Core-shell structure vascularized bone organ as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of organoid, and particularly relates to a core-shell structure vascularized bone organoid and a preparation method and application thereof. The MSCs and the ECs are cultured by adopting an organoid culture mode, the cell spheroid with a'core-shell 'structure is constructed by using the MSCs / ECs with a specific cell proportion, a microenvironment for in-vivo growth of cells can be better simulated, cell-cell connection and a complete extracellular matrix are preserved, the cell spheroid has a structure and a function similar to those of source cells or directionally differentiated stem cells, and the cell spheroid has a good application prospect. And the development process of organs is simulated, so that higher self-renewal and proliferation capabilities are achieved, the metabolic capability of cells is improved, the cell viability is improved, and good proliferation, osteogenesis and vascularization effects are achieved. The vascularized bone organ prepared by the method can be widely applied to preparation of bone repair materials.

Description

Technical Field

[0001] This invention belongs to the field of organoid technology, specifically relating to a nucleus-shell structured vascularized bone organoid, its preparation method, and its application. Background Technology

[0002] The jawbone is a vital organ for maintaining facial appearance, chewing, and speech. Large jawbone defects caused by tumors, inflammation, or trauma, exceeding the body's natural healing capacity, often lead to nonunion or delayed healing, impacting patients' quality of life and health, and in severe cases, even endangering their lives. Autologous bone grafting is the "gold standard" for repairing large bone defects. Among these, vascularized autologous bone grafts, compared to non-vascularized autologous bone, have less absorption and stronger resistance to infection, making them the mainstream method for clinical repair of large jawbone defects. However, autologous bone has limitations such as limited availability, significant trauma, and difficulty in shaping, restricting its clinical application. Commonly used bone replacement materials, such as Bio-Oss®, lack osteoinductive properties and are difficult to shape, failing to restore the complex shape and function of large jawbone defects. Furthermore, traditional bone repair materials primarily achieve osseointegration with the recipient bone through "creeping replacement," meaning that as the bone repair material is gradually absorbed, new bone grows in and replaces the original material. The "creeping replacement" process primarily occurs at the recipient bone-repair material interface, gradually progressing inwards or parallel to the bone repair material to achieve complete replacement of the bone repair material, typically taking 3 to 6 months. Due to the excellent osteoconductivity, osteoinductive properties, and bone regeneration capacity of autologous bone, its creeping replacement rate is significantly higher than that of bone replacement materials. Studies have found that using tissue engineering methods and a multi-center osteogenic model can accelerate bone formation and reduce the repair time for large-volume bone defects.

[0003] Bone / cartilage organoids are micro-tissues formed by the self-assembly of stem cells into spherical bodies under 3D culture conditions, which further develop into biomimetic 3D spatial features and organ functions. Bone / cartilage organoids are ideal in vitro models reflecting the complex physiological microenvironment of bone / cartilage and can be used to study bone / cartilage formation, remodeling, and regeneration.

[0004] However, the existing foundation still has the following drawbacks: 1. Traditional 2D cell culture alters the natural growth pattern of cells, resulting in the absence of extracellular matrix. When cells are digested with trypsin to prepare seed cell suspensions, trypsin disrupts integrin β1 on the cell surface and the connections between cells, affecting cell viability and reducing cell repair capabilities.

[0005] 2. Based on the principles of jawbone development, mesenchymal stem cells (MSCs) and endothelial cells exhibit a spatiotemporal distribution of a "nucleus-shell" structure. Current organoid preparation methods directly mix the two cell types together, oversimplifying their interaction and communication. Whether the optimal ratio and spatial structure of MSCs and ECs can be determined by adjusting cell culture methods to construct vascularized bone organoids remains unexplored.

[0006] 3. No studies have yet compared the osteogenic and vascularization abilities of spheroidal cells with "nucleo-shell" structures with those of homogeneous spheroidal cells. Summary of the Invention

[0007] The first objective of this invention is to provide a method for preparing vascularized bone organoids.

[0008] The second aspect of the present invention is to provide a vascularized bone organoid.

[0009] The third objective of this invention is to provide a preparation method for the first aspect of this invention and the application of vascularized bone organoids in the preparation of bone repair materials.

[0010] The fourth aspect of this invention is to provide a bone repair material.

[0011] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a method for preparing vascularized bone organoids, comprising the following steps: 1) Preparation of mesenchymal stem cell spheroids; 2) Seed the endothelial cell suspension onto the surface of mesenchymal stem cell spheroids; 3) Incubation, forming a core-shell structure; 4) Osteogenic induction, forming vascularized osteoids.

[0012] In some embodiments of the present invention, the method for preparing the mesenchymal stem cell spheroids is as follows: Mesenchymal stem cell suspensions were seeded into 3D cell culture plates and cultured to obtain mesenchymal stem cell spheroids.

