Preparation method of bone organ-derived extracellular vesicles and application thereof in promoting angiogenesis

Abstract

The application discloses a preparation method of bone organ-derived extracellular vesicles and application of the bone organ-derived extracellular vesicles in promoting angiogenesis. The application comprises the following steps: culturing bone marrow mesenchymal stem cells after osteogenic induction in a three-dimensional culture to construct bone organs, transfecting Hif-1 alpha plasmid, collecting bone organ culture supernatants of different culture time, separating and extracting extracellular vesicles through ultracentrifugation to obtain bone organ-derived extracellular vesicles (BOEVs) rich in BMP-2, TGF-beta 1, HIF-1 alpha and VEGF. The BOEVs are co-cultured with HUVEC cells, and the results show that the BOEVs can significantly promote the lumen formation and branching of the HUVEC cells by about 5 times, and up-regulate the expression of angiogenesis-related genes VEGF and CCN2, indicating that the BOEVs can promote angiogenesis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to a method for preparing extracellular vesicles derived from bone organoids and their application in promoting angiogenesis. Background Technology

[0002] Insufficient blood supply to tissues caused by ischemic vascular disease, impaired wound healing, chronic wounds, or congenital vascular malformations is a global clinical challenge. As the core structure maintaining tissue oxygenation, nutrient supply, and metabolic waste removal, insufficient angiogenesis can directly lead to ischemic tissue necrosis, functional loss, and even death. Existing clinical methods for promoting angiogenesis and improving tissue blood supply, such as local injection of growth factors, cell transplantation, and vascular bypass surgery, have drawbacks including short half-lives of bioactive factors, poor targeting, low cell transplant survival rates, significant surgical trauma, and poor long-term patency. Therefore, these strategies are not the optimal treatment for promoting angiogenesis and improving the prognosis of ischemic tissues; more effective techniques are needed to regulate the angiogenesis process and improve its efficiency and stability.

[0003] With the development of regenerative medicine, organoid technology has become a promising solution and has attracted widespread attention. Organoids are cell clusters formed from stem cells using in vitro three-dimensional culture technology. They possess self-renewal and self-organization capabilities, constituting a type of microphysiological system with spatial structure and physiological function similar to their source tissue. Bone organoids are three-dimensional, miniaturized, and simplified versions of bone tissue created in vitro using stem cells or progenitor cells. They are meticulously designed to mimic the structure, function, and cellular complexity of natural bone tissue, including key features such as mineralization and vascularization.

[0004] Extracellular vesicles (EVs) are membranous vesicles with a diameter of approximately 30–150 nanometers, widely distributed in the secretions of various cell types, and play a crucial role in intercellular signaling. Compared to traditional two-dimensional culture, organoids form self-organized 3D cell clusters, with cell contact more closely resembling the in vivo state. Importantly, organoid-derived EVs share 96% RNA similarity with serum-derived EVs, and 3D culture yields 1.5–4.5 times more EVs than 2D culture. Therefore, organoid-derived extracellular vesicles (OEVs) possess superior physiological and therapeutic potential compared to traditional EVs. However, current research on bone organoid-derived extracellular vesicles has not received sufficient attention, and their role in promoting angiogenesis has not been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem of insufficient research on extracellular vesicles derived from bone organoids in the prior art, and to provide a method for preparing extracellular vesicles derived from bone organoids.

[0006] A second objective of the present invention is to provide bone organoid-derived extracellular vesicles obtained by the above preparation method.

[0007] A third objective of the present invention is to provide an application of the bone organoid-derived extracellular vesicles in the preparation of a drug for promoting angiogenesis.

[0008] The above objectives of the present invention are achieved by the following technical solutions: The present invention first provides a preparation method of bone organoid-derived extracellular vesicles, comprising the following steps: S1. Subjecting bone marrow mesenchymal stem cells after osteogenic induction culture to three-dimensional culture for construction to obtain bone organoids; S2. Constructing a plasmid for stable overexpression Hif-1α of the gene, transfecting the plasmid into the bone organoids constructed in step S1, performing culture, collecting the supernatant, and performing isolation and extraction to obtain extracellular vesicles, that is, the bone organoid-derived extracellular vesicles (extracellular vesicles rich in Hif-1α (BOEVs)) are obtained.

