Use of uterine mesenchymal stem cell exosomes in preparation of drugs for preventing and treating osteoporosis

Abstract

The application provides application of uterine mesenchymal stem cell exosomes in preparation of drugs for preventing and treating osteoporosis, and belongs to the technical field of biological medicine. The uterine mesenchymal stem cell-derived exosomes provided by the application have extremely low immunogenicity and good biological safety, avoid the immune rejection and adverse reactions possibly caused by traditional drugs or cell therapy, can be effectively targeted to the skeletal system, especially the diseased bone marrow microenvironment, and realize precise drug delivery; the exosomes not only can effectively increase bone density and improve bone microstructure, but also can improve the biomechanical strength of the skeleton, significantly reduce the risk of fracture; in addition, the preparation process is mature and stable, and as a natural cell-free therapeutic product, the exosomes avoid the ethical and safety concerns related to live cell transplantation, and provide a new strategy with great clinical application potential for the prevention and treatment of osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of uterine mesenchymal stem cell exosomes in the preparation of drugs for the prevention and treatment of osteoporosis. Background Technology

[0002] Osteoporosis (OP) is a systemic metabolic bone disease characterized by decreased bone mass and deterioration of bone microstructure, leading to increased bone fragility and a significantly elevated risk of fractures. With the deepening aging of society, this disease has become a major health problem affecting the quality of life of middle-aged and elderly people and increasing the social healthcare burden.

[0003] Currently, clinical treatments for osteoporosis mainly revolve around two pathways: one is inhibiting bone resorption, such as using bisphosphonates and RANKL inhibitors (e.g., denosumab). These drugs can effectively slow down bone loss, but long-term use may lead to adverse reactions such as osteonecrosis of the mandible and atypical femoral fractures, and they cannot repair damaged bone microstructures. The other is promoting bone formation, such as parathyroid hormone analogs (e.g., teriparatide). While these can stimulate new bone formation, there are concerns about limited treatment cycles, high treatment costs, and potential tumor risks. Furthermore, traditional drugs often have a single mechanism of action, making it difficult to simultaneously coordinate the dynamic balance between bone formation and bone resorption, thus limiting their therapeutic effects. In recent years, although biologics and small-molecule targeted drugs have made progress, they still generally face problems such as inconvenient administration, uncertain long-term safety, or high cost. Therefore, developing a novel therapeutic product that is highly efficient, safe, and capable of multi-target regulation of bone metabolism balance remains a pressing clinical challenge in the fields of orthopedics and endocrinology. Against this backdrop, exploring new treatment strategies that are biocompatible, low in toxicity, and can comprehensively improve bone quality is of great practical significance. Summary of the Invention

[0004] The purpose of this invention is to provide the application of uterine mesenchymal stem cell exosomes in the preparation of drugs for the prevention and treatment of osteoporosis, overcoming the shortcomings of existing osteoporosis treatment drugs, which generally have large side effects and can only delay bone loss but are difficult to promote bone regeneration and repair.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of uterine mesenchymal stem cell-derived exosomes in the preparation of drugs for the prevention and treatment of osteoporosis.

[0007] Preferably, in the above applications, the exosomes derived from uterine mesenchymal stem cells are prepared by the following method: culturing uterine mesenchymal stem cells, collecting their supernatant, and performing gradient centrifugation and purification on the supernatant to obtain the exosomes.

[0008] Preferably, in the above applications, gradient centrifugation includes sequential centrifugation at 300g for 10 minutes, centrifugation at 2000g for 10 minutes, centrifugation at 10000g for 30 minutes, and two centrifugations at 100000g for 70 minutes each.

[0009] Preferably, in the above applications, the diameter of the exosomes is 40 nanometers to 160 nanometers.

[0010] Preferably, in the above applications, exosomes express specific markers CD9, CD63, and TSG101.

[0011] The present invention also provides the use of a pharmaceutical composition in the preparation of a medicament for the prevention and treatment of osteoporosis, said pharmaceutical composition comprising uterine mesenchymal stem cell-derived exosomes and a pharmaceutically acceptable carrier.

[0012] Preferably, in the above applications, the pharmaceutically acceptable carrier is selected from one or more of physiological saline, salicylic acid, phosphate buffer, starch tablets, and capsules.

[0013] Preferably, in the above applications, the concentration of exosomes in the pharmaceutical composition is from 800 micrograms / mL to 1000 micrograms / mL.