[0013] In some embodiments of the present invention, the method for preparing mesenchymal stem cell spheroids further includes the step of treating 3D cell culture plates with an anti-adhesion washing solution.

[0014] In some embodiments of the present invention, the 3D cell culture plate includes a hanging drop plate, a Nunclon Sphera™ ultra-low adsorption plate, and an AggreWell plate. TM Culture plate.

[0015] In some embodiments of the present invention, the ratio of the number of mesenchymal stem cells to endothelial cells is 1:(2~10); more preferably, it is 1:5.

[0016] In some embodiments of the present invention, the incubation time is 6 to 12 hours.

[0017] In some embodiments of the present invention, the incubation culture medium is a mixture of osteogenic induction culture medium and endothelial cell culture medium at a cell ratio of 1:(2~10).

[0018] In some embodiments of the present invention, the endothelial cell culture medium comprises DMEM high glucose medium of 8-12% FBS.

[0019] In some embodiments of the present invention, the osteogenic induction culture medium comprises 8-12% FBS, 8-12 mmol / L sodium β-glycerophosphate, 40-60 mg / L vitamin C, and (0.5-1.5) 10 -7 DMEM high-glucose medium containing mol / L dexamethasone.

[0020] In some embodiments of the present invention, the endothelial cell culture medium and osteogenic induction culture medium further include bispecific antibodies.

[0021] Specifically, the incubation conditions are as follows: at AggreWell TM In the 400 culture plates, the culture medium used for cell incubation is a mixture of DMEM high glucose medium containing 8-12% fetal bovine serum and 1% penicillin and antibiotics and endothelial cell medium according to the corresponding cell number.

[0022] Specifically, the osteogenic induction conditions are as follows: osteogenic induction medium and endothelial cell medium are mixed at appropriate cell ratios and then used for organoid culture. The osteogenic induction medium contains 10% FBS, 1% penicillin-dextrin, 10 mmol / L sodium β-glycerophosphate, 50 mg / L vitamin C, and 10... -7 DMEM high-glucose medium containing mol / L dexamethasone.

[0023] In a second aspect, the present invention provides a vascularized bone organoid prepared by the preparation method described in the first aspect of the present invention.

[0024] A third aspect of the present invention provides a preparation method for the first aspect of the present invention and the application of vascularized bone organoids of the second aspect in the preparation of bone repair materials.

[0025] In some embodiments of the present invention, the bone repair material is used for jawbone and skull repair.

[0026] In a fourth aspect, the present invention provides a bone repair material comprising the vascularized bone organoids described in the second aspect of the present invention.

[0027] In some embodiments of the present invention, the bone repair material further includes pharmaceutically acceptable excipients.

[0028] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0029] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0030] The present invention has the following beneficial effects: 1. 3D cell culture method: This invention uses organoid culture to culture MSCs and ECs, which can better simulate the microenvironment of cell growth in vivo, preserve cell-cell connections and complete extracellular matrix, have similar structure and function to the source cells or directed differentiation stem cells, and simulate the development process of organs, with higher self-renewal and proliferation capabilities, and improve cell metabolism and cell activity.

[0031] 2. The "nucleo-shell" structured spheroids constructed from MSCs / ECs exhibit good proliferation, osteogenic, and angiogenic effects, with an optimal cell ratio of 1:5. Attached Figure Description

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1The expression of stemness-related genes was detected in 3D spheroidal and 2D cultured cells after 4 days of in vitro culture. The 6:0 group consisted of MSCs; the 4:2 group consisted of a 4:2 mixture of MSCs and ECs co-cultured. * P <0.05; **: P <0.01; ***: P <0.001; ****: P <0.0001.

[0033] Figure 2 Expression of osteogenic and angiogenic genes in 3D spheroidal and 2D cultured cells after 4 (A) and 7 (B) days of in vitro osteogenic induction. The 6:0 group consisted of MSCs; the 4:2 group consisted of a 4:2 mixture of MSCs and ECs co-cultured. * P <0.05; **: P <0.01; ***: P <0.001; ****: P <0.0001.

[0034] Figure 3 Expression of osteogenic and angiogenic-related proteins in 3D spheroidal and 2D cultured cells after 7 days of in vitro osteogenic induction. A: Western blotting (WB) grayscale images and quantitative analysis of osteogenic-related proteins; B: Western blotting (WB) grayscale images and quantitative analysis of angiogenic-related proteins. The 6:0 group consisted of MSCs; the 4:2 group consisted of MSCs and ECs co-cultured at a 4:2 ratio. *: P <0.05; **: P <0.01; ***: P <0.001; ****: P <0.0001.

[0035] Figure 4 After 1, 4, and 7 days of in vitro osteogenic induction for 3D spheroids and 2D cultured cells, ALP staining was used to detect the osteogenic differentiation capacity of the cells (scale bar 300 μm). The 6:0 group consisted of MSCs; the 4:2 group consisted of MSCs and ECs co-cultured at a 4:2 ratio.