[0009] In the present invention, by introducing hypoxia-inducible factor-1α (Hif-1α) into bone organoids, the release and secretion of extracellular vesicles can be further promoted, and the yield of extracellular vesicles can be increased. Meanwhile, Hif-1α causes downstream angiogenic differentiation factors to drive the expression of downstream pathways related to vascular differentiation and osteogenesis, upregulates VEGF expression, and promotes angiogenesis.

[0010] Preferably, the bone marrow mesenchymal stem cells described in step S1 are derived from the femur of SD rats.

[0011] Preferably, the osteogenic induction culture described in step S1 is to culture bone marrow mesenchymal stem cells in an α-MEM medium containing 10 mM β-sodium glycerophosphate, 50 μM ascorbic acid and 100 nM dexamethasone for 5 to 8 days. The preferred culture time is 7 days.

[0012] Preferably, the culture plate used for the three-dimensional culture described in step S1 is an ultra-low adsorption culture plate, and the cell seeding density is 6×10 5 cells / mL. The preferred three-dimensional culture time is 1 day.

[0013] Preferably, the stable overexpression in step S2 Hif-1α the backbone of the gene plasmid is the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector. That is, Hif-1α the gene is introduced into the MCS of the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector to construct the pCDH-CMV- Hif-1α -EF1-copGFP-T2A-Puro plasmid.

[0014] Preferably, preferably, step S2 is described Hif-1α The gene's accession number is NM_001243084.2.

[0015] Preferably, the culture time in step S2 is 1 to 14 days, more preferably 6 to 8 days, and even more preferably 7 days. The culture is a two-dimensional culture.

[0016] Preferably, the separation and extraction method in step S2 is differential centrifugation: first, centrifuge at 300 g for 20 min to remove cells, then centrifuge at 2000 g for 20 min to remove cell debris, then centrifuge at 10000 g for 30 min to remove large particles, and finally centrifuge at 100000 g for 70 min to collect extracellular vesicle precipitate.

[0017] The present invention also provides extracellular vesicles (Hif-1α-rich extracellular vesicles (BOEVs)) derived from bone organoids prepared by any of the above-described preparation methods.

[0018] This invention further describes the application of bone organoid-derived extracellular vesicles (Hif-1α-rich extracellular vesicles (BOEVs)) to human umbilical vein endothelial cells (HUVECs) to examine their ability to promote angiogenesis. The results showed that BOEVs significantly promoted lumen formation and branching in HUVECs by approximately 5-fold, and upregulated the expression of angiogenesis-related genes VEGF and CCN2.

[0019] Therefore, the present invention also provides the use of the above-mentioned extracellular vesicles derived from bone organoids in the preparation of drugs that promote angiogenesis.

[0020] Specifically, the drug promotes angiogenesis by stimulating lumen formation and branching of human umbilical vein endothelial cells (HUVECs) and upregulating the expression of angiogenesis-related genes VEGF and CCN2.

[0021] Furthermore, it can be used in the preparation of drugs for insufficient tissue blood supply caused by ischemic vascular diseases, impaired wound healing, chronic wounds, or congenital vascular developmental abnormalities.

[0022] The present invention also provides a drug for promoting angiogenesis, comprising extracellular vesicles derived from bone organoids as described above.

