Mesoporous bioactive glass drug-loaded bone repair composite material, particle-hydrogel, preparation method and application

Abstract

The present application relates to a kind of mesoporous bioactive glass drug-loaded bone repair composite material, particle-hydrogel, preparation method and application, the mesoporous bioactive glass drug-loaded bone repair composite material uses mesoporous bioactive glass as drug-loading framework, with quercetin derivative carbon dot and human amniotic membrane mesenchymal stem cell-derived extracellular vesicle as active ingredient.The present application uses MBG as inorganic porous bone repair matrix, QCDs as antioxidant and redox regulation functional component, hAMSC-EVs as cell signal regulation component, using the way of first MBG loading quercetin, then in situ generation quercetin derivative carbon dot, then load hAMSC-EVs, preparation forms same composite nano / micro-nano platform, for the local filling of osteoporotic bone defect area, active component retention and bone regeneration microenvironment regulation.

Description

Technical Field

[0001] This invention belongs to the field of bone repair materials, specifically relating to a mesoporous bioactive glass drug-loaded bone repair composite material, a particle-hydrogel, its preparation method, and its application. Background Technology

[0002] Osteoporosis and related fragility fractures have a high incidence in the aging population. Bone defect repair in osteoporotic states is often accompanied by decreased osteogenic capacity, increased osteoclast activity, persistent chronic inflammation, and elevated local oxidative stress levels. These pathological factors collectively weaken the tissue regeneration capacity of the bone defect area, making it difficult for conventional osteoconductive materials to adequately regulate the complex bone metabolic imbalance microenvironment.

[0003] Existing clinical and experimental bone repair materials are mostly based on filling, support, or simple osteoconduction, and generally lack the ability to regulate the local microenvironment of osteoporotic bone defects in multiple dimensions. For example, while single inorganic materials can provide a certain mineralization scaffold and ion release, their ability to regulate excessive inflammation, reactive oxygen species accumulation, and excessive osteoclast activity is limited; single drugs or active nanocomponents are prone to problems such as insufficient local retention, uncontrollable release behavior, or decreased bioactivity. Furthermore, existing materials struggle to simultaneously address issues such as promoting bone differentiation, inhibiting osteoclast formation, regulating macrophage inflammatory phenotypes, alleviating oxidative stress, and improving the local retention capacity of active ingredients.

[0004] Therefore, the development of mesoporous bioactive glass drug-loaded bone repair composite materials, particle-hydrogels, preparation methods, and applications that combine osteoconduction, antioxidant, immunomodulatory, osteopromoting, and osteoclast-inhibiting functions has high research and translational value. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects in the prior art and provide a mesoporous bioactive glass drug-loaded bone repair composite material, particle-hydrogel, preparation method and application, which can effectively improve the problems of insufficient osteogenic formation, enhanced osteoclastosis, inflammatory imbalance and oxidative stress in osteoporotic bone defects, and can be used to prepare drugs for osteoporotic bone defects.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A mesoporous bioactive glass-based drug-loaded bone repair composite material uses mesoporous bioactive glass as the drug-loaded framework and quercetin-derived carbon dots and extracellular vesicles derived from human amniotic mesenchymal stem cells as active ingredients.

[0007] As a further technical solution, its forms include granular, lyophilized powder, sterile suspension liquid, hydrogel, scaffold, or coating.

[0008] A method for preparing a mesoporous bioactive glass drug-loaded bone repair composite material includes the following steps: S1. Loading of quercetin or its derivatives: Mesoporous bioactive glass particles were added to solvent B and dispersed until uniform. Then, quercetin or its derivatives were added and dispersed until the quercetin or its derivatives were adsorbed into the pores of the mesoporous bioactive glass particles. Then, solid-liquid separation was performed to remove unloaded free quercetin. The solid product was washed and dried to obtain MBG@Que loaded with quercetin or its derivatives. S2. In-situ hydrothermal carbonization of quercetin or its derivatives: MBG@Que is dispersed in solvent C and subjected to a hydrothermal reaction at 120-220℃ (preferably 160-200℃) to generate quercetin-derived carbon dots. The reaction time is 2-24h (6-12h). After the reaction is completed, the mixture is naturally cooled to room temperature. After solid-liquid separation and washing of the precipitate, it is dialyzed to remove free small molecules, unreacted quercetin or its derivatives and free carbon dots. Then, the precipitate is collected by solid-liquid separation and dried to obtain MBG@QCDs loaded with quercetin-derived carbon dots. S3. Loading of hAMSC-EVs: MBG@QCDs were added to solvent E and dispersed evenly. Then, hAMSC-EVs suspension was added to make the final concentration 100 μg / mL. The mixture was incubated at 0-25℃ with shaking for 2-24 h to allow hAMSC-EVs to be physically adsorbed and fixed on the surface of MBG@QCDs, forming MBG@QCDs@EVs. After shaking incubation, unbound hAMSC-EVs were washed away to obtain quercetin-derived carbon dots and MBG@QCDs@EVs loaded with hAMSC-EVs, which is the mesoporous bioactive glass drug-loaded bone repair composite material.

[0009] The mesoporous bioactive glass particles (MBG) of this invention are preferably bioactive glass particles containing Si, Ca, and P elements. They can be spherical, near-spherical, porous, or irregular in shape, and preferably have radial mesopores, interconnected mesopores, or pore-surface interconnected structures. The pore structure provides a spatial basis for quercetin pre-adsorption, in-situ carbonization to form QCDs, and subsequent adsorption on the surface of EVs; its inorganic components such as Si, Ca, and P can serve as osteoconductive matrix in the bone repair environment and provide material support for osteoblast adhesion, proliferation, differentiation, and matrix mineralization.

[0010] The quercetin-derived carbon dots (QCDs) of this invention are derived from quercetin or its pharmaceutically acceptable derivatives. In MBG@QCDs@EVs composite materials, the QCDs of this invention serve as redox modulators, participating in reactive oxygen species scavenging, oxidative stress relief, and the construction of local antioxidant microenvironments. They can also work with the porous structure of MBG to improve the material's loading and local retention capacity of bioactive components.

[0011] The introduction of EVs in this invention enables the material to further possess the potential for regulating the immune microenvironment and cell function, in addition to its inorganic osteoconduction and antioxidant functions.

[0012] As a further technical solution, the dialysis bag used for dialysis has a molecular weight cutoff of 3.5 kDa, the dialysis time is 24 hours, and the water is changed every 4 hours.

[0013] As a further technical solution, the mesoporous bioactive glass particles are prepared by any one of the following methods: CTAB / TEA-mediated sol-gel method, template method, spray drying method, and microemulsion method; among which, the CTAB / TEA-mediated sol-gel method has the best effect.

[0014] As a further technical solution, the method for preparing the mesoporous bioactive glass particles includes the following steps: The soft template agent hexadecyltrimethylammonium bromide was added to solvent A and stirred to disperse it evenly. Then, the catalyst and morphology modifier triethanolamine was added and stirred evenly at 40-80℃ to obtain the template solution. A silicon source and a phosphorus source are added to an organic solvent to prepare an oil phase solution; The oil phase solution was added to the template solution and stirred to carry out the hydrolysis and condensation reaction. After the reaction was completed, the solid and liquid phases were separated and the precipitate was collected. Then, the unreacted precursor, free surfactant and residual small molecules were washed to remove them and dried to obtain silicon phosphorus mesoporous particles. The mesoporous silica-phosphorus particles and calcium source were added to anhydrous ethanol and stirred for 24 hours. The solid and liquid were separated, the precipitate was collected, washed, and then calcined to remove the CTAB template. After cooling and grinding, mesoporous bioactive glass particles MBG were obtained.

