CD301b < + >-EXO, preparation method of CD301b < + >-EXO, CD301b < + >-EXO-loaded bone targeting microspheres as well as preparation method and application of CD301b < + >-EXO-loaded bone targeting microspheres

Abstract

The invention discloses a CD301b < + >-EXO, a preparation method of the CD301b < + >-EXO, a CD301b < + >-EXO loaded bone targeting microsphere as well as a preparation method and application of the CD301b < + >-EXO loaded bone targeting microsphere. The CD301b < + >-EXO loaded bone targeting microsphere adopts an alendronate modified functional component, is endowed with excellent bone targeting ability, can significantly improve the enrichment and retention of exosomes in a bone defect area, and realizes continuous controllable release in a bone defect environment. According to the invention, the triple repair function of CD301b < + >-EXO is fully exerted, and the bone targeting and slow release characteristics of the microspheres are combined, so that the synergistic promotion of the whole bone healing process is realized, the repair efficiency and quality are remarkably improved, and the clinical transformation value is very high.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a CD301b + -EXO and a preparation method thereof, and a CD301b + -EXO, a bone-targeting microsphere of the CD301b BACKGROUND

[0002] Bone defect is a common major problem in clinic, and its causes include trauma, tumor resection, infection and orthopedic surgery. Minor injury can be healed through self-repair of the body, but critical size or disease-related bone defects often exceed the natural regeneration limit of bone tissue, and thus require exogenous intervention. At present, autologous bone transplantation is still the gold standard for bone repair, and has good biocompatibility and osteoconductive properties, but is limited by donor shortage, large surgical trauma and infection risk, and thus has obvious limitations in clinical application. Allogeneic bone and artificial bone replacement materials can partially alleviate the shortage of donors, but still cannot meet the clinical needs due to defects such as immune rejection, infection and insufficient osteogenesis.

[0003] The existing bone repair materials generally have deficiencies in terms of bioactive ingredients, and most of them only have a single function, such as only anti-inflammatory, only pro-angiogenic or only osteogenic. However, bone healing is an orderly and complex process involving “inflammation regulation — angiogenesis — bone formation”, and each stage requires specific bioactive effects to support. Traditional strategies often use a combination of multiple active factors or cells to cover different repair links, but this not only increases the complexity and cost of preparation, but also may bring uncontrollable risks due to the interaction between components, affecting the repair effect.

[0004] Recent studies have shown that successful bone repair depends on the coordinated regulation of the bone conductive properties of the material and the local immune microenvironment. Macrophages, as core immune regulatory cells, play a key role in inflammation resolution, angiogenesis and osteogenic differentiation. Although the classical M1 / M2 two-part model explains the functional diversity of macrophages to some extent, it still cannot fully cover their complex behavior in bone regeneration. Recent studies have found that CD301b + Macrophages exhibit significant advantages in immune regulation, pro-angiogenic and osteogenic induction, which are superior to traditional M2-type macrophages. However, direct use of CD301b + Macrophages for cell therapy still faces many bottlenecks, including limited cell sources, difficult in vitro expansion, low survival rate after transplantation and poor targeted delivery efficiency, which seriously restricts its clinical translation value.

[0005] As a kind of nanoscale vesicles (30-150 nm) secreted by cells, EXO (exosome) can carry various active molecules such as proteins and nucleic acids, and can inherit and transmit the functions of parent cells, thus showing the advantages of cell-free therapy in regenerative medicine. Studies have shown that macrophages mainly regulate bone homeostasis by secreting exosomes. SUMMARY

[0006] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present application is to provide a preparation method of CD301b + -EXO (exosome derived from CD301b + macrophages.

[0007] The second purpose of the present application is to provide CD301b + -EXO (exosome derived from CD301b + macrophages). The present application first identifies and systematically clarifies the exosomes derived from CD301b + macrophages (CD301b + -EXO). The exosomes naturally inherit the core functions of their parent cells, and have multiple biological activities such as immune regulation, pro-angiogenesis and osteogenic induction, and can cover the key links of bone repair with a single source, avoiding the complexity of multi-component synergistic strategies, and showing outstanding application advantages and important clinical translation potential. In addition to direct application, CD301b + -EXO can also be used in combination with various biomaterials to further improve its bone repair effect. It can be loaded in natural or synthetic polymer hydrogels, microspheres, nanofiber scaffolds or inorganic / organic composite scaffolds, and the three-dimensional structure of the material can achieve precise positioning, stable delivery and controlled release, while maintaining the biological activity of the exosomes, and relying on the mechanical and microenvironment regulation characteristics of the material itself, further amplifying its multiple functions. For example, hydrogels have good injectability and self-healing properties, and are suitable for plastic filling of irregular bone defects; microspheres can achieve controllable degradation and time-release, which is highly matched with the dynamic process of bone repair. In order to better utilize CD301b + -EXO in the field of regenerative medicine, the present application also prepares a bone-targeting microsphere (A-SCH / OSA@EXO microsphere) loaded with CD301b + -EXO.