[0014] The present invention also provides the use of uterine mesenchymal stem cell-derived exosomes in the preparation of reagents for increasing any one of the following: trabecular bone number, trabecular bone thickness, bone volume, bone surface area, or bone volume fraction.

[0015] The present invention also provides the use of a kit in the preparation of a drug for the prevention and treatment of osteoporosis, the kit comprising uterine mesenchymal stem cell-derived exosomes and an application device, the application device comprising an instrument for administering uterine mesenchymal stem cell-derived exosomes by intravenous injection.

[0016] The beneficial effects of this invention are: The exosomes derived from uterine mesenchymal stem cells provided by this invention have extremely low immunogenicity and good biocompatibility, largely avoiding the immune rejection and adverse reactions that may be caused by traditional drugs or cell therapies. They can effectively target the skeletal system, especially the diseased bone marrow microenvironment, to achieve precise drug delivery. These exosomes can simultaneously exert a dual regulatory effect of promoting bone formation and inhibiting bone resorption, fundamentally improving bone metabolism imbalance. They not only effectively increase bone density and improve bone microstructure, but also enhance the biomechanical strength of bones and significantly reduce the risk of fractures. In addition, their preparation process is relatively mature and stable, and as a cell-free therapeutic product from a natural source, it avoids the ethical and safety concerns associated with live cell transplantation, providing a novel and clinically promising strategy for the prevention and treatment of osteoporosis. Attached Figure Description

[0017] Figure 1 Flowchart for the isolation and purification of exosomes derived from uterine mesenchymal stem cells; Figure 2 Transmission electron micrograph of exosomes; Figure 3 Western blot results for exosome markers CD9, CD63, and TSG101; Figure 4 Micro-CT three-dimensional imaging comparison of mice treated with exosomes for osteoporosis; Figure 5 This is a comparison of bone mineral density between the exosome treatment group and the control group; Figure 6 This is a TRAP staining image showing the inhibition of osteoclast formation by exosomes. Detailed Implementation

[0018] This invention provides the application of exosomes derived from uterine mesenchymal stem cells (uMSCs) in the preparation of drugs for the prevention and treatment of osteoporosis.

[0019] In this invention, "exosomes derived from uterine mesenchymal stem cells" refers to nanoscale extracellular vesicles secreted by mesenchymal stem cells derived from the uterus. Mesenchymal stem cells are a type of adult stem cell with self-renewal and multi-lineage differentiation potential, and uterine mesenchymal stem cells are a specific tissue source among them, possessing advantages such as easy acquisition and strong proliferative capacity. Exosomes are membrane-bound vesicles with a diameter of 40-160 nanometers, containing various bioactive substances such as proteins, nucleic acids (e.g., mRNA, miRNA), and lipids. They are important carriers of intercellular communication and can mediate the biological functions of blast cells. Applying these exosomes to the prevention and treatment of osteoporosis utilizes their natural bioactive components to regulate bone metabolism balance, thereby promoting bone formation and / or inhibiting bone resorption. This application provides a new strategy and drug source for the treatment of osteoporosis.

[0020] In a preferred embodiment, the exosomes derived from uterine mesenchymal stem cells are prepared by culturing uterine mesenchymal stem cells, collecting their conditioned culture supernatant, and subjecting the supernatant to gradient centrifugation and purification to obtain the exosomes.

[0021] Specifically, the preparation method includes steps such as cell culture, supernatant collection, differential centrifugation, and purification. Cell culture is typically carried out in a culture medium suitable for stem cell growth, such as DMEM or DMEM / F12 media supplemented with serum or specific growth factors. The preferred culture conditions are a humid environment of 37°C and 5% CO2. When the cells grow to a suitable density (e.g., approximately 80% confluence), to obtain serum-free exosomes, the culture medium can be replaced and cultured for a period of time (e.g., 24-72 hours, preferably 48 hours), after which the supernatant from this culture is collected. The "gradient centrifugation" is a series of key technical steps based on different centrifugal forces to remove impurities and enrich target exosomes. In a specific embodiment, the gradient centrifugation includes sequential low-speed centrifugation to remove cell debris, medium-speed centrifugation to remove large particles such as apoptotic bodies, and ultracentrifugation to precipitate exosomes. More preferably, as shown in the embodiments of the present invention, the gradient centrifugation includes sequential centrifugation at 300g for 10 minutes, at 2000g for 10 minutes, at 10000g for 30 minutes, and twice at 100000g for 70 minutes. The 300g centrifugation primarily removes intact cells; the 2000g centrifugation primarily removes large cell debris; the 10000g centrifugation primarily removes organelles and larger vesicles (such as microvesicles); and the final 100000g ultracentrifugation is the key step in precipitating exosomes, with a centrifugation time of 60-90 minutes, preferably 70 minutes. The purification step may include resuspending the precipitate obtained from ultracentrifugation in sterile buffer (such as PBS), followed by washing with ultracentrifugation again to remove co-precipitated impurity proteins, and finally, possibly filtering through a 0.22 μm filter membrane for sterilization to obtain a high-purity exosome preparation.