[0015] As a further technical solution, solvent A includes any one of deionized water and aqueous ethanol solution; As a further technical solution, the silicon source is tetraethyl orthosilicate (TEOS). As a further technical solution, the phosphorus source is triethyl phosphate (TEP). As a further technical solution, the temperature of the hydrolysis-condensation reaction is 50-70℃ (preferably 60℃), the time is 18-28h (preferably 24h), and the stirring speed is 120-180rpm (preferably 150rpm).

[0016] As a further technical solution, the calcium source is a soluble calcium salt, which includes one or more of calcium nitrate, calcium chloride, and calcium acetate. As a further technical solution, in the preparation process of mesoporous bioactive glass particles, the solvent used for washing includes any one of deionized water, anhydrous ethanol, or an aqueous solution of ethanol. As a further technical solution, the drying includes any one of vacuum drying, freeze drying, and oven drying; As a further technical solution, the calcination temperature is 500-700℃ and the time is 3-8 h.

[0017] As a further technical solution, in S1, the solvent B includes one or more of anhydrous ethanol, deionized water, PBS buffer solution, and aqueous ethanol solution; As a further technical solution, in S1, the dispersion treatment and the continued dispersion treatment adopt one or more of ultrasonic dispersion, stirring, and oscillation incubation.

[0018] As a further technical solution, in S1, the dispersion process is carried out by first ultrasonic treatment, and then incubation in a shaker at 37 degrees Celsius for 24 hours.

[0019] As a further technical solution, in S1, the solvent used for washing includes one or more of anhydrous ethanol, deionized water, ethanol solution, and PBS buffer solution.

[0020] As a further technical solution, in S2, the preparation method of the hAMSC-EVs includes: culturing human amniotic mesenchymal stem cells, first removing cells by low-speed centrifugation of the culture medium, then removing cell debris and large particles by medium-speed centrifugation, and then purifying by ultracentrifugation to obtain hAMSC-EVs, then washing the hAMSC-EVs with PBS buffer, then ultracentrifuging again, and resuspending them in solvent D to obtain an hAMSC-EVs suspension.

[0021] As a further technical solution, in S2, the rotation speed of the low-speed centrifugation is 300 × g, and the time is 10 min. As a further technical solution, in S2, the rotation speed of the medium-speed centrifugation is 2,000 × g, and the time is 20 min; As a further technical solution, in S2, the rotation speed of the ultracentrifugation is 100,000 × g, and the time is 30 min; As a further technical solution, in S2, the rotation speed of the second ultracentrifugation is 100,000 × g, and the time is 70 min; As a further technical solution, in S2, the solvent D includes one or more of PBS buffer solution and serum-free culture medium.

[0022] As a further technical solution, the solvent C includes deionized water or an aqueous solution of ethanol.

[0023] As a further technical solution, the solid-liquid separation includes centrifugation, filtration, dialysis, etc.

[0024] As a further technical solution, the drying includes any one of vacuum drying, freeze drying, and oven drying.

[0025] A mesoporous bioactive glass drug-loaded bone repair particle-hydrogel, the preparation method of which includes the following steps: The quercetin-derived carbon dots and hAMSC-EVs-loaded MBG@QCDs@EVs prepared by the above preparation method were mixed with the photocrosslinking hydrogel precursor solution and then sterilized to obtain the precursor solution; the photocrosslinking hydrogel precursor solution contains a photoinitiator. After the precursor solution is injected into the bone defect site, it is irradiated with ultraviolet, blue, or visible light to form a gel in situ, resulting in a mesoporous bioactive glass-loaded bone repair hydrogel. This invention employs an in-situ gelation method to prepare particle-hydrogels, which enables the MBG@QCDs@EVs composite material to achieve better local retention at the defect site and to act as the main bioactive component in regulating the bone repair microenvironment.

[0026] As a further technical solution, the irradiation time is 10-120 s.

[0027] As a further technical solution, the concentration of photoinitiator in the photocrosslinking hydrogel precursor solution is 0.05-1 wt%; the photocrosslinking hydrogel precursor solution also includes GelMA (methacrylamide gelatin) at a concentration of 5-20 wt%.

[0028] A mesoporous bioactive glass drug-loaded bone repair hydrogel, the preparation method of which includes the following steps: The quercetin-derived carbon dots and hAMSC-EVs-loaded MBG@QCDs@EVs prepared by the above preparation method are mixed evenly with the hydrogel material to obtain a mesoporous bioactive glass drug-loaded bone repair hydrogel.

[0029] As a further technical solution, the hydrogel material includes one or more of gelatin, hyaluronic acid, alginate, chitosan or PEG-based hydrogels.

[0030] A mesoporous bioactive glass drug-loaded bone repair scaffold, the preparation method of which includes the following steps: loading quercetin-derived carbon dots and hAMSC-EVs-loaded MBG@QCDs@EVs prepared by the preparation method onto the scaffold to obtain a mesoporous bioactive glass drug-loaded bone repair scaffold, wherein the scaffold includes any one of porous scaffolds, 3D printed scaffolds, metal implants, ceramic scaffolds or biodegradable polymer scaffolds.

[0031] The application of the mesoporous bioactive glass drug-loaded bone repair composite material or the mesoporous bioactive glass drug-loaded bone repair hydrogel in the preparation of drugs for preventing and treating osteoporotic bone injury.

[0032] The prevention and treatment of osteoporotic bone injury includes one or more of the following: promoting bone differentiation, inhibiting osteoclast formation, regulating macrophage inflammatory phenotype, and alleviating oxidative stress.

[0033] Furthermore, the prevention and treatment of osteoporotic bone injury includes one or more of the following: promoting osteogenic differentiation and matrix mineralization of hBMSCs, reducing RANKL-induced osteoclast-related phenotypes, promoting macrophage transformation to repair-related phenotypes, and reducing intracellular ROS levels under H2O2 stimulation model.

[0034] As a further technical solution, the mesoporous bioactive glass drug-loaded bone repair composite material, the mesoporous bioactive glass drug-loaded bone repair particles-hydrogel, etc., all need to be sterilized before use. The sterilization methods include ultraviolet irradiation, sterile filtration, ethylene oxide treatment, etc.

[0035] In this invention, Quercetin is a class of natural flavonoid bioactive molecules with a polyphenolic hydroxyl structure, possessing potential for antioxidant, anti-inflammatory, and bone metabolism regulation. However, free quercetin suffers from limitations such as poor water solubility and insufficient bioavailability. Converting quercetin into quercetin-derived carbon dots (QCDs) can improve its dispersibility and nanoscale cell interactions while retaining its redox regulatory advantages.

[0036] Extracellular vesicles (EVs) can participate in intercellular signaling by carrying active components such as proteins, nucleic acids, and lipids. Human amniotic mesenchymal stem cell-derived EVs (hAMSC-EVs) are derived from placental-related tissues and have the advantages of relatively convenient sourcing, avoiding invasive sourcing, and low immunogenicity, making them suitable as bioactive regulatory components in bone repair materials.