[0036] Figure 5 After 1, 4, and 7 days of in vitro osteogenic induction in 3D spheroidal and 2D cultured cells, ALP was quantitatively assessed to determine the osteogenic differentiation capacity of the cells. The 6:0 group consisted of MSCs; the 4:2 group consisted of a 4:2 mixture of MSCs and ECs co-cultured. * P <0.05; **: P <0.01; ***: P <0.001; ****: P <0.0001.

[0037] Figure 6 After 4, 7, and 21 days of in vitro osteogenic induction in 3D spheroidal and 2D cultured cells, the osteogenic differentiation capacity of the cells was quantitatively assessed using ARS. The 6:0 group consisted of MSCs; the 4:2 group consisted of a 4:2 mixture of MSCs and ECs co-cultured. * P <0.05; **: P <0.01; ***: P <0.001; ****: P <0.0001.

[0038] Figure 7 Morphology, size, and cell viability of spheroids with different proportions of nucleo-shell structures; A: Schematic diagram of nucleo-shell structure preparation; B: Morphology of spheroids with different proportions of nucleo-shell structures observed under a light microscope; C: Diameter of nucleo-shell structure spheroids; D: Cell viability of nucleo-shell structure spheroids; ns: No statistically significant difference.

[0039] Figure 8 The morphology of CS spheroids at different proportions was observed using fluorescence microscopy and confocal microscopy; green: Dio-labeled MSCs. red: Dil-labeled ECs.

[0040] Figure 9 Expression of osteogenic (OSX, OPN, and BMP2) and angiogenic (PDGFA and PGFFB) related genes after 3 and 7 days of culture;* P <0.05,** P <0.01, *** P <0.001.

[0041] Figure 10 To detect the liveness and death staining (A) and proliferative capacity (B) of three types of spheroids. * P <0.05,** P <0.01, *** P <0.001.

[0042] Figure 11 Osteogenic activity of MSCs, UFs, and CSs was detected by ALP and ARS staining. A: ALP staining. B&C: Quantitative detection of ALP activity in globular bodies at 3 and 7 days. D&E: ARS staining and quantitative detection. UF: Homogeneous globular bodies; CS: Core-shell globular bodies. * P <0.05,** P <0.01, *** P <0.001.

[0043] Figure 12The results of Matrigel lumen formation assay: A: Microvascular formation observed under light and fluorescence microscopy; green: Calcine AM staining; B: Statistical analysis of the number of intervascular crossings, total trunk length, and mesh index. P <0.05,** P <0.01, *** P <0.001.

[0044] Figure 13 This study investigated the expression of osteogenic genes (ALP, OPN, RUNX2, COL1, TGFβ, DMP1, OCN, and SOST) and angiogenesis-related genes (KDR, VEGF, PDGFA, and PDGFB) in UF and CS globules after osteogenic induction at 3, 7, 14, and 21 days. UF: homogeneous globules; CS: nucleo-shell globules. P <0.05,** P <0.01, *** P <0.001.

[0045] Figure 14 Immunofluorescence staining was used to detect MSC differentiation within CS globules. Blue: DAPI-labeled nuclei; Green: FITC-phallane-labeled F-actin; Pink: target proteins, RUNX2 (1 d), ALP (3 d), DMP1 (21 d), SOST (28 d); Red: target protein, CD31. CS: nucleoshell globules.

[0046] Figure 15 To assess the effectiveness of micro-CT in repairing critical bone defects in rat skulls using UF and CS spheroids, A: 3D reconstruction and coronal section, with red circles representing defect location and size; B: Bone volume / total volume; C: Trabecular separation; D: Bone mineral density; UF: Uniform spheroid; CS: Core-shell spheroid. P <0.05,** P <0.01, *** P <0.001.

[0047] Figure 16 H&E staining was used to assess the repair of skull defects in rats. A: H&E staining; B: Percentage of new bone tissue and total tissue area. HB: Host bone; NB: New bone; CT: Connective tissue. P <0.05, *** P <0.001. Detailed Implementation

[0048] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0049] The primer information involved in this invention is shown in Table 1.

[0050] Table 1. Names and primer sequences of genes related to stem cell, osteogenic, and angiogenic activity.

[0051] Example 1: Comparative Study of the Performance of MSCs / ECs Cell Spheroids and 2D Cultured Cells 1. Experimental Methods Construction of MSCs / ECs spheroids: MSCs and ECs were seeded at a ratio of 4:2 in AggreWells treated with anti-adhesion washing solution. TM 400 culture plates, total cell concentration 1.2 × 10⁴ 6 Cells / well. Incubate overnight at 37°C with DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics at 5% CO2. Displace the spheroids in the wells, collect the suspension, and count them. Centrifuge at 500 rpm for 5 min and collect the precipitate. Resuspend in 5% GelMA hydrogel and seed into low-adhesion plates. Cure the cells using a visible blue light curing lamp (405 nm wavelength) for 20 s, add DMEM complete medium, and return to the incubator. For 2D cell culture, the same number of MSCs and ECs were directly seeded into culture plates and cultured under the same conditions for 4 days. RT-PCR was used to detect the expression of stemness-related genes OCT-4, SOX-2, and NANOG to compare the stemness and differentiation potential of spheroid (3D) and 2D cultured cells.