[0023] Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable excipients.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing extracellular vesicles derived from bone organoids. The method involves constructing bone organoids through three-dimensional culture of bone marrow mesenchymal stem cells induced by osteogenic induction, followed by the construction of a stable overexpression... Hif-1α The gene plasmid was transfected into the cells, cultured, and the supernatant was collected. Extracellular vesicles were then isolated and extracted to obtain bone organoid-derived extracellular vesicles (BOEVs) rich in BMP-2, TGF-β1, HIF-1α, and VEGF. This invention, by introducing Hif-1α into bone organoids, can further promote the release and secretion of extracellular vesicles, increase extracellular vesicle production, and simultaneously drive the expression of downstream pathways related to angiogenesis differentiation, upregulate VEGF expression, and promote angiogenesis. The results of this invention show that these BOEVs can significantly promote lumen formation and branching in HUVEC cells by approximately 5-fold and upregulate the expression of angiogenesis-related genes. This invention successfully achieves in vitro angiogenesis promotion using extracellular vesicles derived from bone organoids, providing a new therapeutic strategy for promoting angiogenesis. Attached Figure Description

[0025] Figure 1 Bright-field images of bone marrow mesenchymal stem cells in a 3D culture plate at 0, 12, and 24 hours in Example 1.

[0026] Figure 2 This is a bright-field image of the free-floating bone organoids in Example 1.

[0027] Figure 3 For the qPCR analysis of osteogenic-related mRNA expression in bone organoids in Example 1, n=3, *: p<0.05, **: p<0.01, ***: p<0.001.

[0028] Figure 4 The measurements and frequency distribution of bone organoids based on scanning electron microscopy images in Example 1, as well as the quantitative determination of the number of bone organoids, are n=3, *p<0.05, **:p<0.01, ***p<0.001.

[0029] Figure 5 This shows the mRNA sequencing results of bone organoids in Example 1. Among them, Figure 5 In Example 1, A represents a heatmap of differentially expressed genes (DEGs) from the mRNA sequencing of bone organoids. Figure 5 In the figure, B represents the gene ontology (GO) functional enrichment analysis of differentially expressed genes; Figure 5 C in the figure represents the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes.

[0030] Figure 6 Immunofluorescence staining of VEGFR in Example 1.

[0031] Figure 7 For the quantitative analysis of Alp, Hif-1a, and Rab27amRNA expression in Example 1, n=3, *: p<0.05, **: p<0.01, ***: p<0.001.

[0032] Figure 8 The relative mRNA expression level of Hif-1α in Example 2, n=3, *: P <0.05, **: P <0.01, ***: P <0.001, ns: no significant.

[0033] Figure 9 For the BCA analysis of EV protein concentration in Example 2, n=10, *: p<0.05, **: p<0.01, ***: p<0.001. ns: p>0.05.

[0034] Figure 10 NTA analysis of EV size and concentration in Example 2.

[0035] Figure 11 This is a Western blot analysis of the EVs markers in Example 2.

[0036] Figure 12 TEM observation of the morphology of EVs in Example 2.

[0037] Figure 13 For the ELISA analysis of the concentrations of Bmp2, Tgf-B1, Hif-1a, Vegf and Sdc4 in EVs in Example 2, n=7, *p<0.05, **:p<0.01, ***p<0.001, ns:p>0.05.

[0038] Figure 14 Fluorescence imaging analysis of PKH67-labeled extracellular vesicle-induced vascular-like structures in Example 3.

[0039] Figure 15 For the quantitative analysis of the total length and number of nodes of the vascular-like structure in Example 3, n=5, *: p<0.05, **: p<0.01, ***: p<0.001, ns: p>0.05.

[0040] Figure 16 The relative mRNA expression levels of VEGF, CCN2, and FN in Example 3 are shown. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0042] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0043] Example 1: Construction and Characterization of Bone Organoids I. Experimental Materials SD rats (2-week-old males) were purchased from the Experimental Animal Center of Southern Medical University; DMEM basal medium, fetal bovine serum (FBS), and penicillin-streptomycin antibiotics were purchased from Gibco; osteogenic induction medium components: sodium β-glycerophosphate, ascorbic acid, and dexamethasone were purchased from Sigma; and three-dimensional culture plates were purchased from Heidolph.