[0022] In a preferred embodiment, the exosomes have a diameter of 40 to 160 nanometers. More preferably, their diameter ranges from 50 to 150 nanometers, and even more preferably from 80 to 120 nanometers; for example, a typical diameter of about 100 nanometers is observed by transmission electron microscopy. This size range is one of the important physical characteristics of exosomes and can be characterized by techniques such as nanoparticle tracking analysis, dynamic light scattering, or transmission electron microscopy.

[0023] In another preferred embodiment, the exosomes express specific biomarkers CD9, CD63, and TSG101. CD9 and CD63 are members of the four-transmembrane protein superfamily, commonly enriched on the exosome membrane, and are widely recognized positive biomarkers for exosomes. TSG101 (tumor susceptibility gene 101 protein) is a key protein in the ESCRT (endosome sorting and transporting complex) pathway, involved in exosome biogenesis, and is also one of the characteristic biomarkers of exosomes. The expression of these biomarkers can be detected by techniques such as Western blotting, flow cytometry, or enzyme-linked immunosorbent assay (ELISA). Exosomes typically do not express or express low levels of certain negative biomarkers (such as Calnexin, an endoplasmic reticulum biomarker), which helps to further confirm their purity.

[0024] The present invention also provides the use of a pharmaceutical composition in the preparation of a medicament for the prevention and treatment of osteoporosis, said pharmaceutical composition comprising a therapeutically effective amount of exosomes derived from uterine mesenchymal stem cells and a pharmaceutically acceptable carrier.

[0025] The term "pharmaceutically acceptable carrier" refers to a diluent, excipient, or medium that is non-toxic and harmless to the body and administered co-administered with the active ingredient (i.e., exosomes). Its selection depends on the intended route of administration and dosage form. The carrier can be selected from a variety of forms, including but not limited to: solvents used in solutions or injections (such as water for injection, physiological saline, phosphate buffer); excipients used in oral solid dosage forms, such as fillers (such as starch, microcrystalline cellulose, lactose), binders (such as polyvinylpyrrolidone, hydroxypropyl methylcellulose), disintegrants (such as croscarmellose sodium, low-substituted hydroxypropyl cellulose), lubricants (such as magnesium stearate, talc) for preparing tablets, capsules (such as hard capsules, soft capsules); and possible stabilizers, preservatives, etc. Preferably, the carrier is selected from one or more of physiological saline, salicylic acid, phosphate buffer, starch tablets, and capsules. Physiological saline is more preferred because it has high compatibility with the human physiological environment and is particularly suitable for preparing injectable formulations.

[0026] In a preferred embodiment, the concentration of the exosomes in the pharmaceutical composition is from 800 μg / mL to 1000 μg / mL. More preferably, the concentration is 850-950 μg / mL, for example, about 900 μg / mL. This concentration range is determined based on effective doses in animal experiments, ensuring that a sufficient total amount of exosomes to produce a significant preventive and therapeutic effect is provided when a reasonable volume of the drug is administered. For example, when administered via tail vein injection, a single dose may contain about 100 μg of exosomes.

[0027] The present invention also provides the use of exosomes derived from uterine mesenchymal stem cells in the preparation of reagents for increasing one or more of the following: the number of bone trabeculae, the thickness of bone trabeculae, the volume of bone, the surface area of ​​bone, or the volume fraction of bone.

[0028] Trabecular bone number, trabecular bone thickness, bone volume, bone surface area, and bone volume fraction are all key morphometric indicators for evaluating bone microstructure quality and bone mass, and are typically analyzed quantitatively using microcomputed tomography (MCT). Trabecular bone number (Tb.N) refers to the number of trabeculae per unit length, reflecting trabecular continuity; trabecular bone thickness (Tb.Th) reflects the fineness of the trabeculae; bone volume (BV) is the total volume of bone tissue within the region of interest; bone surface area (BS) is the total surface area of ​​bone tissue; and bone volume fraction (BV / TV) is the ratio of bone volume to total tissue volume, a direct indicator of bone mass. Increases in these indicators signify improved bone microstructure, enhanced bone density and strength, and thus effective combating osteoporosis. The exosomes of this invention have been experimentally proven to significantly enhance these indicators, and therefore can serve as the core active ingredient in the preparation of reagents or diagnostic kits with these specific biological functions.

