling the bone organoid culture process to achieve a balance between matrix stability and cell growth and development. In the method for using the bone organoid hydrogel material for 3D printing, the specific printing method is suspension extrusion 3D printing, in which the hydrogel serves as a suspension support bath material, supporting the suspended structure of the bio-ink print and ensuring stable and uniform distribution of the blended cells in space. The bio-ink used for printing is a 12% gelatin solution in PBS. It is in a gel state at room temperature for printing, but rapidly melts and flows out at cell culture temperatures, forming a hollow structure. In addition, the density of human bone marrow mesenchymal stem cells (hBMSC) and human umbilical vein endothelial cells (HUVEC) in the support bath and bio-ink were 4×10 7 cells / mL and 1×10 7 cells / mL.

[0109] Example 1 Preparation of a hydrogel support bath for suspending 3D printing media and vascularized bone organoid culture

[0110] S11) 4.5% GelMA, 0.5% HAMA, 5% glycerol, and 90% PBS buffer were mixed in a proportion and added to a centrifuge tube. The mixture was incubated in a water bath at 60° C. for 4 h until all materials in the centrifuge tube were completely dissolved to obtain a GHG hydrogel solution.

[0111] S12) sterilizing the GHG hydrogel solution by pasteurization and keeping it warm at 37° C.

[0112] S13) preparing a working solution of LAP photoinitiator and PBS solvent at a concentration of 40 mg / mL, incubating the solution in a water bath at 50°C for 15 minutes with several shakes, sterilizing the solution through a 0.22 μm filter, and storing in the dark.

[0113] S14) In a clean bench, add the LAP working solution to the GHG hydrogel solution in proportion, mix thoroughly, and keep warm at 37°C.

[0114] The mass ratios of GelMA, HAMA, glycerol, and PBS buffer are as follows:

[0115] S15) Digest and resuspend human bone marrow mesenchymal stem cells into a suspension at 4×10 7 cells / mL density was thoroughly mixed with the GHG hydrogel solution and added to the well plate according to the required amount to obtain a support bath.

[0116] Example 2 Preparation of biological ink:

[0117] S21) preparing a gelatin solution (preferably 12%) at a mass ratio of 10%-15% using PBS buffer as a solvent, incubating in a water bath at 60° C. until completely dissolved, sterilizing the gelatin solution by pasteurization, and keeping warm at 37° C.

[0118] S22) Human umbilical vein endothelial cells were digested and resuspended into a suspension at 1×10 7 cells / mL was thoroughly mixed with the gelatin solution to obtain the bio-ink.

[0119] Example 3

[0120] S31) The bio-ink is loaded into the printing cartridge, and after setting the print head height, suspended printing can be performed in the support bath. The printing temperature is 22-26°C, preferably 25°C, and the printing extrusion pressure is 1.0-1.8 bar, preferably 1.5 bar. A suspended vascularized network is constructed in the support bath according to the model.

[0121] S32) After printing is completed, the orifice plate is irradiated with an ultraviolet light source with a wavelength of 365-405 nm (preferably 365 nm) for 10-20 seconds (preferably 15 seconds) to cure and crosslink.

[0122] S33) After adding culture medium, place the cells in a cell culture incubator. The gelatin bio-ink rapidly melts out at 37°C, and the endothelial cells carried by the bio-ink adhere to the wall of the hollow tubes left behind to form a vascularized co-culture structure. Dead cells and residual gelatin in the tubes can be removed by PBS injection and flushing. Fresh culture medium is replaced every 2-3 days for long-term culture.

[0123] Figure 2 illustrates the method for constructing a printed vascularized bone organoid in an embodiment. A vascularized bone organoid model was constructed using a suspension 3D printing method. The gelatin solution exhibits a gel state at room temperature and a liquid state at 37°C during cell culture. It is unaffected by UV crosslinking with a photoinitiator, making it an excellent temperature-sensitive sacrificial ink. HUVEC cells transfected with red fluorescent protein (HUVEC-RFP) and mouse embryonic osteoblast precursor cells transfected with green fluorescent protein (MC3T3-E1-GFP) replaced the original HUVEC and hBMSC cells, respectively, and were mixed into the bio-ink and support bath for positioning. Fluorescence microscopy demonstrated that the HUVEC-RFP cells (red fluorescence) were able to adhere to the walls of the gelatin ink-melted tubes and were precisely distributed with the MC3T3-E1-GFP cells (green fluorescence) in the support bath according to the model settings, demonstrating the formation of a multicellular co-printed macrostructure.