[0044] II. Experimental Methods 1. Isolation and culture of bone marrow mesenchymal stem cells (BMSCs) In accordance with the ethical guidelines for laboratory animals, 2-week-old male SD rats were anesthetized by intraperitoneal injection of an excessive dose of 3% sodium pentobarbital and then disinfected by immersion in 75% ethanol for 5 minutes. Under aseptic conditions, the tibia and femur were removed, and the bone marrow tissue was flushed to obtain a cell suspension. After incubation with erythrocyte lysis buffer for 5 minutes, the cells were collected by centrifugation at 600 g. The cells were cultured in DMEM medium containing 10% exosome-free fetal bovine serum and 1% penicillin-streptomycin for 24 hours; purified bone marrow mesenchymal stem cells (BMSCs) were obtained after medium replacement, and the medium was changed every two days thereafter. After passage to the third generation, the cells were harvested for subsequent experiments.

[0045] 2. Construction of bone organoids (BO) After 7 days of induction in osteogenic induction medium (DMEM basal medium supplemented with 10 mM β-glycerophosphate, 50 μM ascorbic acid, and 100 nM dexamethasone) in conventional culture plates, bone marrow mesenchymal stem cells were digested with 0.25% trypsin-EDTA, washed with PBS, and resuspended in osteogenic induction medium. Cells were then cultured at a rate of 6 × 10⁶ cells / year. 5 Cells were seeded at a density of [number] cells / mL in three-dimensional culture plates. Cell growth was observed under a microscope at 0 h, 12 h, and 24 h post-seeding. Cells seeded under the same conditions and cultured in conventional culture plates were used as two-dimensional cultured bone marrow mesenchymal stem cells.

[0046] 3. Grouping and Culture of Bone Organoids Successfully constructed osteoid organoids were transferred to conventional culture plates for further culture. Based on culture time, they were divided into three groups: BO-1d (1 day), BO-7d (7 days), and BO-14d (14 days). Osteogenesis-related genes Runx2, Sp7, Collar, and Ocn were detected, with GAPDH used as an internal control gene. Specific primers are shown in Table 1. Thermal cycling conditions: pre-denaturation at 95℃ for 5 minutes, followed by 40 cycles (denaturation at 95℃ for 30 seconds, extension at 60℃ for 30 seconds, and digestion at 72℃ for 30 seconds). Relative gene expression levels were calculated using the 2^(-ΔΔCt) method. Osteogenesis morphology was observed using electron microscopy. All groups used osteogenic induction medium, with medium changes every 3 days.

[0047] Table 1. RT-qPCR primers used for osteogenic differentiation analysis

[0048] 4. mRNA sequencing analysis Three-dimensionally cultured bone organoids and two-dimensionally cultured bone marrow mesenchymal stem cells were collected separately. Total RNA was extracted using TRIzol reagent, and RNA quality and purity were assessed using a NanoDrop spectrophotometer. High-quality RNA was reverse transcribed into cDNA, and cDNA libraries were prepared. High-throughput sequencing was performed on the Illumina platform. Sequencing data underwent quality control, reference genome alignment, gene expression quantification, and differentially expressed gene identification. GO and KEGG enrichment analyses were performed on differentially expressed key genes.

[0049] 5. RT-qPCR validation of key genes sequenced Design Alp 、 Primers for Hif-1α and Rab27a were used, with GAPDH as an internal reference gene. Thermal cycling conditions: pre-denaturation at 95℃ for 5 minutes, followed by 40 cycles (denaturation at 95℃ for 30 seconds, extension at 60℃ for 30 seconds, and digestion at 72℃ for 30 seconds). Relative gene expression levels were calculated using the 2^(-ΔΔCt) method. Specific primers are shown in Table 2.