[0029] The present invention also provides the use of a kit in the preparation of a drug for the prevention and treatment of osteoporosis, the kit comprising exosomes derived from uterine mesenchymal stem cells and an application device, the application device comprising an instrument for administering the exosomes derived from uterine mesenchymal stem cells by intravenous injection.

[0030] The "kit" refers to a packaged form that integrates the active ingredient (exosomes) and / or related auxiliary components required for treatment, facilitating storage, transportation, and use. In addition to the core exosome components, the kit may also include: a buffer solution (such as sterile PBS) for resuspending or diluting the exosomes, a measuring instrument for measuring the prescribed dose, and an "application device" for administering the medication. The application device is preferably suitable for intravenous injection, such as a disposable sterile syringe, an injection needle (e.g., a 1ml syringe with a 27G needle), and alcohol swabs. Intravenous injection is preferred. The instructions detail the resuscitation method for the exosomes, recommended dosage (e.g., micrograms per kilogram of body weight), injection frequency (e.g., once a week), and treatment duration (e.g., for 8 consecutive weeks). This kit facilitates standardized treatment of osteoporosis.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example This embodiment provides an application of exosomes derived from uterine mesenchymal stem cells in the preparation of drugs for the prevention and treatment of osteoporosis: Exosome preparation method The exosomes are prepared by cell culture, supernatant extraction, exosome acquisition, and exosome purification steps, specifically including: Cell culture: P4 generation uterine mesenchymal stem cells were seeded in DMEM-F12 medium containing 10% FBS at a seeding density of 1. 10 6 Cells per 10cm culture dish. When the cells reach about 80% confluence, wash twice with PBS, replace with serum-free culture medium (Youkang NC0103), and continue culturing for 48 hours.

[0033] 2. Supernatant Extraction and Cell Passage: Collect the supernatant after 48 hours of culture for subsequent centrifugation. Pass cells at a confluence density of 90%-100% from cell line 1 to cell line 3, discarding the old culture medium, and add 10 ml of the first-stage supernatant. Wash twice with PBS, add 4 ml of 0.05% trypsin solution, place in a 37°C incubator, digest for 4 min, add 1 ml of DMEM-F12 medium containing 10% FBS to stop digestion, transfer the cell suspension in the culture dish to a 15 ml sterile centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the centrifuge tube and discard the supernatant, resuspend in 3 ml of DMEM-F12 medium containing 10% FBS, then add to a culture dish containing 9 ml of DMEM-F12 medium containing 10% FBS, shake well and place in a 37°C, 5% CO2 incubator for culture.

[0034] 3. Obtaining exosomes: Place the supernatant in a centrifuge and centrifuge using a gradient centrifuge.

[0035] • Centrifuge the collected supernatant at 4°C and 300g for 10 minutes, and discard the precipitate. • Centrifuge the obtained supernatant at 4°C, 2000g for 10 minutes, and discard the precipitate. • Centrifuge the obtained supernatant at 4°C, 10000g for 30 minutes, and discard the precipitate. • Filter the supernatant after low-speed centrifugation using a 0.22µm filter membrane. • Place the supernatant in an ultra-high speed centrifuge and centrifuge at 4°C and 100,000g for 70 minutes. • The resulting precipitate was resuspended in an appropriate amount of sterile PBS and then centrifuged twice at 4°C and 100,000g for 70 minutes. • After removing the supernatant, the resulting precipitate is the exosome derived from uterine mesenchymal stem cells. 4. Purify exosomes: Resuspend the precipitate in 1 ml of sterile PBS, filter through a 0.22 μm filter membrane, and set aside for later use.

[0036] The exosomes are extracellular vesicles secreted by cells with a diameter between 40-160 nm, encapsulating biologically active components such as proteins, DNA, and RNA, and expressing specific markers CD9, CD63, and TSG101.