[0052] In addition, to compare the differences in osteogenic differentiation capacity between 3D and 2D cells, osteogenic induction medium (containing 10% FBS, 1% penicillin and antibiotic, 10 mmol / L sodium β-glycerophosphate, 50 mg / L vitamin C, and 10 mg / L sodium β-glycerophosphate) was used. -7 After culturing cells in DMEM high-glucose medium (containing mol / L dexamethasone) for 4 and 7 days, the expression of osteogenic and angiogenic genes ALP, COLⅠ, RUNX2, VEGF, PDGF-α, and PDGF-β was detected. Seven days after osteogenic induction, Western blotting was used to detect the expression of osteogenic and angiogenic proteins under different culture conditions and different MSC / EC ratios (3D 6:0, 3D 4:2, 2D 6:0, and 2D 4:2 groups). The 6:0 group contained only MSC cells.

[0053] After osteogenic induction for 1, 4, and 7 days, ALP staining was used to detect ALP protein expression in the 3D 6:0, 3D 4:2, 2D 6:0, and 2D 4:2 groups. The specific method was as follows: The culture medium was discarded, and the samples were washed three times with 10% PBS, followed by fixation with 4% paraformaldehyde for 1 h. After washing three more times, the ALP staining working solution was prepared by mixing BCIP solution, NBT solution, and alkaline phosphatase staining buffer at a ratio of 1:2:300. 300 μL of staining solution was added to each well, and the samples were incubated at room temperature in the dark for 30 min (observing the staining effect every 5 min after 15 min). Once the staining effect was obvious, incubation was stopped, the working solution was discarded, and the samples were washed three times with PBS for 2 min each time. The samples were then photographed using a stereomicroscope while immersed in PBS (to prevent hydrogel reflection).

[0054] Simultaneously, ALP protein expression in the 3D 6:0, 3D 4:2, 2D 6:0, and 2D 4:2 groups was detected using an ALP quantitative detection kit. The culture medium was discarded, and 600 μL of hydrogel lysis buffer was added. After obtaining cell pellets, 100 μL of 1% Triton lysis buffer was added to each group, and the mixture was repeatedly pipetted and placed on ice for 30 min for lysis. Then, the cells were centrifuged at 12000 rpm / min for 10 min at 4 ℃. After centrifugation, the supernatant was transferred to a new 600 μL EP tube, and 1 μL was used for BCA total protein detection. A 96-well plate was prepared, and samples were added according to the product instructions. The absorbance of each well was measured at 520 nm using a microplate reader. A standard curve was plotted to calculate the ALP concentration of the sample. ALP activity (King's units / gprot) = ALP concentration of sample (mg / mL) ÷ Protein concentration of sample (gprot / mL).

[0055] After osteogenic induction for 7, 14, and 21 days, the formation of calcium nodules, late-stage bone markers, was quantitatively detected by Alizarin Red S (ARS) staining. The culture medium was discarded, and the nodules were washed three times with PBS, fixed with 4% paraformaldehyde for 1 h, washed three times with PBS, and stained with 500 μL of 1% pH 4.2 Alizarin Red staining solution for 10-15 min, observing the staining status at 5-min intervals. Staining was stopped when a difference in staining was observed. The staining solution was discarded, and the nodules were washed three times with PBS for 3 min each time. After washing, the mineralized nodules were dissolved with 10% hexadecylpyridinium chloride monohydrate (CPC), and the absorbance was measured at 562 nm using a microplate reader and statistically analyzed.

[0056] 2. Experimental Results Compared to traditional 2D cell culture, prevascularized spheroids constructed from MSCs / ECs highly express stem cell-related genes such as OCT-4, SOX-2, and NANOG, indicating that the cells within the spheroids have stronger stem cell and differentiation potential. Figure 1 Furthermore, in vitro PCR, Western blotting, and ALP assays revealed that 3D globular cells exhibited significantly superior osteogenic and angiogenic capacity compared to 2D cells. Figure 2-6 ).

[0057] Example 2: Construction and Performance Study of Core-shell (CS) Structured Vascularized Cell Spheroids 1. Experimental Methods Construction of vascularized spheroidal cells with a "core-shell" (CS) structure: Both MSCs and ECs used in the experiments were purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd. MSCs and ECs were cultured in mesenchymal stem cell culture medium and endothelial cell culture medium, respectively. When the cell confluence reached 80%-90%, the cells were digested with 0.25% trypsin / EDTA and then passaged. Cells from passages P4 to P6 were used for subsequent experiments.