[0037] Mesoporous bioactive glass (MBG) has an ordered or radial mesoporous structure, which can serve as a local delivery matrix for drugs, nano-active components and biomacromolecules. At the same time, it can release bioactive ions such as silicon, calcium and phosphorus, providing a favorable inorganic microenvironment for bone repair.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses MBG as an inorganic porous bone repair matrix, QCDs as antioxidant and redox regulatory components, and hAMSC-EVs as intercellular signaling regulatory components. A single composite nano / micro / nano platform is prepared by first loading quercetin onto MBG, then generating quercetin-derived carbon dots in situ, and finally loading hAMSC-EVs. This platform is used for local filling of osteoporotic bone defects, retention of active components, and regulation of the bone regeneration microenvironment. Compared with existing technologies, it has the following advantages: 1. This invention constructs a single composite material platform by combining MBG, QCDs and hAMSC-EVs, which concentrates osteoconduction, antioxidant, immunomodulatory, osteopromoting and osteoclast-inhibiting functions in the same local delivery system, helping to overcome the problem of single function in traditional bone repair materials.

[0039] 2. This invention uses MBG to provide a porous inorganic matrix and a source of bioactive ions, which can serve as a loading platform for QCDs and EVs, and also provide a favorable microenvironment for osteoblast differentiation and matrix mineralization.

[0040] 3. The quercetin-derived carbon dots of this invention are derived from natural active molecules, which can improve the water dispersibility and nanoscale utilization of quercetin, and play a role in free radical scavenging and redox regulation in composite materials.

[0041] 4. The hAMSC-EVs of this invention can participate in osteogenic differentiation, immune microenvironment remodeling, and intercellular regulation as a biological signaling regulatory component. Compared with inorganic materials alone, the introduction of EVs helps to enhance the material's comprehensive regulatory ability on the complex microenvironment of osteoporotic bone defects.

[0042] 5. This invention can be used in the form of injectable hydrogel, particle filling, scaffold loading or implant surface coating, etc., which makes it easy to select the appropriate product form according to different bone defect morphologies.

[0043] 6. In vitro experimental results show that MBG@QCDs@EVs of the present invention can promote osteogenic differentiation and matrix mineralization of hBMSCs, reduce RANKL-induced osteoclast-related phenotype, promote the transformation of macrophages to repair-related phenotype, and reduce intracellular ROS levels under H2O2 stimulation model.

[0044] 7. Animal bone defect experiments show that the MBG@QCDs@EVs material of this invention, when combined with GelMA, can be locally retained at the bone defect site and improve trabecular bone reconstruction, matrix deposition and defect repair performance in osteoporotic bone defect models. Attached Figure Description

[0045] Figure 1This is a schematic diagram illustrating the preparation principle of the MBG@QCDs@EVs composite material of the present invention; Figure 2 This is a basic structural characterization diagram of the MBG@QCDs@EVs composite material of the present invention; exist Figure 2 In the image, A: Western blot identification of hAMSCs and hAMSC-EVs; B: TEM image of EVs; C: Particle size distribution of hAMSC-EVs; D: TEM image of MBG; E: TEM image of QCDs; F: Enlarged view of E; G: TEM image of MBG@QCDs@EVs. Figure 3 HAADF-STEM images, elemental mappings, and EDS spectra of MBG@QCDs@EVs; Figure 4 The physicochemical properties of the MBG@QCDs@EVs composite material are shown in the diagram. exist Figure 4 In the image, A: XRD patterns of MBG, MBG@QCDs, and MBG@QCDs@EVs; B: Nitrogen adsorption-desorption curves and pore size distribution of MBG; C: Hydrated particle size distribution; D: Zeta potential diagram; E: FTIR spectrum; F: XPS full spectrum. Figure 5 The diagram shows the free radical scavenging effect of different composite materials of the present invention. In the diagram, A: ABTS free radical; B: H2O2; C: hydroxyl free radical; Figure 6 This is a confocal fluorescence image of MBG@QCDs@EVs captured by hBMSCs in this invention; Figure 7 The image shows the CCK-8 detection results of hBMSCs and RAW264.7 in this invention. Figure 8 The results of live / dead cell staining of hBMSCs from different groups in this invention; Figure 9 This is a diagram showing the effect of different composite material treatments on the expression levels of osteogenic-related genes in this invention; A: Quantitative analysis of osteogenic-related gene expression; B: Osteogenic gene heatmap; C: Immunofluorescence images of OCN and COL1A1; Figure 10 The effect of different composite material treatments on the activity of ALP and ARS in this invention; exist Figure 10 In the diagram, A: staining map; B: ARS quantitative analysis map; C: ALP quantitative analysis map; Figure 11 This is a graph showing the effect of different composite material treatments on the expression levels of RUNX2 and COL1A1 proteins in this invention; exist Figure 11 In the Chinese text, A: RUNX2; B: COL1A1; Figure 12 This is a graph showing the effects of different composite material treatments on the expression levels of Nfatc1, Ctsk, c-Fos, and Acp5 genes in this invention. Figure 13 This is a diagram showing the effect of different composite material treatments on osteoclasts in this invention; exist Figure 13 In the image, A: staining map; B: F-actin quantitative analysis map; C: TRAP quantitative analysis map. Figure 14 This is a graph showing the quantitative analysis of NFATc1, CTSK, and c-FOS proteins by different composite material treatments in this invention; Figure 15 This is a flow cytometry analysis of CD86-positive and CD206-positive macrophages treated with different composite materials in this invention. exist Figure 15 In the Chinese dictionary, A: CD86; B: CD206; Figure 16 This is a graph showing the effects of different composite material treatments on the expression levels of Arg1, CD206, Il10, and iNos genes in this invention. Figure 17 Volcano diagrams and thermograms showing the differences in genetic makeup between different composite materials used in the invention; exist Figure 17 In the image, A: Volcano plot of differentially expressed genes; B: Heatmap of differentially expressed genes; Figure 18 This is a diagram showing the effect of different composite material treatments on the enrichment of KEGG and GO according to the present invention; exist Figure 18 In, A: KEGG; B: GO; Figure 19 This is a graph showing the effect of different composite material treatments on p-PI3K / PI3K and p-AKT / AKT in this invention; Figure 20 Graphs showing quantitative analysis of NRF2 and HO-1 in the core of MBG@QCDs@EVs composite material under H2O2 stimulation. Figure 21 This is an NRF2 immunofluorescence staining image of the MBG@QCDs@EVs composite material of the present invention under H2O2 stimulation conditions; Figure 22 The image shows intracellular ROS fluorescent probe staining and bright-field plot of the MBG@QCDs@EVs composite material of this invention under H2O2 stimulation conditions; Figure 23These are Micro-CT three-dimensional reconstruction images of the distal femur and defect area in different groups treated with different composite materials in this invention; Figure 24 These are in vivo fluorescence imaging images of different composite material treatments in this invention; Figure 25 This is a quantitative analysis diagram of BMD, BV / TV, and Tb.Th under different composite material treatments in this invention; Figure 26 These are H&E, Masson, and TRAP staining images of the defective areas after different composite material treatments in this invention. Figure 27 Quantitative analysis of N.Oc / BS and Oc.S / BS for different composite material treatments in this invention. Figure 28 The images show the H&E staining of the main organs in the different composite materials of this invention. Detailed Implementation

[0046] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0048] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field and are commercially available.

[0049] Example 1: 1. A method for preparing mesoporous bioactive glass drug-loaded particles for preventing and treating osteoporotic bone defects, see [link to relevant documentation]. Figure 1 Specifically, it includes the following steps: Step 1: Preparation of mesoporous bioactive glass particles: Mesoporous bioactive glass particles (MBG) were prepared using a CTAB / TEA-mediated biphase or single-phase sol-gel method.