[0037] Experimental verification animal experiments In the SMAP8 6-month-old mouse model (purchased from Beijing Speford Laboratory Animal Technology Co., Ltd.) In our study, 6-month-old female SMAP8 mice were randomly divided into two groups: an exosome treatment group (receiving exosome suspension via tail vein injection) and a placebo control group (receiving an equal volume of sterile PBS via tail vein injection). Exosomes derived from uterine mesenchymal stem cells were suspended in sterile PBS at a concentration of 100 μg / 200 μL. The PBS control group used the same batch of sterile PBS. Dosage and route: 200 μL of the solution was administered to each mouse via tail vein injection. The PBS group received an equal volume of PBS. Frequency and cycle: Injections were given once a week for 8 weeks. After eight weeks, Micro-CT (SkyScan 1276) examination was performed to analyze changes in femoral bone mass (CTAn). We found that after eight weeks of exosome treatment in 6-month-old female SMAP8 mice, the number, thickness, surface area, and volume of trabeculae in the femur were significantly increased compared to the control group.

[0038] The experimental results are as follows: Figure 1 The exosome extraction process is a schematic diagram illustrating the process of isolating and culturing uterine mesenchymal stem cells (hEMSCs) from uterine tissue and extracting their exosomes. First, cells are obtained from uterine tissue and cultured. The culture supernatant is collected and then subjected to differential centrifugation at 300g for 10 min, 2000g for 20 min, 10000g for 30 min, and 100000g for 70 min, ultimately yielding purified exosomes.

[0039] Figure 2 Electron microscopy morphology of exosomes: Scanning electron microscopy images of exosomes, scale bar at 1000 nm. Typical cup-shaped or vesicle-shaped structures are visible in the images, which are characteristic morphologies of exosomes.

[0040] Figure 3 Identification of exosome markers Figure 3 These are Western blotting results, used to validate characteristic markers of exosomes. The proteins detected include: - CD9 (24kDa): positively expressed in hEMSC_EXOs (exosomes derived from uterine mesenchymal stem cells) samples, but no obvious band was observed in hEMSC (uterine mesenchymal stem cell) samples.

[0041] - CD63 (35kDa): Positive expression was found only in hEMSC_EXOs samples.

[0042] - TSG101 (44kDa): Strongly expressed in hEMSC_EXOs samples and weakly expressed in hEMSC samples.

[0043] These results are consistent with the biomarker expression characteristics of exosomes.

[0044] Figure 4 Micro-CT 3D and 2D images of bone tissue Figure 4 The images show Micro-CT scans of the femur in experimental animals, including overall three-dimensional reconstructed images of the long bone and two-dimensional cross-sectional images at different levels, used to observe the overall bone structure and the microscopic morphology of the trabeculae.

[0045] Figure 5 Statistical analysis of bone morphometric parameters The differences between the PBS control group and the EVS (exosome treatment group) were compared using bar charts of bone morphometric parameters: - Trabecular bone thickness (Tb.Th): The EVS group was significantly higher than the PBS group (P=0.0486).

[0046] - Bone volume (BV): The EVS group was significantly higher than the PBS group (P<0.0001).

[0047] - Bone surface area (BS): The EVS group was significantly higher than the PBS group (P=0.0394).

[0048] - Trabecular bone number (Tb.N): The EVS group was significantly higher than the PBS group (P=0.0025).

[0049] These results indicate that exosome treatment can significantly improve bone microstructure.

[0050] Figure 6 Bone tissue pathological staining results Figure 6 The images show pathologically stained sections of bone tissue, including low-magnification panoramic views and high-magnification magnified views, comparing the morphology of bone tissue in the PBS control group (left) and the EVS-treated group (right). The bar chart on the right shows that the positive staining signal (blue bars) in the EVS group is stronger than that in the PBS group (yellow bars), suggesting that the relevant biological activities of bone tissue are enhanced after exosome treatment.

[0051] The experimental results showed that: 1. This invention successfully completes the extraction and purification of exosomes derived from uterine mesenchymal stem cells: Uterine mesenchymal stem cells (hEMSCs) are isolated and cultured from uterine tissue, and the cell culture supernatant is collected and purified stepwise by differential centrifugation steps of 300g for 10min, 2000g for 20min, 10000g for 30min, and 100000g for 70min to obtain hEMSC_EXOs.

[0052] 2. hEMSC_EXOs exhibit typical exosome morphological characteristics: Scanning electron microscopy results show that the extracted hEMSC_EXOs present the characteristic cup-shaped / vesicle-shaped structure of exosomes, which conforms to the morphological standards of exosomes (scale bar 1000nm).