[0058] First, treat AggreWell with an anti-adhesion wetting solution. TM 400 culture plates. Different concentrations of MSCs were seeded into wells of the plates to determine the relationship between cell number and globular size. To determine the optimal conditions for preparing globular cells with nucleoshell morphology, gradient concentrations of MSCs were first seeded into the wells, i.e., 0.2 × 10⁻⁶. 6 / mL, 0.4 × 10 6 / mL, 0.6 × 10 6 / mL, 0.8 ×10 6 / mL, 1.0 × 10 6 / mL, 1.2 × 10 6 / mL. After 6 hours, inoculate sequentially with 1.0 × 10⁹ / mL. 6 / mL, 0.8 × 10 6 / mL, 0.6 × 10 6 / mL, 0.4 × 10 6 / mL, 0.2 × 10 6 / mL ECs, with the two cell ratios being 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0 respectively. The final total number of cells per well was 1.2 × 10⁶. 6Based on the ratio of MSCs to ECs, the culture medium used in each group was a mixture of DMEM high-glucose medium and endothelial cell culture medium (the mixing ratio was the same as the cell number ratio). After culturing in 5% CO2 at 37 °C for 12 h, the wells were pipetted and the culture medium was aspirated. After centrifugation at 700 rpm for 5 min, the supernatant was removed. The spheroid pellet was resuspended in 5% GelMA hydrogel, seeded into low-adhesion plates, irradiated with blue light (405 nm wavelength) for 17 s to solidify, and then cultured in 5% CO2 at 37 °C. The morphology, size, and cell spread of the spheroids were observed using a phase-contrast microscope.

[0059] Cell viability was assessed using the AlamarBlue assay kit: After culturing spheroids for 3 days, the culture medium and reagents were mixed at a 10:1 ratio and added to the sample. After incubation at 37 °C in the dark for 1 h, the absorbance was measured at 570 nm and 600 nm using a microplate reader. The blank control wells contained cell-free culture medium. Cell proliferation rate was calculated using the formula: (%) = 117216 × A570 - 80586 × A600 (sample) / 117216 × A570 - 80586 × A600 (control) × 100%. A cell proliferation rate curve was plotted: the ordinate (Y-axis) represents the proliferation rate (%); the abscissa (X-axis) represents the time points.

[0060] To investigate the distribution of spherical cells with different proportions of nucleo-shell structures and whether ECs could completely encapsulate MSCs, MSCs and ECs were labeled with Dio and Dil, respectively, before seeding cells into well plates for subsequent observation. Specifically, MSCs were digested, centrifuged, resuspended in Dio working solution, incubated at 37 °C for 15 min, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and FBS was added to bind excess dye. After centrifugation again, the cells were resuspended in DMEM complete medium and counted. The Dil labeling process for ECs was the same as for MSCs. After incubation, the cells were resuspended in endothelial cell culture medium and counted. The labeled cell types were then seeded in AggreWells at appropriate ratios. TM The 400 culture plates were spherically formed, resuspended in hydrogel, and then inoculated into well plates for observation.

[0061] Expression of osteogenic and angiogenic genes in "nuclear-shell" spheroids with different proportions: After culturing "nuclear-shell" spheroids in mixed culture medium (a mixture of osteogenic induction medium and endothelial cell culture medium in proportion to cell number) for 3 and 7 days, the expression of osteogenic and angiogenic genes was detected by qRT-PCR: expression of osteogenic-associated transcription factor (Osterix, OSX), osteopontin (OPN), bone morphogenetic protein 2 (BMP2), platelet-derived growth factor A (PDGFA), and platelet-derived growth factor B (PDGFB) was screened to select mixed cell proportions with better osteogenic and angiogenic performance.

[0062] 2. Experimental Results When MSCs and ECs were mixed at ratios of 6:0, 5:1, 4:2, 3:3, 2:4, and 1:5 (total cell count 1.2 × 10⁻⁶), 6 When CS spheroids were formed in each well, the morphology and size were relatively uniform across groups. AlamarBlue assay showed good cell viability with no significant differences between groups. P >0.05)( Figure 7 Fluorescence microscopy revealed that as the number of ECs increased to five times that of MSCs, the MSCs were essentially encapsulated by the ECs. Confocal microscopy further showed that the surface of the 1:5 group of spherical bodies was almost entirely composed of ECs labeled with Dil. Figure 8 ).

[0063] After culturing CS spheroids in GelMa hydrogels at different ratios for 3 and 7 days, RT-PCR analysis showed that with the increase of ECs ratio within the spheroids, the expression of osteogenic-related genes, such as OSX, OPN, and BMP2, and angiogenic-related genes, such as PDGFA and PDGFB, increased. Therefore, a 1:5 ratio was chosen for subsequent comparison of the performance differences between homogeneous and core-shell structured spheroids. Figure 9 ).