[0050] Table 2. RT-qPCR primers for Alp, Hif-1α, and Rab27a

[0051] III. Experimental Results 1. Successful construction of bone organoids In a three-dimensional culture plate, cells aggregate and grow to form multicellular spheres. The growth status of bone spheres on the 3D culture plate was observed under a microscope at 0, 12, and 24 hours post-inoculation. At 0 h post-inoculation, cells were suspended in the 3D culture plate; after 12 h, cells began to aggregate significantly and gradually form small cell clusters; at 24 h, the early structure of bone organoids was initially established. Figure 1 After one day of 3D culture, the bone organoids were transferred to conventional culture plates. BO-1d spheroids were numerous and small in size; as the culture time increased, the size of BO-7d and BO-14d spheroids gradually increased, but their numbers gradually decreased due to unintentional aspiration during medium changes and possible cell death and spheroid disintegration. Figure 2 ).

[0052] 2. Bone organoids express osteogenic-related genes. qPCR analysis showed that Runx2 and Ocn were significantly upregulated in BO-7d compared to BO-1d, and further increased in BO-14d; Sp7 and Col1a1 peaked in BO-7d, but decreased in BO-14d. Figure 3 Analysis of sphere size distribution showed that the average size of BO-1d was approximately 115.58 ± 25.36 μm, increasing to approximately 148.62 ± 35.72 μm for BO-7d, and further increasing to approximately 203.71 ± 45.30 μm for BO-14d, generally showing an increasing trend over time. Figure 4 The quantitative results of the number of spheres show that the number gradually decreases from BO-1d to BO-14d. Figure 4 This may be due to cell loss during the fluid exchange process and potential spheroid disintegration.

[0053] 3. mRNA sequencing reveals the association between bone organoids and osteoogenesis, hypoxia, and vesicle transport. mRNA sequencing was performed on two-dimensional cultured BMSCs, BO-1d, BO-7d, and BO-14d, identifying a total of 270 differentially expressed genes. A heatmap was generated to analyze these differentially expressed genes. Figure 5 A). GO enrichment analysis showed that in biological processes (BP), in addition to being significantly associated with osteoblast differentiation pathways, there were also highly significant differences in the response to hypoxia, especially in the BO-7d and BO-14d groups; in cellular component (CC) analysis, cell-cell junctions and focal adhesions were highly enriched; furthermore, there was a significant correlation with transport vesicles and transport vesicle membranes; in molecular function (MF) analysis, functions such as cadherin binding and GTPase binding were highly enriched. Figure 5 The above analysis indicates that these differentially expressed genes may not only promote osteogenic formation but also be related to angiogenesis and have some association with extracellular vesicles (EVs) of bone organoids (BOs). KEGG analysis showed significant enrichment of signaling pathways regulating the actin cytoskeleton and stem cell pluripotency, including the hypoxia-induced pathway (HIF-1 signaling pathway) and its downstream angiogenesis-related pathway (VEGF signaling pathway). Figure 5 The presence of C in the figure suggests that it may play a role in promoting angiogenesis.

[0054] 4. Bone organoids gradually express osteogenic and angiogenic factors over time. Based on mRNA sequencing results, we experimentally verified the presence of osteoglobulins (BOs). Immunofluorescence staining showed that the distribution and intensity of Vegfr fluorescence signals in cells gradually increased with the extension of osteogenic induction time. Figure 6 qPCR analysis showed that, compared with two-dimensionally cultured BMSCs, the expression levels of Alp, Hif-1a, and Rab27a in BO gradually increased over time. Figure 7 The expression level was consistent with the mRNA sequencing results. This indicates that bone organoids not only undergo osteogenic differentiation during culture, but also experience hypoxic changes and promote the release of extracellular vesicles.

[0055] These results demonstrate that we identified differentially expressed genes between two-dimensionally cultured bone marrow mesenchymal stem cells (BMSCs) and three-dimensionally cultured bone organoid (BO) cells. Further investigation of these differentially expressed genes suggests they may be associated with osteogenic differentiation, angiogenesis, and extracellular vesicle release. These results indicate that extracellular vesicles extracted from BOs at different culture times may not only promote bone differentiation but also angiogenesis.