[0008] The third purpose of the present application is to provide a preparation method of a bone-targeting microsphere (A-SCH / OSA@EXO microsphere) loaded with CD301b + -EXO. The preparation method loads CD301b + -EXO in the bone-targeting microsphere (A-SCH / OSA@EXO microsphere).The implementation of the EXO-loaded bone-targeting microspheres is achieved. The preparation method utilizes microfluidic technology and pH-responsive Schiff base bonds formed by grafting alendronate onto succinylated chitosan (A-SCH) and oxidized sodium alginate (OSA), to achieve intelligent and controllable release of exosomes in an acidic microenvironment, prolong the action time and maintain biological activity, thereby significantly improving the local retention and functional exertion of exosomes.

[0009] The fourth object of the present application is to provide the CD301b + EXO-loaded bone-targeting microspheres (A-SCH / OSA@EXO microspheres). The CD301b + The bone-targeting microspheres (A-SCH / OSA@EXO microspheres) of the EXO have uniform particle size and stable dispersion, and the material is endowed with bone-targeting properties by introducing bisphosphonate groups.

[0010] The fifth object of the present application is to provide the CD301b + Application of the bone-targeting microspheres (A-SCH / OSA@EXO microspheres) of the EXO. The A-SCH / OSA@EXO microspheres prepared by the present application are applied in the field of bone defects, which not only improves the enrichment efficiency of exosomes in the bone defect area, but also realizes delayed release, significantly prolongs the biological action time. Through this "CD301b + The combination of the multiple biological functions of the EXO and the targeted delivery ability of the material overcomes the limitations of single activity, complex components, and uncoordinated regulation of existing materials, and provides a new strategy for bone defect repair, which is efficient, stable, and has good clinical transformation prospects. The A-SCH / OSA@EXO microspheres of the present application realize the synergistic effect of material delivery advantage and exosome multifunctionality, effectively cover the whole process of bone repair, and show better repair effect than traditional single active factor or multi-component combination, and have higher clinical transformation value.

[0011] The primary object of the present application is achieved by the following technical solutions: A CD301b + EXO (derived from CD301b + Macrophage exosomes) preparation method, comprising the following steps, (1) CD301b + Preparation of macrophages: RAW264.7 macrophages are cultured in the medium after being stimulated by interleukin-4, the single cell suspension is prepared by collecting the cells, the single cell suspension is mixed with the antibody mixture and incubated at room temperature in the dark, the supernatant is discarded after centrifugation, repeated washing, resuspension, filtration, and sorting to obtain CD301b + Macrophages; (2), CD301b + Preparation of EXO: the CD301b + After culturing the macrophages in the medium after interleukin-4 stimulation, differential centrifugation was sequentially performed in a low-temperature environment, and then the supernatant was taken, the first ultracentrifugation was performed to enrich the exosomes, the obtained precipitate was pre-cooled and resuspended, the second ultracentrifugation was performed, and the obtained precipitate was resuspended to prepare CD301b + EXO, i.e., CD301b + EXO of macrophages; The mass concentration of interleukin-4 in steps (1) and (2) is 20 ng / mL; the medium is high-glucose DMEM complete medium containing 10% (v / v) exosome-free FBS and 1% (v / v) penicillin-streptomycin; The specific steps of sequentially performing differential centrifugation in a low-temperature environment in step (2) are as follows: first, remove living cells by centrifugation at 300-400 x g for 10-15 minutes at 4-10℃, then remove dead cells by centrifugation at 2000-3000 x g for 10-15 minutes, and then remove cell debris by centrifugation at 10000-12000 x g for 30-40 minutes, and collect the supernatant.