[0064] Example 3: Comparison of organoid performance using different culture methods 1. Experimental Methods MSCs and ECs were mixed at a ratio of 1:5 and then inoculated into AggreWell. TM 400 culture plates (cell count 1.2 × 10⁶) 6Collected after 12 hours (number per well), these were designated as uniform (UF) spheroids. CS spheroids were prepared using the same method as in Example 2. Furthermore, spheroids collected after seeding MSCs in the same manner and number were designated as mesenchymal stem cell (MSC) spheroids.

[0065] Cell viability assay: After 7 and 21 days of culture, MSC, UF, and CS spheroids were stained using a calcein AM / propidium iodide (PI) live / dead cell staining kit. Cell viability and growth were observed using confocal microscopy. Specifically, the cells were first gently washed 2 to 3 times with PBS, followed by adding an appropriate amount of live / dead cell staining working solution to cover the sample. The cells were incubated for 15 min. Subsequently, the cells were washed 3 times with PBS, and observation and recording were performed using confocal microscopy.

[0066] Cell proliferation assay: After 1, 3 and 7 days of culture of MSCs, UFs and CS spheroids, the proliferation of the three types of spheroid cells was detected using CCK8 reagent.

[0067] Three types of spheroids were cultured in osteogenic induction medium for 3 and 7 days, and ALP protein expression was detected using an ALP staining kit. Specifically: after removing the medium, the samples were washed three times with PBS and fixed with 4% paraformaldehyde for 1 h. After washing three times with PBS, the ALP staining working solution was prepared by mixing BCIP solution, NBT solution, and alkaline phosphatase staining buffer at a ratio of 1:2:300. 300 μL of staining solution was added to each well, and the samples were incubated at room temperature in the dark. Incubation was stopped after significant staining, the working solution was discarded, and the samples were washed three times with PBS. Images were taken using a stereomicroscope. Twenty-one days after osteogenic induction, Alizarin Red staining was used to detect the formation of calcium nodules, a late marker of osteogenic development. Specifically: after removing the medium, the samples were washed three times with PBS, fixed with 4% paraformaldehyde for 1 h, washed with PBS, and stained with 500 μL of Alizarin Red staining solution for 10 min. The staining solution was discarded, and the samples were washed three times with PBS, 3 min each time. After cleaning, the mineralized nodules were dissolved using 10% hexadecylpyridinium chloride monohydrate (CPC), and the absorbance values ​​were detected at 562 nm using an ELISA reader and statistically analyzed.

[0068] After osteogenic induction for 3 and 7 days, the expression of ALP protein in three types of globular cells was quantitatively detected. Specifically, the culture medium was removed, and 200 μL of hydrogel lysis buffer was added. After complete lysis, the cells were centrifuged to obtain cell pellets. 100 μL of 1% Triton lysis buffer was added to each group, and the cells were repeatedly pipetted and placed on ice for 30 min for lysis. The cells were then centrifuged at 12000 rpm for 10 min at 4 ℃. After centrifugation, the supernatant was transferred to new EP tubes, and samples were added according to the manufacturer's instructions. The absorbance of each well was measured at 520 nm using a microplate reader. In addition, the protein concentration of the samples was detected using a BCA kit. The calculation formula was: ALP activity (King's units / gprot) = ALP concentration of the sample (mg / mL) ÷ Protein concentration of the sample (gprot / mL).

[0069] Matrigel angiogenesis assay: Under low temperature conditions, 50 μL of Matrigel was added to a pre-cooled 96-well plate and incubated at 37 °C for 30 min to allow it to solidify. 600 spheroids were added to each well, and after incubation for 7 h, Calcine AM staining was used to observe microvascular formation. The ImageJ angiogenesis analysis plugin was used to analyze vascular intersections, trunk length, and mesh index.

[0070] Osteogenesis and angiogenesis-related gene expression detection: After osteogenic induction culture of globular UF and CS for 3 d, 7 d, 14 d and 21 d, respectively, the expression of osteogenic and angiogenesis-related genes was detected by qRT-PCR.

[0071] To determine whether terminal differentiation of osteocytes (CS) globules can form osteoid organoids, immunofluorescence staining was used to detect the expression of osteocyte-specific markers. Specifically, samples were fixed with 4% paraformaldehyde for 30 min, washed with PBS, treated with 0.1% Triton X-100 for 15 min, and blocked with 5% bovine serum albumin solution for 60 min. Primary antibody was then added and incubated overnight at 4 °C. After washing, fluorescent secondary antibody Alexa Fluor® 647 / 555 was added and incubated in the dark for 1 h. After washing with PBS buffer, FITC-labeled phalloidin backbone protein was added for staining for 20 min, followed by DAPI staining and CLSM observation and statistical analysis. Antibody information is shown in Table 2. Table 2 Antibody Name, Species, Source and Concentration

[0072] 2. Experimental Results 1) Proliferative activity After 7 days of in vitro culture, the three types of spheroids showed a large number of green-stained live cells radially distributed outward from the spheroids, with virtually no red-stained dead cells. After 21 days, the hydrogel was filled with green-stained live cells, with a small number of red-stained dead cells visible. Cell proliferation experiments showed that all three types of spheroids had good proliferative activity in the hydrogel. Specifically, CS spheroids were significantly more proliferative than MSCs at 1, 3, and 7 days (P<0.05), and significantly more proliferative than UFs at 3 and 7 days (P<0.05). No significant difference was observed between MSCs and UFs. Figure 10 ).