[0056] Example 2: Isolation and characterization of extracellular vesicles derived from bone organoids overexpressing Hif-1α I. Experimental Methods 1. Gene overexpression in cells Using the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector, a stable expression vector was constructed. Hif-1α The plasmid (Gen accession number: NM_001243084.2) was used to screen for stable Hif-1α-overexpressing two-dimensional cultured bone marrow mesenchymal stem cells (BMSCs) and three-dimensional cultured bone organoid (BO) cell lines.

[0057] 2. Isolation of extracellular vesicles BOs that were never transfected with the Hif-1α plasmid WTEVs were isolated from the culture supernatant of two-dimensional cultured BMSCs overexpressing Hif-1α and three-dimensional cultured BO-7d and BO-14d groups overexpressing Hif-1α. After cells reached 80% confluence, the conditioned medium was collected, and the cells were subjected to differential centrifugation: 300 g for 20 min, 2000 g for 20 min to remove dead cells and cell debris; the supernatant was centrifuged at 10000 g for 30 min to remove large particles; finally, the supernatant was ultracentrifuged at 100000 g for 70 min to collect the precipitate. The precipitate was washed with pre-chilled PBS and centrifuged again at 100000 g for 70 min. The final precipitate was resuspended in pre-chilled PBS to obtain 2D-EVs and BOEVs, respectively. WT BOEV-7d and BOEV-14d, stored at -80°C for subsequent experiments.

[0058] 3. BCA protein concentration determination Prepare a 0.5 mg / mL protein standard solution. Mix BCA reagents A and B at a 50:1 volume ratio to prepare the working solution. Add the standard and sample to a 96-well plate, adding 200 μL of working solution to each well, and incubate at 37°C for 20–30 min. Measure the absorbance at 562 nm using a microplate reader, and calculate the sample protein concentration based on the standard curve.

[0059] 4. Nanoparticle Tracking Analysis (NTA) NTA analysis was performed using a ZetaView PMX 110 instrument. Each group of EVs was appropriately diluted with particle-free PBS and loaded into the sample cell. Particle size distribution and concentration were measured at 405 nm.

[0060] 5. Transmission electron microscopy (TEM) observation Add 10 μL of EV suspension to the center of the Parafilm membrane, invert the copper mesh onto the sample, and let it stand for 10 min. Then invert the membrane onto 2% phosphotungstic acid and let it stand for 5 min. Use filter paper strips to absorb excess staining solution from the edge of the copper mesh, and dry it at 15 cm under an incandescent lamp. Image it using a Hitachi H-7650 transmission electron microscope at 80 kV.

[0061] 6. ELISA detection of growth factors The concentrations of Bmp-2, Tgf-β1, Hif-1α, Vegf, and Sdc4 proteins in EV lysates were quantitatively determined using a species-specific ELISA kit. EV pellets were resuspended in pre-chilled PBS and lysed using RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor. After protein concentration determination using the BCA method, the protein concentrations of each EV lysate were standardized to 50 μg / mL. The assay was performed according to the kit instructions, measuring absorbance at 450 nm, and concentrations were calculated based on the standard curve.

[0062] II. Experimental Results 1. Gene overexpression in cells A plasmid stably expressing Hif-1α was successfully constructed. Stable overexpressing cells were screened after lentiviral infection, and RNA was extracted from the stable cell lines for RT-qPCR to detect the overexpression efficiency. Results are as follows: Figure 8 As shown, compared with the control group, the mRNA level of Hif-1α in the stably overexpressing cells was higher, which preliminarily indicates that the two-dimensional cultured bone marrow mesenchymal stem cells (BMSCs) stably overexpressing the Hif-1α gene and the three-dimensional cultured bone organoid (BO) cell line overexpressing the Hif-1α gene were successfully constructed.

[0063] 2. Isolation of extracellular vesicles We successfully isolated extracellular vesicles. BCA analysis showed no significant difference in EV protein concentration between the BOEV-7d (85.11±10.95 μg / mL) and BOEV-14d (92.33±14.05 μg / mL) groups, but the EV protein concentrations were significantly higher than those of BOEVs not transfected with the Hif-1α plasmid. WT (60.36±6.49 μg / mL). Compared with 2D-EV (27.08±3.31 μg / mL), the EV protein concentration in the BOEV group was 2-3 times higher. Figure 9 ).