[0050] 12g of the soft template agent hexadecyltrimethylammonium bromide was added to 108mL of solvent A (deionized water), and after stirring and dispersing evenly, 0.36mL of the catalyst and morphology regulator triethanolamine was added. The mixture was stirred at 60℃ and 150 rpm for 1h until it was homogeneous, thus obtaining the template solution. 12 mL of TEOS, 0.912 mL of triethyl phosphate (TEP), and 48 mL of cyclohexane were mixed to form an oil phase solution; The oil phase solution was added to the template solution and stirred at 60℃ and 150 rpm for 24 h to carry out hydrolysis and condensation reaction. After the reaction was completed, the oil phase was removed, the aqueous phase was centrifuged to collect the precipitate, and then washed with anhydrous ethanol to remove unreacted precursors, free surfactants and residual small molecules. After vacuum drying, the silicophosphorus mesoporous particles were obtained. Then, 1 g of silica-phosphorus mesoporous particles were dispersed in 40 mL of anhydrous ethanol containing 0.704 g of calcium nitrate tetrahydrate, stirred at room temperature for 24 h, centrifuged, and the precipitate was washed with anhydrous ethanol. The CTAB template was removed by calcination, and then cooled and ground to obtain mesoporous bioactive glass particles MBG.

[0051] Step 2, Quercetin loading: 1.0 g of mesoporous bioactive glass particles were added to 25 mL of solvent B (anhydrous ethanol), and ultrasonically dispersed until homogeneous. Then, 0.10 g of quercetin was added, and ultrasonic dispersion was continued. The mixture was then incubated in a shaker at 37 °C for 24 h to allow quercetin to adsorb into the pores of the mesoporous bioactive glass particles. The unloaded free quercetin was then removed by filtration. The solid product was washed with anhydrous ethanol and dried to obtain quercetin-loaded MBG@Que. Step 3: In-situ hydrothermal carbonization of quercetin: MBG@Que was dispersed in solvent C (deionized water) and subjected to a hydrothermal reaction at 180°C to generate quercetin-derived carbon dots. The reaction time was 8 hours. After the reaction was completed, the mixture was naturally cooled to room temperature, filtered to collect the precipitate, washed with deionized water, dialyzed, centrifuged to collect the precipitate, and then freeze-dried to obtain MBG@QCDs loaded with quercetin-derived carbon dots. Step 4, Preparation of hAMSC-EVs: Human amniotic mesenchymal stem cells were cultured. The resulting culture medium was first centrifuged at low speed (300 × g for 10 min) to remove cells, and then centrifuged at medium speed (2,000 × g for 20 min) to remove cell debris and large particles. After purification, hAMSC-EVs were obtained by ultracentrifugation (100,000 × g for 30 min). The hAMSC-EVs were then washed with PBS buffer, ultracentrifuged again (100,000 × g for 70 min), and resuspended in solvent D (PBS buffer solution) to obtain hAMSC-EVs suspension. Step 5: Loading hAMSC-EVs: Add 10 mg of MBG@QCDs to 1 mL of solvent E (sterile PBS buffer solution), sonicate until evenly dispersed, then add hAMSC-EVs suspension to a final concentration of 100 μg / mL, and incubate at 4 °C with shaking for 24 h. This allows hAMSC-EVs to be immobilized on the surface of MBG@QCDs through physical adsorption, electrostatic interaction, or surface interaction, forming MBG@QCDs@EVs. After incubation, centrifuge at low speed (5,000 × g for 10 min), wash to remove unbound hAMSC-EVs, and filter to obtain quercetin-derived carbon dots and MBG@QCDs@EVs loaded with hAMSC-EVs, which are the mesoporous bioactive glass drug-loaded particles.

[0052] 2. A mesoporous bioactive glass-loaded drug precursor formulation for the prevention and treatment of osteoporotic bone defects, the preparation method of which includes the following steps: GelMA (methacrylamide gelatin) was dissolved in sterile PBS buffer, and LAP photoinitiator was added. After mixing evenly, a photocrosslinking hydrogel precursor solution with a GelMA concentration of 10 wt% and an LAP concentration of 0.5 wt% was prepared. Mesoporous bioactive glass drug-loaded particles were added to the photocrosslinking hydrogel precursor solution and ultrasonically dispersed uniformly under light-protected conditions to prepare a 5% (w / v, i.e., 50 mg / mL) mesoporous bioactive glass drug-loaded precursor formulation. 3. A mesoporous bioactive glass drug-loaded particle-hydrogel composite formulation, the preparation method of which includes the following steps: After sterilization, the mesoporous bioactive glass drug-loaded precursor formulation is injected into the bone defect site. After the mesoporous bioactive glass drug-loaded precursor formulation is injected into the bone defect site, it is irradiated with 405nm blue light for 10-120 s to form an in-situ gel, thus obtaining a mesoporous bioactive glass drug-loaded particle-hydrogel composite formulation.

[0053] 4. Structural characterization 1) Immunoblot detection of hAMSC-EVs and hAMSCs; such as Figure 2 Western blot results for A showed that extracellular vesicle positive markers such as CD63 and CD9 could be detected in the hAMSC-EVs group, while the endoplasmic reticulum-related negative marker Calnexin was mainly present in the hAMSCs lysate. This indicates that the obtained vesicles have typical EVs protein marker characteristics.

[0054] 2) Perform TEM observation on hAMSC-EVs; such as Figure 2 As shown in B, hAMSC-EVs exhibit a round or near-round vesicle-like morphology; 3) The NTA was used to evaluate the hydrated particle size and the particle size distribution of EVs. Figure 2 The results showed that the particle size of hAMSC-EVs was mainly concentrated in the range of 100-200 nm, which is consistent with the distribution characteristics of nanoscale composite particle systems.

[0055] 3) TEM was used to observe the morphology of MBG, QCDs, and MBG@QCDs@EVs; such as Figure 2 DG analysis revealed that MBGs exhibited relatively uniform spherical or near-spherical particles with a radial mesoporous structure, providing channels and surface sites for quercetin loading, in-situ carbonization, and EV adsorption. Discrete QCDs with a size of approximately 20 nm were observed after hydrothermal carbonization. Figure 2 HRTEM of F showed lattice fringes of approximately 0.22 nm, indicating that quercetin formed a carbon dot structure with certain crystalline carbon domains after hydrothermal treatment. After loading QCDs and EVs, MBG@QCDs@EVs still maintained the overall particle morphology, but the particle surface was denser and the boundaries were more blurred than those of bare MBG, suggesting that the surface had been modified with active components.

[0056] 4) Elemental mapping analysis of Si, O, Ca, P, N, etc., in different materials was performed using HAADF-STEM and EDS; for example... Figure 3 The results showed that bioactive glass framework elements such as Si, O, Ca, and P were distributed within the particles, and N element signals were also detected, which can be attributed to the introduction of EVs-related proteins or other organic components.

[0057] 4) The composite material was systematically characterized using XRD, nitrogen adsorption-desorption, DLS, potential analysis, and XPS. Figure 4 XRD results for A showed that MBG, MBG@QCDs, and MBG@QCDs@EVs all exhibited broad diffuse peaks in the range of approximately 20-35°, consistent with the characteristics of amorphous bioactive glass frameworks.

[0058] Figure 4 The nitrogen adsorption-desorption curve of B shows that MBG has typical mesoporous material adsorption behavior and pore size distribution within the mesoporous range, indicating that the porous bioactive glass matrix was successfully constructed.