[0073] 2) Comparison of osteogenic capacity After osteogenic induction for 3 and 7 days, ALP staining showed that the globular bodies and surrounding extended cells were bluish-purple, with a darker color in the center of the globular bodies. CS staining was darker than MSC and UF. Quantitative ALP detection showed that ALP activity in the CS group was significantly higher than that in the UF and MSC groups (P<0.01). At day 7, there was no significant difference between the UF and MSC groups. ARS staining showed deep red calcified nodules in all three groups (MSC, UF, and CS). Quantitative detection showed that the absorbance value of the CS group was significantly higher than that of MSC and UF (P<0.01). Calcium ion deposition in the UF group was significantly higher than that in the MSC group (P<0.05). Figure 11 ).

[0074] 3) Comparison of angiogenic capacity After culturing the spheroids on Matrigel surfaces for 7 h, both light and fluorescence microscopy revealed almost no tubular connections between the MSC spheroids. In the UF group, only a small number of spheroids showed interconnections. In the CS group, the spheroids aggregated, and tubular connections between them were more pronounced. Statistical analysis showed that the number of junctions, total segment length, and mesh index in the MSC group were significantly lower than the other two groups (P<0.001). The number of junctions, total segment length, and mesh index in the CS group were significantly higher than those in the UF group (P<0.05). Figure 12 ).

[0075] 4) Expression of genes related to osteogenic and angiogenic processes in globular bodies with different structures: After osteogenic induction culture of UF and CS globules for 3, 7, 14, and 21 days, differential expression of osteogenic and angiogenic genes was observed. After 3 days of induction, the expression of osteogenic genes ALP, OPN, RUNX2, COL1, and TGFβ in CS globules was significantly higher than that in UF (P<0.001); the expression of angiogenic genes KDR, PDGFA, and PGFFB in CS globules was significantly higher than that in UF (P<0.001). There was no significant difference in VEGF expression between the two globule types. After 7 days of induction, the expression of osteogenic genes ALP, OPN, and RUNX2 in CS globules was significantly higher than that in UF (P<0.05); the expression of COL1 and OCN was not significantly different. The expression of angiogenic genes KDR, PDGFA, and PGFFB in CS globules was significantly higher than that in UF (P<0.01). There was no significant difference in VEGF expression between the two groups. After 14 days of induction, the expression of osteogenic genes OPN, OCN, DMP1, and TGFβ in CS globules was significantly higher than that in UF (P<0.05); ALP expression showed no significant difference. The expression of angiogenesis-related gene KDR in CS globules was significantly higher than that in UF (P<0.05). There was no significant difference in the expression of VEGF, PDGFA, and PGFFB between the two groups. After 21 days of induction, the expression of osteogenic genes OCN, DMP1, TGFβ, and SOST in CS globules was significantly higher than that in UF (P<0.05); OPN expression showed no significant difference. The expression of angiogenesis-related gene KDR in CS globules was significantly higher than that in UF (P<0.05). There was no significant difference in the expression of VEGF, PDGFA, and PGFFB between the two groups. Figure 13 ).

[0076] 5) CS spheroids form vascularized osteoid organoids. like Figure 14 As shown, after 1 day of induction, immunofluorescence staining revealed that CS spheroids highly expressed the osteogenic progenitor cell marker RUNX2; after 3 days of induction, they expressed the osteogenic cell marker ALP; at 21 days of induction, the early osteogenic cell marker DMP1 was highly expressed; and at 28 days, the mature osteoblast marker SOST was expressed and, along with red-stained CD31, was widely distributed throughout the microtissue, indicating that vascularized bone organoids had been formed.