[0064] 3. Characterization of extracellular vesicles NTA analysis confirmed that the peak particle size of all EV groups was around 100 nm. Particle concentration trends showed that BOEV-7d and BOEV-14d had the highest vesicle numbers, followed by BOEV... WT 2D-EV lowest ( Figure 10 To further characterize BOEVs, we detected classic EV marker proteins using Western blotting. The results showed that CD63, CD81, and ALIX are present in BOEVs. WT Positive expression was observed in both BOEV-7d and BOEV-14d. Figure 11This confirms that BOEVs conform to the core molecular characteristics of EVs. Transmission electron microscopy (TEM) observation shows that all EV groups are between 50 and 200 nanometers in size, exhibiting typical spherical, membrane-like vesicle characteristics. Figure 12 The above results demonstrate that we successfully isolated extracellular vesicles from two-dimensionally cultured bone marrow mesenchymal stem cells and three-dimensionally cultured bone organoids. Furthermore, the results show that the concentration of extracellular vesicles obtained from three-dimensionally cultured bone organoids (BO) was significantly higher than that from two-dimensionally cultured bone marrow mesenchymal stem cells, indicating that three-dimensional culture better promotes the release and secretion of extracellular vesicles compared to two-dimensional culture. Simultaneously, the results show that BO overexpressing Hif-1α yielded a significantly higher concentration of extracellular vesicles compared to wild-type (WT) BO. Figure 9 and Figure 10 This indicates that overexpression of Hif-1α can further promote the release and secretion of extracellular vesicles.

[0065] 4. BOEVs are rich in osteogenic and angiogenic factors. Guided by previous mRNA sequencing results, ELISA was used to further quantify the protein levels of osteogenic factors and angiogenic factors in each EV group. ELISA results showed that the concentrations of Bmp-2 (314.00±33.09 pg / mL), Tgf-β1 (1500.67±315.94 pg / mL), Hif-1α (223.10±114.51 pg / mL), and Vegf (566.8±96.78 pg / mL) were the highest among all groups in the BOEV-7d group. The BOEV-14d group showed no significant difference in Bmp-2, Tgf-β1, and Hif-1α levels compared to BOEV-7d, but its Vegf level (422.8±100.62 pg / mL) was significantly lower. WT The levels of Bmp-2, Hif-1α, and Vegf in 2D-EV were significantly lower than those in BOEV-7d and BOEV-14d. Notably, the levels of these factors in 2D-EV were very low, showing a statistically significant difference compared to the BOEV group. SDC4 was an exception, exhibiting a high protein level of 224.33 ± 62.03 pg / mL in 2DEV. This protein is closely related to cell adhesion and morphology maintenance and typically shows lower expression levels in 3D culture. Figure 13 ).

[0066] The above findings indicate that BOs overexpressing Hif-1α can effectively secrete EVs rich in high levels of osteogenic and angiogenic factors (VEGF). This suggests that BOEVs, especially BOEV-7d, may have the potential to promote osteogenic differentiation and angiogenesis.

[0067] Example 3: BOEVs promote angiogenesis in HUVEC cells I. Experimental Methods 1. Cell Culture Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC. Cells were cultured in medium containing 10% SBI, 100 μg / mL streptomycin, and 100 U / mL penicillin in a 37°C, 5% CO2 incubator.

[0068] 2. Lumen formation experiment HUVECs in the logarithmic growth phase were fed at 3×10 4 Cells were seeded at a density of 100 cells / well in Matrigel-coated 96-well plates. BOEVs (BoV16 cells / well) were added to the corresponding wells immediately after seeding. WT BOEV-7d, BOEV-14d, 2D-EV (negative control), or pre-cooled PBS (control group) were used. The final concentration of EVs in each treatment group was normalized to 10 μg / mL using NTA. The incubator was incubated at 37℃ and 5% CO2 for 6–8 h. After incubation, the vascular-like luminal structures were observed and photographed using an inverted phase-contrast microscope. The total length and number of nodes of the vascular network were quantitatively analyzed using ImageJ software.