[0059] Figure 4 The DLS results for C showed that the hydrated particle size of MBG was approximately 156.2 nm, MBG@QCDs increased to approximately 169.6 nm, and MBG@QCDs@EVs further increased to approximately 237.4 nm. The gradual increase in particle size is consistent with the sequential loading of QCDs and EVs.

[0060] Figure 4Potential detection at D showed that all samples were negatively charged, indicating that the material has certain surface charge characteristics in the aqueous system.

[0061] Figure 4 XPS results for F further support the surface modification process: N 1s signal can be detected after EVs loading, indicating that the organic components related to EVs have been bound to the MBG@QCDs surface.

[0062] The aforementioned changes in structure, particle size, and chemical composition collectively demonstrate that MBG, QCDs, and hAMSC-EVs have formed a common composite material platform.

[0063] Example 2 1. A method for preparing mesoporous bioactive glass drug-loaded particles for preventing and treating osteoporotic bone defects, see [link to relevant documentation]. Figure 1 Specifically, it includes the following steps: Step 1: Preparation of mesoporous bioactive glass particles: Mesoporous bioactive glass particles (MBG) were prepared using a CTAB / TEA-mediated biphase or single-phase sol-gel method.

[0064] Add 12g of the soft template agent hexadecyltrimethylammonium bromide to 110mL of solvent A (deionized water), stir and disperse evenly, then add 0.36mL of the catalyst and morphology regulator triethanolamine, and stir at 60℃ and 150 rpm for 1h until the mixture is homogeneous to obtain the template solution; 12 mL of TEOS, 0.912 mL of triethyl phosphate (TEP), and 48 mL of cyclohexane were mixed to form an oil phase solution; The oil phase solution was added to the template solution and stirred at 65℃ and 170 rpm for 24 h to carry out hydrolysis and condensation reaction. After the reaction was completed, the oil phase was removed, the aqueous phase was centrifuged to collect the precipitate, and then washed with anhydrous ethanol to remove unreacted precursors, free surfactants and residual small molecules. After vacuum drying, the silicophosphorus mesoporous particles were obtained. Then, 1 g of silica-phosphorus mesoporous particles were dispersed in 40 mL of anhydrous ethanol containing 0.704 g of calcium nitrate tetrahydrate, stirred at room temperature for 24 h, centrifuged, and the precipitate was washed with anhydrous ethanol. The CTAB template was removed by calcination, and then cooled and ground to obtain mesoporous bioactive glass particles MBG.

[0065] Step 2, Quercetin loading: 1.0 g of mesoporous bioactive glass particles were added to 25 mL of solvent B (anhydrous ethanol), and ultrasonically dispersed until uniform. Then, 0.10 g of quercetin was added, and ultrasonic dispersion was continued. The mixture was then incubated in a shaker at 37 °C for 28 h to allow quercetin to adsorb into the pores of the mesoporous bioactive glass particles. The unloaded free quercetin was then removed by filtration. The solid product was washed with anhydrous ethanol and dried to obtain quercetin-loaded MBG@Que. Step 3: In-situ hydrothermal carbonization of quercetin: MBG@Que was dispersed in solvent C (deionized water) and subjected to a hydrothermal reaction at 170°C to generate quercetin-derived carbon dots. The reaction time was 9 hours. After the reaction was completed, the mixture was naturally cooled to room temperature, filtered to collect the precipitate, washed with deionized water, dialyzed, centrifuged to collect the precipitate, and then freeze-dried to obtain MBG@QCDs loaded with quercetin-derived carbon dots. Step 4, Preparation of hAMSC-EVs: Human amniotic mesenchymal stem cells were cultured. The resulting culture medium was first centrifuged at low speed (300 × g for 10 min) to remove cells, and then centrifuged at medium speed (2,000 × g for 20 min) to remove cell debris and large particles. After purification, hAMSC-EVs were obtained by ultracentrifugation (100,000 × g for 30 min). The hAMSC-EVs were then washed with PBS buffer, ultracentrifuged again (100,000 × g for 70 min), and resuspended in solvent D (PBS buffer solution) to obtain hAMSC-EVs suspension. Step 5: Loading hAMSC-EVs: Add 10 mg of MBG@QCDs to 1 mL of solvent E (sterile PBS buffer solution), sonicate until evenly dispersed, then add hAMSC-EVs suspension to a final concentration of 100 μg / mL, and incubate at 4 °C with shaking for 28 h. This allows hAMSC-EVs to be immobilized on the surface of MBG@QCDs through physical adsorption, electrostatic interaction, or surface interaction, forming MBG@QCDs@EVs. After incubation, centrifuge at low speed (5,000 × g for 10 min), wash to remove unbound hAMSC-EVs, and filter to obtain quercetin-derived carbon dots and MBG@QCDs@EVs loaded with hAMSC-EVs, i.e., the mesoporous bioactive glass drug-loaded particles.

[0066] 2. A mesoporous bioactive glass-loaded drug precursor formulation for the prevention and treatment of osteoporotic bone defects, the preparation method of which includes the following steps: GelMA (methacrylamide gelatin) was dissolved in sterile PBS buffer, and LAP photoinitiator was added. After mixing evenly, a photocrosslinking hydrogel precursor solution with a GelMA concentration of 12 wt% and an LAP concentration of 0.4 wt% was prepared. Mesoporous bioactive glass drug-loaded particles were added to the photocrosslinking hydrogel precursor solution and ultrasonically dispersed uniformly under light-protected conditions to prepare a 5% (w / v, i.e., 50 mg / mL) mesoporous bioactive glass drug-loaded precursor formulation. 3. A mesoporous bioactive glass drug-loaded particle-hydrogel composite formulation, the preparation method of which includes the following steps: same as in Example 1.

[0067] Example 3 1. A method for preparing mesoporous bioactive glass drug-loaded particles for preventing and treating osteoporotic bone defects, see [link to relevant documentation]. Figure 1 Specifically, it includes the following steps: Step 1: Preparation of mesoporous bioactive glass particles: Mesoporous bioactive glass particles (MBG) were prepared using a CTAB / TEA-mediated biphase or single-phase sol-gel method.

[0068] 12g of the soft template agent hexadecyltrimethylammonium bromide was added to 95mL of solvent A (deionized water), and after stirring and dispersing evenly, 0.36mL of the catalyst and morphology regulator triethanolamine was added. The mixture was stirred at 60℃ and 150 rpm for 1h until it was homogeneous, thus obtaining the template solution. 12 mL of TEOS, 0.912 mL of triethyl phosphate (TEP), and 48 mL of cyclohexane were mixed to form an oil phase solution; The oil phase solution was added to the template solution and stirred at 65℃ and 170 rpm for 24 h to carry out hydrolysis and condensation reaction. After the reaction was completed, the oil phase was removed, the aqueous phase was centrifuged to collect the precipitate, and then washed with anhydrous ethanol to remove unreacted precursors, free surfactants and residual small molecules. After vacuum drying, the silicophosphorus mesoporous particles were obtained. Then, 1 g of silica-phosphorus mesoporous particles were dispersed in 40 mL of anhydrous ethanol containing 0.704 g of calcium nitrate tetrahydrate, stirred at room temperature for 24 h, centrifuged, and the precipitate was washed with anhydrous ethanol. The CTAB template was removed by calcination, and then cooled and ground to obtain mesoporous bioactive glass particles MBG.