[0077] Example 4: Comparison of the effects of different organoids on the repair of skull defects in rats 1. Experimental Methods With the approval of the ethics committee (KY2023-484-03), the experiment used SPF-grade adult male SD rats (6 to 8 weeks old, 250 to 300 g), provided by the Guangdong Provincial Animal Center. The rats were randomly assigned to cages of 4 per cage and housed in a barrier environment. The rats were acclimatized for one week prior to surgery. The animals were anesthetized with isoflurane gas. Once the animals were under anesthesia, the surgical procedure was performed. Specifically, under sterile conditions, the top of the rat's skull was prepared with a 2 cm longitudinal incision. Subcutaneous tissue was bluntly dissected to expose the bone surface and remove the periosteum. A bone defect was created using a 5 mm diameter bone harvesting ring and cooled with sterile saline. Two holes were prepared in the skull of each rat. Samples from each group, induced in vitro for 14 days, were then implanted into the round bone defects and sutured in layers. After the rats recovered, they were placed back in their cages and allowed to resume drinking and eating. Four weeks post-surgery, the rats were euthanized due to excessive CO2 inhalation, and the samples were harvested and fixed.

[0078] After 24 hours of sample fixation, micro-CT was used to assess bone defect repair. The scan parameters were: aluminum filter, 4-frame overlay, angle gain of 0.72 degrees, voltage of 70 kV, current of 100 µA, exposure time of 100 ms, and slice thickness of 20 μm. The sample was rotated one full turn to complete the scan. Data was imported into Micro-CT Reconstruction, and regions of interest were selected along the skull defect. Images were processed using Medproject software, and thresholds of 50-255 were set to analyze the percentage of new bone volume (BV / TV), bone mineral density (BMD), and trabecular separation (Tb.Sp). Subsequently, the samples were decalcified, stained with Hematoxylin and eosin (H&E), and the area of ​​new bone was measured and statistically analyzed using Image J 1.54g.

[0079] 2. Experimental Results One month after implantation, significant bone regeneration was observed in both the CS and UF groups, with some new bone formation on the lateral wall of the Blank group. Coronal images showed only minimal bone regeneration at the Blank defect site, while in the CS group, new bone connected the entire defect, but bone density was reduced. In the UF group, only partial bone connection was observed. Two months after implantation, new bone in the CS group almost completely covered the defect, while some defects in the UF group remained unrepaired. The defect in the Blank group remained relatively large. Statistical analysis showed that the BV / TV ratio in the CS group was significantly higher than that in the Blank and UF groups (P<0.05), and the BV / TV ratio in the UF group was significantly higher than that in the Blank group (P<0.05). At 1 month, the Tb.Sp ratio in the Blank group was significantly higher than that in the CS and UF groups (P<0.001), while the BMD ratio was significantly lower than that in the CS and UF groups (P<0.01). At 2 months, the Tb.Sp ratio in the Blank group was significantly higher than that in the CS group (P<0.001), while the BMD ratio was significantly lower than that in the CS group (P<0.01). Histological staining at 1 minute showed a clear boundary between new bone (NB) and host bone (HB) at the edge of the Blank defect, with significant new bone regeneration on both sides of the defect, but no bone regeneration in the middle. In the UF and CS groups, significant new bone regeneration and increased angiogenesis were observed in the middle of the defect. Statistical analysis of new bone content showed that the percentage of bone volume in both the UF and CS groups was significantly higher than that in the Blank group (P<0.001), while the new bone content in the CS group was significantly higher than that in the UF group (P<0.05). Figure 15 , 16 ).

[0080] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing vascularized bone organoids, comprising the following steps: 1) Preparation of mesenchymal stem cell spheroids; 2) Seed the endothelial cell suspension onto the surface of mesenchymal stem cell spheroids; 3) Incubation, forming a core-shell structure; 4) Osteogenic induction, forming vascularized bone organoids.

2. The preparation method according to claim 1, characterized in that: The method for preparing the mesenchymal stem cell spheroids is as follows: Mesenchymal stem cell suspensions were seeded into 3D cell culture plates and cultured to obtain mesenchymal stem cell spheroids.

3. The method according to claim 1, characterized in that: The ratio of mesenchymal stem cells to endothelial cells is 1:(2~10).

4. The preparation method according to claim 1, characterized in that: The incubation time is 6-12 hours.

5. The preparation method according to claim 4, characterized in that: The incubation medium is a mixture of osteogenic induction medium and endothelial cell medium at a cell ratio of 1:(2~10).

6. The preparation method according to claim 5, characterized in that: The endothelial cell culture medium includes DMEM high glucose medium containing 8-12% FBS; The osteogenic induction culture medium contains 8-12% FBS, 8-12 mmol / L sodium β-glycerophosphate, 40-60 mg / L vitamin C, and (0.5-1.5) 10 -7 DMEM high-glucose medium containing mol / L dexamethasone; Preferably, the endothelial cell culture medium and osteogenic induction culture medium further include dual antibodies.

7. A vascularized osteoid organoid, prepared by the preparation method according to any one of claims 1 to 6.

8. The preparation method according to any one of claims 1 to 6 and the application of the vascularized bone organoid according to claim 7 in the preparation of oral bone repair materials.

9. A bone repair material comprising the vascularized bone organoid of claim 7.

10. The bone repair material according to claim 9, characterized in that: The bone repair material also includes pharmaceutically acceptable excipients.

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