[0053] 3. The expression of characteristic biomarkers of hEMSC_EXOs met the identification criteria: Western blotting of exosome core biomarkers showed that CD9 (24kDa) and CD63 (35kDa) were positively expressed only in hEMSC_EXOs samples, TSG101 (44kDa) was strongly positively expressed in hEMSC_EXOs samples, and there were no obvious bands of CD9 and CD63 in hEMSC samples, only TSG101 was weakly positively expressed, which is consistent with the biomarker expression characteristics of exosomes.

[0054] 4. hEMSC_EXOs can significantly improve the microstructure of bone tissue: Micro-CT scan results of femur in experimental animals showed that, compared with the PBS control group, the overall three-dimensional structure of bone tissue in the hEMSC_EXOs (EVS) treatment group was more complete, the two-dimensional morphology of bone trabeculae cross sections was more dense, and the bone microstructure was significantly improved.

[0055] 5. hEMSC_EXOs can significantly improve key bone morphometric indicators: Statistical analysis of bone morphometric indicators showed that, compared with the PBS control group, the EVS-treated group had significantly increased trabecular thickness (Tb.Th, P=0.0486), bone volume (BV, P<0.0001), bone surface area (BS, P=0.0394), and trabecular number (Tb.N, P=0.0025), and the differences in each indicator were statistically significant.

[0056] 6. hEMSC_EXOs can enhance bone tissue-related biological activities: Bone tissue pathological staining results showed that the EVS-treated group had stronger positive staining signals compared with the PBS control group; quantitative analysis results further confirmed that the staining signal intensity of the EVS-treated group was significantly higher than that of the PBS control group, suggesting that hEMSC_EXOs can significantly enhance bone tissue-related biological activities.

[0057] In summary, this invention successfully extracted and identified exosomes derived from uterine mesenchymal stem cells. These exosomes can significantly improve bone microstructure, enhance bone morphometric indicators, and increase the biological activity of bone tissue, demonstrating their clear preventive and therapeutic effects on osteoporosis.

[0058] As demonstrated by the above embodiments, this invention successfully established and optimized an extraction and identification system for exosomes derived from uterine mesenchymal stem cells. The obtained exosomes conform to internationally recognized morphological and molecular marker characteristics. In an aged osteoporosis mouse model, this exosome treatment regimen exhibited remarkable preventive and therapeutic efficacy, significantly reversing bone loss, improving bone microstructure, and enhancing morphological indicators and biological activity of the skeleton, fully demonstrating its feasibility and effectiveness as a novel drug for the prevention and treatment of osteoporosis.

[0059] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of exosomes derived from uterine mesenchymal stem cells in the preparation of drugs for the prevention and treatment of osteoporosis.

2. The application according to claim 1, characterized in that, The uterine mesenchymal stem cell-derived exosomes were prepared using the following method: Uterine mesenchymal stem cells were cultured, and their supernatant was collected. The supernatant was then subjected to gradient centrifugation and purification to obtain the exosomes.

3. The application according to claim 2, characterized in that, The gradient centrifugation includes sequential centrifugation at 300g for 10 minutes, at 2000g for 10 minutes, at 10000g for 30 minutes, and twice at 100000g for 70 minutes.

4. The application according to claim 1 or 2, characterized in that, The diameter of the exosomes ranges from 40 nanometers to 160 nanometers.

5. The application according to claim 1 or 2, characterized in that, The exosomes express specific biomarkers CD9, CD63, and TSG101.

6. The use of a pharmaceutical composition in the preparation of a medicament for the prevention and treatment of osteoporosis, characterized in that, It contains exosomes derived from uterine mesenchymal stem cells and pharmaceutically acceptable carriers.

7. The application according to claim 6, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of physiological saline, salicylic acid, phosphate buffer, starch tablets, and capsules.

8. The application according to claim 6, characterized in that, The concentration of the exosomes in the pharmaceutical composition is from 800 μg / mL to 1000 μg / mL.

9. Application of uterine mesenchymal stem cell-derived exosomes in the preparation of reagents for increasing any one of the following: trabecular bone number, trabecular bone thickness, bone volume, bone surface area, or bone volume fraction.

10. The use of a reagent kit in the preparation of a drug for the prevention and treatment of osteoporosis, characterized in that, The invention comprises uterine mesenchymal stem cell-derived exosomes and an application device, the application device including instruments for administering uterine mesenchymal stem cell-derived exosomes via intravenous injection.

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