[0069] Step 2, Quercetin loading: 1.0 g of mesoporous bioactive glass particles were added to 25 mL of solvent B (anhydrous ethanol), and ultrasonically dispersed until homogeneous. Then, 0.10 g of quercetin was added, and ultrasonic dispersion was continued. The mixture was then incubated in a shaker at 37 °C for 20 h to allow quercetin to adsorb into the pores of the mesoporous bioactive glass particles. The unloaded free quercetin was then removed by filtration. The solid product was washed with anhydrous ethanol and dried to obtain quercetin-loaded MBG@Que. Step 3: In-situ hydrothermal carbonization of quercetin: MBG@Que was dispersed in solvent C (deionized water) and subjected to a hydrothermal reaction at 150°C to generate quercetin-derived carbon dots for 10 hours. After the reaction was completed, the mixture was naturally cooled to room temperature, filtered to collect the precipitate, washed with deionized water, dialyzed, centrifuged to collect the precipitate, and then freeze-dried to obtain MBG@QCDs loaded with quercetin-derived carbon dots. Step 4, Preparation of hAMSC-EVs: Human amniotic mesenchymal stem cells were cultured. The resulting culture medium was first centrifuged at low speed (300 × g for 10 min) to remove cells, and then centrifuged at medium speed (2,000 × g for 20 min) to remove cell debris and large particles. After purification, hAMSC-EVs were obtained by ultracentrifugation (100,000 × g for 30 min). The hAMSC-EVs were then washed with PBS buffer, ultracentrifuged again (100,000 × g for 70 min), and resuspended in solvent D (PBS buffer solution) to obtain hAMSC-EVs suspension. Step 5: Loading hAMSC-EVs: Add 10 mg of MBG@QCDs to 1 mL of solvent E (sterile PBS buffer solution), sonicate until evenly dispersed, then add hAMSC-EVs suspension to make a final concentration of 100 μg / mL, and incubate at 4 °C with shaking for 22 h to allow hAMSC-EVs to be immobilized on the surface of MBG@QCDs through physical adsorption, electrostatic interaction, or surface interaction, forming MBG@QCDs@EVs. After incubation, centrifuge at low speed (5,000 × g for 10 min), wash to remove unbound hAMSC-EVs, filter, and obtain quercetin-derived carbon dots and MBG@QCDs@EVs loaded with hAMSC-EVs, i.e., the mesoporous bioactive glass drug-loaded particles.

[0070] 2. A mesoporous bioactive glass-loaded drug precursor preparation for preventing and treating osteoporotic bone defects, the preparation method of which includes the following steps: same as in Example 1.

[0071] 3. A mesoporous bioactive glass drug-loaded particle-hydrogel composite formulation, the preparation method of which includes the following steps: same as in Example 1.

[0072] Application Example 1: ROS Removal Performance Evaluation MBG, MBG@QCDs, and MBG@QCDs@EVs prepared in Example 1 were dispersed in PBS buffer solution. The antioxidant capacity (scavenging capacity for ABTS radicals, H2O2, and ·OH) of different material samples was evaluated using an ABTS radical scavenging kit, an H2O2 scavenging kit, and a hydroxyl radical scavenging kit. After incubating the samples with the corresponding working solutions, they were centrifuged, and the absorbance of the supernatant was read at the wavelength recommended by the kit. The radical scavenging rate was then calculated. The results are shown below. Figure 5 .

[0073] Depend on Figure 5It can be seen that bare MBG only exhibits weak free radical scavenging ability; after introducing QCDs, the scavenging ability of MBG@QCDs against ABTS radicals, H2O2, and ·OH is significantly enhanced, indicating that quercetin-derived carbon dots can participate in free radical scavenging as the main antioxidant functional component. After further loading of EVs, MBG@QCDs@EVs still maintains ROS scavenging performance similar to that of MBG@QCDs, suggesting that the surface modification of EVs does not significantly weaken the antioxidant effect of QCDs.

[0074] Application Example 2: Evaluation of Cellular Uptake and Biocompatibility 1. To observe the ability of the composite material to enter cells, hAMSC-EVs were labeled with PKH26 lipid membrane dye and then compounded with MBG@QCDs to obtain fluorescently labeled MBG@QCDs@EVs. After co-culturing with hBMSCs, intracellular red fluorescence signals were observed using confocal microscopy to evaluate the cellular uptake of the composite material or EV components. The results are shown in [Figure number missing]. Figure 6 .

[0075] from Figure 6 The results showed that co-culturing hBMSCs with MBG@QCDs@EVs resulted in a clear distribution of red fluorescence signals within the cells, indicating that the EV-related components carried by the material could be taken up by hBMSCs. This result provides a basis for subsequent studies on regulating osteoblast behavior through the delivery of bioactive signals via EVs.

[0076] 2. Regarding cell compatibility, hBMSCs or RAW264.7 cells were co-cultured with different material groups, and cell compatibility was assessed using CCK-8 and Live / Dead staining methods. Results are shown below. Figure 7-8 .

[0077] from Figure 7 CCK-8 assays showed that the OD450 values ​​of hBMSCs in each group gradually increased with the extension of culture time, and no significant decrease was observed with material treatment.

[0078] Figure 8 The results of live / dead cell staining showed that hBMSCs and RAW264.7 cells maintained a high proportion of live cells in all material treatment groups, and no large number of red dead cell signals were observed.

[0079] The above results indicate that, under the experimental conditions used, MBG@QCDs@EVs have good in vitro compatibility with hBMSCs and RAW264.7 cells, and can serve as a material basis for subsequent osteogenic, osteoclast, and immunomodulatory experiments.

[0080] Application Example 3: Evaluation of bone differentiation and matrix mineralization To evaluate the effect of MBG@QCDs@EVs on osteogenic differentiation of hBMSCs, hBMSCs were co-cultured with CON, MBG, MBG@QCDs, and MBG@QCDs@EVs groups under osteogenic induction conditions. The expression of osteogenic-related genes such as Runx2, Col1a1, Ocn, and Opn was detected by RT-qPCR; early osteogenic differentiation was evaluated by ALP staining and ALP activity assay; mineralization nodule formation was evaluated by ARS staining and quantitative analysis; and the expression levels of osteogenic-related proteins such as RUNX2 and COL1A1 were detected by Western blot. The results are shown in [Figure number missing]. Figure 9-11 ; Figure 9 RT-qPCR results showed that, compared with the CON group, the expression of osteogenic-related genes such as Col1a1, Runx2, Ocn, and Opn was increased in the MBG group; the expression was further increased in the MBG@QCDs group; and the expression level was highest in the MBG@QCDs@EVs group. This trend indicates that with the gradual introduction of QCDs and hAMSC-EVs, the promoting effect of the composite material on osteogenic-related transcriptional programs is gradually enhanced.

[0081] Figure 10 ALP and ARS staining results showed that they were consistent with gene expression trends. ALP staining and quantification showed that the higher the degree of material functionalization, the stronger the early osteogenic differentiation activity; ARS staining showed that the formation of mineralized nodules gradually increased, with the most obvious red calcium deposition in the MBG@QCDs@EVs group.

[0082] from Figure 11 The gray-scale quantitative results showed that the expression of RUNX2 and COL1A1 proteins was higher in the material treatment groups than in the CON group, and the highest expression was observed in the MBG@QCDs@EVs group.

[0083] The above results indicate that MBG@QCDs@EVs can simultaneously promote early osteogenic differentiation and late matrix mineralization of hBMSCs.