[0069] 3. qPCR detection of angiogenesis-related genes Total RNA was extracted from HUVEC cells in each group, reverse transcribed into cDNA, and then detected by qPCR. Primers for VEGF, CCN2, and FN genes were designed (Table 3). GAPDH was used as an internal reference gene, and the relative gene expression levels were calculated using the 2^(-ΔΔCt) method.

[0070] Table 3. RT-qPCR primers for angiogenesis-related genes

[0071] II. Experimental Results 1. BOEVs promote the formation of vascular-like structures in HUVEC cells. After internalization of fluorescently labeled BOEVs, HUVECs exhibited green fluorescence and showed a tendency to form vascular-like lumens in Matrigel. Compared with the control group and the 2D-EV group, the BOEVs group showed a more extensive and tightly connected vascular-like network structure, with the most significant lumen formation in the BOEV-7d group. Figure 14 ).

[0072] 2. BOEVs significantly increase vascular network length and number of nodes. Quantitative analysis showed that the total vessel length and number of nodes in the BOEV group were significantly higher than those in the control group and the 2D-EV group, with the highest values ​​observed in the BOEV-7d group (total vessel length 5182.60±958.11 μm, number of nodes 307.40±44.42). Figure 15 This indicates that BOEVs promote the formation of angiogenic structures, with the most significant effect observed after 7 days of culture. The pro-angiogenic effect did not increase monotonically with prolonged BOEV culture time; although BOEVs WT It showed comparable effects to BOEV-14d, but its potency was significantly lower than that of BOEV-7d.

[0073] 3. BOEVs upregulate the expression of angiogenesis-related genes. qPCR analysis showed that the mRNA expression level of the angiogenesis gene VEGF was significantly higher in the BOEV-7d group than in other groups. There was no significant difference in CCN2 levels among the BOEV groups, while FN expression was highest in the BOEV-14d group. Figure 16 All three mRNAs positively regulate the adhesion, migration, and lumen formation of HUVECs.

[0074] These results indicate that extracellular vesicles derived from three-dimensionally cultured bone organoids (BO) with stable overexpression of Hif-1α possess pro-angiogenic properties, confirming the upregulation of angiogenic factors observed in our previous mRNA sequencing data.

Claims

1. A method for preparing extracellular vesicles derived from bone organoids, characterized in that, Includes the following steps: S1. Bone organoids were constructed by three-dimensional culture of bone marrow mesenchymal stem cells after osteogenic induction culture; S2. Constructing a stable overexpression Hif-1α The gene plasmid was transfected into the bone organoids constructed in step S1, cultured, the supernatant was collected, and extracellular vesicles were separated and extracted to obtain extracellular vesicles derived from bone organoids. The osteogenic induction culture described in step S1 involves culturing bone marrow mesenchymal stem cells for 7 days in α-MEM medium containing 10 mM β-glycerophosphate sodium, 50 μM ascorbic acid and 100 nM dexamethasone. Step S2 Hif-1α The gene's accession number is NM_001243084.2; The culture time described in step S2 is 7 to 14 days; The separation and extraction method described in step S2 is differential centrifugation: first, centrifuge at 300 g for 20 min to remove cells, then centrifuge at 2000 g for 20 min to remove cell debris, then centrifuge at 10000 g for 30 min to remove large particles, and finally centrifuge at 100000 g for 70 min to collect extracellular vesicle precipitate.

2. The preparation method according to claim 1, characterized in that, The stable overexpression described in step S2 Hif-1α The base plasmid for the gene is the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector.

3. Extracellular vesicles derived from bone organoids prepared by the preparation method according to claim 1 or 2.

Images