[0124] The structure and characterization of the hollow vascular network in the embodiment are shown in Figure 3. A cell-free vascular network model was printed in a support bath using a suspension 3D printing method and UV crosslinked and cured. After gelatin melted at 37°C, it was rinsed with PBS, injected with contrast agent, and stored in a 4°C refrigerator. Micro-CT 3D reconstruction confirmed the formation of the hollow channel network.

[0125] Figure 4 illustrates the cell viability of the examples. Live-dead staining demonstrated the high viability of two different cell distributions: HUVEC (A, B, scale 100 μm) in gelatin ink and hBMSC (C, scale 1000 μm) in the support bath. HUVEC adhered to the four walls of the hollow tube (B) and proliferated rapidly, while hBMSC, encapsulated within the hydrogel, proliferated more slowly but maintained a high survival rate. This demonstrates that the extrusion printing method and LAP-induced UV crosslinking have little effect on cell viability.

[0126] The cell proliferation capacity of the embodiment is shown in Figure 5. The proliferation of cells in the vascularized bone organoids was verified by CCK-8 cytotoxicity experiments. The hBMSC group was a GHG-supported bath hydrogel mixed with only bone marrow mesenchymal stem cells, which was only UV-cured without printing; while the co-printing group was a co-culture structure constructed by suspension printing of bone marrow mesenchymal stem cells and vascular endothelial cells. The hBMSC cells in three-dimensional culture conditions continued to proliferate for 14 days, demonstrating the ability of GHG-supported bath hydrogel as a carrier for long-term cell culture; HUVEC cells rapidly proliferated and grew full and self-assembled into endothelial-like structures on the tube wall, demonstrating that vascularized bone organoids can proliferate for a long time under three-dimensional culture conditions and maintain the preset vascularized structural characteristics.

[0127] The osteogenic differentiation capacity of the bone organoids in this example is shown in Figure 6 (scale 50 μm). Immunofluorescence experiments verified the feasibility of bone organoid differentiation into osteoblasts. Significant expression of two osteogenic differentiation genes, Runx-2 and OCN, was observed 10 days after induction, demonstrating that the suspension 3D printing method and the GHG-supported bath hydrogel can effectively meet the requirements for culturing and inducing bone organoid differentiation.

[0128] The osteogenic mineralization performance of the examples is shown in Figure 7 (scale bar, 750 μm). Vascularized bone organoids were induced in osteogenic induction medium for 14 days, and frozen sections were then stained with Alizarin Red. Alizarin Red chelates with calcium salts in mineralized nodules to form a reddish-brown complex. Observation revealed that the stained mineralized nodules were uniformly distributed over a large area across the sections, further demonstrating the successful differentiation of the bone organoids into osteoblasts.

[0129] Example 4 Supporting the selection of bath hydrogels

[0130] The method of Example 3 was used to prepare a vascularized bone organoid model. The supporting bath hydrogels were 5% GelMA hydrogel solution and GHG hydrogel solution (i.e., the GHG hydrogel solution of Example 1) for comparison, wherein the bio-ink was a 12% gelatin solution. The rheological characterization of the bio-ink and supporting bath hydrogel in the example is shown in Figure 1. A temperature sweep was performed at 1% strain and 1 Hz frequency. The results are shown in Figure 1A. The experimental results show that both the bio-ink and the two supporting bath hydrogels transformed from solid to viscoelastic fluid as the temperature increased, showing excellent temperature sensitivity. For the two supporting bath materials, the shear rate was changed and alternating strain was applied at 20°C. The results are shown in Figure 1B and C. The experimental results show that the GHG supporting bath material has excellent shear-thinning properties and self-recovery properties. At the same time, its viscosity is significantly higher than that of the 5% GelMA hydrogel commonly used for three-dimensional cell culture. It can provide better support for cells and bio-inks and is highly suitable for suspended 3D printing.

Claims

1. A suspension printing support bath for preparing vascularized bone organoids, characterized in that: The support bath material comprises methacrylated gelatin, methacrylated hyaluronic acid, glycerol, a photoinitiator, bone marrow mesenchymal stem cells and a first solution; Preferably, the photoinitiator is selected from 2,4,6-trimethylbenzoyl phosphate lithium salt; Preferably, the density of bone marrow mesenchymal stem cells is 4×10 7 cells / mL; Preferably, the components of the hydrogel material are composed by mass: Preferably, the method for preparing a suspension printing support bath comprises the following steps: S11) mixing methacrylated gelatin, methacrylated hyaluronic acid, glycerol, and the first solution in a certain mass ratio and adding the mixture to a container, heating and stirring until the mixture is completely dissolved to obtain a GHG hydrogel solution; S12) sterilizing the GHG hydrogel solution; S13) preparing a working solution with the photoinitiator and the first solution and sterilizing the solution; S14) mixing the working solution and the GHG hydrogel solution uniformly; S15) Digest and resuspend human bone marrow mesenchymal stem cells into a suspension, and seed them at 4×10 7 The density of cells / mL was fully mixed with the GHG hydrogel solution to obtain a suspended printing support bath.