[0084] Application Example 4: Evaluation of Inhibition of Osteoclast Formation To evaluate the regulatory effect of composite materials on osteoclast differentiation, a RANKL-induced osteoclast differentiation model was established using RAW264.7 cells. Cells were co-cultured with CON, MBG, MBG@QCDs, and MBG@QCDs@EVs groups, respectively. RT-qPCR was used to detect the expression of osteoclast-related genes such as Nfatc1, Ctsk, c-Fos, and Acp5. F-actin staining was used to observe the actin ring structure. TRAP staining was used to evaluate the formation of TRAP-positive multinucleated cells. Western blot was used to detect the expression of proteins such as NFATC1, CTSK, and c-FOS.

[0085] Figure 12 RT-qPCR results showed that the expression levels of osteoclast-related genes such as Nfatc1, Ctsk, c-Fos, and Acp5 gradually decreased after material treatment, with the lowest levels observed in the MBG@QCDs@EVs group. Since Nfatc1 and c-FOS are key transcriptional regulators in osteoclast differentiation, and CTSK and ACP5 are closely related to osteoclast bone resorption, these results indicate that the composite material can inhibit RANKL-induced osteoclast differentiation-related transcriptional programs.

[0086] Figure 13 Morphological staining further supports the above results. Quantitative analysis of F-actin and TRAP showed no significant difference between the CON group and the MBG group, while the MBG@QCDs and MBG@QCDs@EVs groups showed significant reductions, with the MBG@QCDs@EVs group showing the lowest reduction. The MBG@QCDs@EVs group exhibited the most significant inhibitory effect.

[0087] Figure 14 The results showed that the expression of NFATC1, CTSK, and c-FOS proteins decreased after material treatment.

[0088] The above results indicate that MBG@QCDs@EVs inhibit osteoclast differentiation and related phenotypes.

[0089] Application Example 5: Macrophage Polarization and PI3K / Akt Correlation Signal Analysis Osteoporotic bone defects are often accompanied by persistent inflammation, and changes in macrophage phenotypes have a significant impact on osteogenic and osteoclast-reactive balance. RAW264.7 macrophages were co-cultured with different materials. Flow cytometry was used to detect macrophage phenotypic markers such as CD86 and CD206; RT-qPCR was used to detect the expression of genes such as Arg1, CD206, Il10, and iNos; transcriptome sequencing was used to analyze differentially expressed genes and pathways in macrophages after material treatment; and Western blot was used to detect the expression of proteins such as p-PI3K, PI3K, p-AKT, and AKT.

[0090] Figure 15 Flow cytometry results showed that only minor changes were observed in the MBG group; the proportion of CD86 positivity decreased and the proportion of CD206 positivity increased in both the MBG@QCDs and MBG@QCDs@EVs groups, with the most significant changes observed in the MBG@QCDs@EVs group.

[0091] Figure 16 The RT-qPCR results showed that there was no significant difference between the CON group and the MBG group, while Nos2 was downregulated and Mrc1, Arg1 and Il10 were upregulated in the MBG@QCDs and MBG@QCDs@EVs groups. This indicates that the material treatment is associated with the transformation of macrophages to the M2-like repair-related phenotype, and MBG@QCDs@EVs can promote the transformation of macrophages from a pro-inflammatory state to a repair-related state.

[0092] Figure 17-18 The results showed that the MBG@QCDs@EVs treatment group had 204 upregulated genes and 146 downregulated genes compared with the CON group; the differentially expressed genes were mainly enriched in the PI3K-Akt, focal adhesion and extracellular matrix-related pathways.

[0093] Figure 19 The results showed that the p-PI3K / PI3K and p-AKT / AKT ratios increased after MBG@QCDs@EVs treatment, which demonstrates that changes in macrophage phenotype are accompanied by enhanced PI3K / Akt-related signaling.

[0094] Application Example 6: Analysis of the Responsive Effects of Oxidative Stress Relief on NRF2 / HO-1 To simulate the pathological microenvironment of elevated ROS in osteoporotic bone defect areas, an oxidative stress model was established in RAW264.7 cells stimulated with H2O2. Groups were set up including CON, H2O2, H2O2+MBG, H2O2+MBG@QCDs, and H2O2+MBG@QCDs@EVs. Intracellular ROS levels were detected using a ROS fluorescent probe; the expression of antioxidant-related proteins such as NRF2 and HO-1 in the nucleus was detected by Western blot; and NRF2 nuclear translocation was evaluated using immunofluorescence.

[0095] Figure 20 The results showed that H2O2 treatment reduced the expression of HO-1 and nuclear NRF2 to lower levels than the CON group; MBG treatment did not significantly reverse this change, while MBG@QCDs and MBG@QCDs@EVs increased the expression of both proteins, with the MBG@QCDs@EVs group showing the highest expression. NRF2 immunofluorescence showed enhanced nuclear signal in the MBG@QCDs and MBG@QCDs@EVs groups.

[0096] Figure 21 Immunofluorescence results showed that the colocalization of NRF2 with the cell nucleus was enhanced in the MBG@QCDs and MBG@QCDs@EVs groups, suggesting increased NRF2 nuclear translocation.

[0097] Figure 22 ROS fluorescent probe staining showed that the intracellular green fluorescence was significantly enhanced after H2O2 stimulation, indicating an increase in ROS levels. After material treatment, the intensity of green fluorescence decreased, with the MBG@QCDs@EVs group showing the most significant decrease.

[0098] The above results indicate that MBG@QCDs@EVs can reduce intracellular ROS accumulation under oxidative stress conditions, and this change is consistent with the enhanced antioxidant response related to NRF2 / HO-1.

[0099] Application Example 7: Animal Experimental Analysis of Osteoporotic Bone Defects I. Experimental Methods: To evaluate the in vivo bone repair effect of MBG@QCDs@EVs, a rat model of osteoporotic bone defects was established. The preferred method was to first establish an osteoporosis model through bilateral ovariectomy, and after model stabilization, standardized bone defects were prepared in the femoral region. Since MBG@QCDs@EVs are particulate materials, they could be mixed with a GelMA / LAP precursor solution and injected into the bone defect area, followed by in-situ curing via photocrosslinking to reduce particle loss and improve local retention. In this system, GelMA mainly serves as a filler and retention carrier, while MBG-based composite particles are the core bioactive component.

[0100] Postoperatively, tissue samples were collected at predetermined time points. In vivo / ex vivo fluorescence imaging was used to observe the local distribution of EV-related signals at the defect site. Micro-CT three-dimensional reconstruction and parameters such as BMD, BV / TV, Tb.Th, and Tb.Sp were used to evaluate bone regeneration. H&E, Masson, and TRAP staining were used to evaluate tissue ingrowth, collagen matrix deposition, and osteoclast-related indicators. If necessary, immunostaining with OCN, OPN, RUNX2, COL1A1, iNOS, ARG1, CD206, NRF2, and HO-1 could be added to evaluate the osteogenic, immune, and antioxidant microenvironment.

[0101] II. Results and Analysis Micro-CT three-dimensional reconstruction 6 weeks postoperatively ( Figure 23 The results showed that the defect area in the CON group still had obvious structural gaps, while the bone structure coverage of the material treatment groups increased to varying degrees. Among them, the defect area in the MBG@QCDs@EVs group was filled most fully, and the trabecular bone network was denser.

[0102] Figure 24 Fluorescence imaging showed that clear EV-related fluorescence signals were detected in the femoral implantation area in the MBG@QCDs@EVs group, while only weak background signals were observed in the CON group, indicating that the EV load constructs had a detectable local distribution in the implantation area.

[0103] Figure 25 Quantitative analysis showed that compared with the CON group, BMD, BV / TV, and Tb.Th increased and Tb.Sp decreased in all material groups, with the MBG@QCDs@EVs group showing the most significant improvement.

[0104] The above results indicate that MBG@QCDs@EVs can enhance bone remodeling and trabecular structure restoration in osteoporotic bone defect areas.

[0105] Figure 26 The histological results were consistent with those of Micro-CT. Figure 26 Masson staining showed that only sparse matrix distribution was observed in the defect area of ​​the CON group, while matrix deposition gradually increased after material treatment, and the collagen matrix of the MBG@QCDs@EVs group was more continuous. Figure 26 H&E staining showed that the MBG@QCDs@EVs group had more obvious tissue ingrowth into the defect area, and the tissue structure in the defect area was denser and more orderly. Figure 26 TRAP staining showed that there were more TRAP-positive cells around the defect area in the CON group, while the overall TRAP-positive signal decreased in the material treatment group. Figure 27 Quantitative analysis of N.Oc / BS and Oc.S / BS showed that: MBG@QCDs@EVs group osteoclast count / bone surface area Both N.Oc / BS and osteoclast surface / bone surface (Oc.S / BS) were significantly reduced.

[0106] Figure 28 H&E staining of major organs showed that the alveolar structure, myocardial fiber arrangement, kidney tissue structure, liver lobule structure, and spleen tissue morphology of each material group were not significantly different from those of the CON group, suggesting that no significant systemic histological damage was found within the observation time and detection range of this study.

[0107] In vivo results suggest that MBG@QCDs@EVs can improve the repair of osteoporotic bone defects by promoting bone remodeling, increasing matrix deposition, and reducing osteoclast-related activity.

[0108] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A mesoporous bioactive glass-loaded bone repair composite material, characterized in that, Mesoporous bioactive glass serves as the drug delivery framework, while quercetin-derived carbon dots and extracellular vesicles derived from human amniotic mesenchymal stem cells are the active ingredients.

2. The mesoporous bioactive glass drug-loaded bone repair composite material according to claim 1, characterized in that, Its forms include granules, lyophilized powders, sterile suspensions, hydrogels, scaffolds, or coatings.

3. A method for preparing a mesoporous bioactive glass drug-loaded bone repair composite material, characterized in that, Includes the following steps: S1. Loading of quercetin or its derivatives: Mesoporous bioactive glass particles were added to solvent B and dispersed until uniform. Then, quercetin or its derivatives were added and dispersed until the quercetin or its derivatives were adsorbed into the pores of the mesoporous bioactive glass particles. Then, solid-liquid separation was performed, and the solid product was washed and dried to obtain MBG@Que loaded with quercetin or its derivatives. S2. In-situ hydrothermal carbonization of quercetin or its derivatives: MBG@Que is dispersed in solvent C and subjected to a hydrothermal reaction at 120-220℃ to generate quercetin-derived carbon dots. The reaction time is 2-24h. After the reaction is completed, the mixture is naturally cooled to room temperature. After solid-liquid separation, the precipitate is washed and dialyzed. Then, solid-liquid separation is performed, the precipitate is collected and dried to obtain MBG@QCDs loaded with quercetin-derived carbon dots. S3. Loading of hAMSC-EVs: MBG@QCDs were added to solvent E and dispersed evenly. Then, hAMSC-EVs suspension was added and incubated at 0-25℃ with shaking for 2-24 h to fix hAMSC-EVs on the surface of MBG@QCDs, forming MBG@QCDs@EVs. After shaking incubation, unbound hAMSC-EVs were washed away to obtain quercetin-derived carbon dots and MBG@QCDs@EVs loaded with hAMSC-EVs, which is the mesoporous bioactive glass drug-loaded bone repair composite material.

4. The method for preparing a mesoporous bioactive glass drug-loaded bone repair composite material according to claim 3, characterized in that, The method for preparing the mesoporous bioactive glass particles includes the following steps: The soft template agent hexadecyltrimethylammonium bromide was added to solvent A and stirred to disperse it evenly. Then, the catalyst and morphology modifier triethanolamine was added and stirred evenly at 40-80℃ to obtain the template solution. A silicon source and a phosphorus source are added to an organic solvent to prepare an oil phase solution; The oil phase solution was added to the template solution and stirred to carry out the hydrolysis and condensation reaction. After the reaction was completed, the solid and liquid phases were separated and the precipitate was collected. Then, the unreacted precursor, free surfactant and residual small molecules were washed to remove them and dried to obtain silicon phosphorus mesoporous particles. The mesoporous silica-phosphorus particles and calcium source were added to anhydrous ethanol and stirred for at least 24 hours. The solid and liquid were separated, the precipitate was collected, washed, and then calcined to remove the CTAB template. After cooling and grinding, mesoporous bioactive glass particles MBG were obtained.

5. The method for preparing a mesoporous bioactive glass drug-loaded bone repair composite material according to claim 4, characterized in that, Solvent A includes either deionized water or an aqueous ethanol solution; The silicon source is tetraethyl orthosilicate; The phosphorus source is triethyl phosphate; The calcium source is a soluble calcium salt, which includes one or more of calcium nitrate, calcium chloride, and calcium acetate. The hydrolysis-condensation reaction was carried out at a temperature of 50-70℃ for 18-28 hours, with a stirring speed of 120-180 rpm. The solvent used for washing includes any one of deionized water, anhydrous ethanol, or an aqueous solution of ethanol. The calcination temperature is 500-700℃, and the time is 3-8 h.

6. The method for preparing a mesoporous bioactive glass drug-loaded bone repair composite material according to claim 3, characterized in that, In S1, Solvent B includes one or more of anhydrous ethanol, deionized water, PBS buffer solution, and aqueous ethanol solution; The dispersion treatment and the continued dispersion treatment employ one or more of the following: ultrasonic dispersion, stirring, and oscillation incubation. The solvent used for washing includes one or more of anhydrous ethanol, deionized water, ethanol solution, and PBS buffer solution.

7. The method for preparing a mesoporous bioactive glass drug-loaded bone repair composite material according to claim 3, characterized in that, In S2, the preparation method of hAMSC-EVs includes: culturing human amniotic mesenchymal stem cells, first removing cells by low-speed centrifugation of the culture medium, then removing cell debris and large particles by medium-speed centrifugation, and then purifying by ultracentrifugation to obtain hAMSC-EVs, then washing hAMSC-EVs with PBS buffer, then ultracentrifuging again, and resuspending in solvent D to obtain hAMSC-EVs suspension.

8. A mesoporous bioactive glass-loaded drug-eluting bone repair hydrogel, characterized in that, Its preparation method includes the following steps: Quercetin-derived carbon dots prepared by any one of claims 3-6 and MBG@QCDs@EVs loaded with hAMSC-EVs are mixed with a photocrosslinking hydrogel precursor solution to obtain a precursor solution; the photocrosslinking hydrogel precursor solution contains a photoinitiator. After the precursor solution is injected into the bone defect site, it is irradiated with ultraviolet light, blue light or visible light to form a gel in situ, thus obtaining a mesoporous bioactive glass drug-loaded bone repair hydrogel.

9. The mesoporous bioactive glass drug-loaded bone repair hydrogel according to claim 8, characterized in that, The photoinitiator concentration in the photocrosslinking hydrogel precursor solution is 0.05-1 wt%; the photocrosslinking hydrogel precursor solution also includes GelMA at a concentration of 5-20 wt%.

10. The use of the mesoporous bioactive glass drug-loaded bone repair composite material according to any one of claims 1-2 or the mesoporous bioactive glass drug-loaded bone repair hydrogel according to any one of claims 8-9 in the preparation of drugs for preventing and treating osteoporotic bone injury.

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