2. A raw material for a suspension printing support bath for preparing vascularized bone organoids, comprising the hydrogel material of claim 1 and a suspension printing bio-ink placed separately, The bio-ink for suspension printing comprises umbilical vein endothelial cells, gelatin and a second solution; Preferably, the density of umbilical vein endothelial cells in the bio-ink for suspension printing is 1×10 7 cells / mL; Preferably, the concentration of gelatin in the bio-ink for suspension printing is 10%-15%; Preferably, the second solution is a buffer solution; Preferably, the method for preparing bio-ink for suspension printing comprises the following steps: S21) preparing a gelatin solution with a mass ratio of 10% to 15% using gelatin and a second solution, heating and stirring until completely dissolved, and sterilizing; S22) Umbilical vein endothelial cells were digested and resuspended into a suspension, and 1×10 7 The density of cells / mL was fully mixed with the gelatin solution to obtain the bio-ink for suspension printing.

3. Use of the hydrogel material for preparing a suspension printing support bath for vascularized bone organoids according to claim 1 as a suspension printing support bath for vascularized bone organoids.

4. A method for preparing vascularized bone organoids, characterized in that: The preparation method comprises the following steps: S1) preparing a hydrogel support bath as described above for use as a suspended 3D printing medium and vascularized bone organoid culture; S2) preparing the bio-ink as described above; S3) Suspension printing of vascularized bone organoids; Wherein step S3) comprises the following steps: S31) performing suspension printing with bio-ink in a support bath, constructing a suspended vascularized network in the support bath according to the model; S32) After printing is completed, irradiating with an ultraviolet light source to complete the curing and cross-linking of the support bath to obtain a cross-linked and cured vascularized bone organoid model precursor; S33) After the culture medium is added to the vascularized bone organoid model precursor, cell culture is performed. The bio-ink inside the vascularized bone organoid model precursor melts and flows out under the cell culture temperature conditions, and the endothelial cells in the bio-ink adhere to the hollow tubes left at the original bio-ink printing site and grow to form a vascular network structure; dead cells and residual gelatin in the tubes are removed by flushing, and fresh culture medium is replaced every 2-3 days for long-term culture.

5. The preparation method according to claim 4, wherein S1) comprises the following steps: S11) mixing methacrylated gelatin, methacrylated hyaluronic acid, glycerol, and the first solution according to a mass ratio and adding the mixture to a centrifuge tube, heating and stirring until completely dissolved to obtain a GHG hydrogel solution; S12) sterilizing the GHG hydrogel solution; S13) preparing a working solution of the photoinitiator with the first solution and sterilizing the solution; S14) mixing the photoinitiator working solution and the GHG hydrogel solution uniformly; S15) Digest and resuspend human bone marrow mesenchymal stem cells into a suspension, and seed them at 4×10 7 cells / mL density was thoroughly mixed with the GHG hydrogel solution to obtain a suspended printing support bath.

6. The preparation method according to claim 4, wherein S2) comprises the following steps: S21) preparing a gelatin solution with a mass ratio of 10% to 15% using gelatin and a second solution, heating and stirring until completely dissolved, and sterilizing; S22) Umbilical vein endothelial cells were digested and resuspended into a suspension, and 1×10 7 cells / mL density was thoroughly mixed with the gelatin solution to obtain the bio-ink for suspension printing; Preferably, in S11), the mass proportions of methacryloylated gelatin, methacryloylated hyaluronic acid, glycerol and the first solution are 4.5%, 0.5%, 5% and 90% respectively.

7. The preparation method according to claim 4, characterized in that The printing temperature in S31) is 22-26°C, Preferably, the printing extrusion pressure in S31) is 1.0-1.8 bar.

8. The preparation method according to claim 4, characterized in that S32) wherein the ultraviolet light source is a 365-405 nm ultraviolet light source, Preferably, the irradiation time of the ultraviolet light source in S32) is 10-20s.

9. The vascularized bone organoid prepared by the preparation method according to any one of claims 4 to 8.

10. Use of the vascularized bone organoid according to claim 9 in the preparation of an implantable medical device for bone repair.

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