[0012] Preferably, the antibody mixture in step (1) is a mixture of APC-labeled anti-mouse F4 / 80 antibody diluted 400 times and PE-labeled anti-mouse CD301b antibody diluted 200 times.

[0013] Preferably, the specific conditions of the first ultracentrifugation and the second ultracentrifugation in step (2) are 4℃-10℃, 100000-120000 x g ultracentrifugation for 70-90 minutes.

[0014] The second object of the application is achieved by the following technical scheme: A CD301b + EXO (CD301b + EXO of macrophages) prepared by the above preparation method.

[0015] The third object of the application is achieved by the following technical scheme: A CD301b + The preparation method of the bone-targeting microspheres (A-SCH / OSA@EXO microspheres) loaded with CD301b (a) Preparation of oxidized sodium alginate (OSA): a sodium periodate aqueous solution with a mass concentration of 0.099 g / mL to 0.12 g / mL is poured into a sodium alginate (SA) anhydrous ethanol solution with a mass concentration of 0.2 g / mL to 0.16 g / mL, and magnetically stirred, dialyzed, and freeze-dried to obtain the oxidized sodium alginate (OSA). (b) Preparation of succinylated chitosan (SCH): chitosan (CH), a methanol solution, and succinic anhydride are sequentially added to a lactic acid aqueous solution, and magnetically stirred at room temperature until uniform, and then NaOH is added dropwise until the pH of the solution is 7.00 to 7.11, and the precipitate is filtered, dissolved in deionized water, dialyzed, and freeze-dried to obtain the succinylated chitosan (SCH), wherein the mass-volume ratio of the chitosan (CH), lactic acid aqueous solution, methanol solution, and succinic anhydride is 0.5 g:44.25 mL:160 mL:150 mg. (c) Synthesis of alendronate-grafted succinylated chitosan (A-SCH): aldehyde-activated alendronate (ALE-CHO) and succinylated chitosan (SCH) are stirred in pure water to obtain a mixture, which is dialyzed, freeze-dried, and obtained as A-SCH, wherein the mass ratio of ALE-CHO to SCH is 50 mg:250 mg. (d) Preparation of bone-targeting microspheres loaded with CD301b + -EXO: the water phase is injected into the microfluidic device at a flow rate of 2 to 4 μL / min, the oil phase is injected into the microfluidic device at a flow rate of 40 to 80 μL / min, and the sodium alginate oxidate (OSA) solution has a mass concentration of 25 to 30 mg / mL; the water phase is a CD301b + -EXO solution; and the oil phase is mineral oil. + -EXO solution; and the oil phase is mineral oil.

[0016] Preferably, the dialysis in step (a) and step (b) is performed for three days, and the dialysis bag used in the dialysis has a molecular weight cut-off of 3500 Da.

[0017] Preferably, the lactic acid aqueous solution in step (b) is a 5% (v / v) lactic acid aqueous solution, and the concentration of NaOH is 8M to 10M.

[0018] Preferably, the alendronate aldehyde (ALE-CHO) in step (c) is prepared by reacting alendronate (ALE) with 50% (v / v) glutaraldehyde at a mass-volume ratio of 1.2 g: 30 mL~35 mL at 45~50°C for 12~16 hours.

[0019] Preferably, the aqueous phase in step (d) contains 0.5 mg / mL CD301b + - A-SCH aqueous solution of EXO, wherein the A-SCH concentration is 10~8 mg / mL; the oil phase is mineral oil containing 5% (v / v) Span 80; the low-temperature crosslinking temperature is 4~8°C, the low-temperature crosslinking time is 12~24 hours, the centrifugal speed is 3000~3500 rpm, and the centrifugal time is 5~10 minutes.

[0020] The fourth object of the present application is achieved by the following technical solution: A bone-targeting microsphere (A-SCH / OSA@EXO microsphere) loaded with CD301b + -EXO.

[0021] The fifth object of the present application is achieved by the following technical solution: A bone-targeting microsphere (A-SCH / OSA@EXO microsphere) loaded with CD301b + application of the bone-targeting microsphere (A-SCH / OSA@EXO microsphere) loaded with CD301b

[0022] The bone-targeting microsphere (A-SCH / OSA@EXO microsphere) loaded with CD301b + The bone-targeting microsphere (A-SCH / OSA@EXO microsphere) loaded with CD301b + The multifunctional characteristics of the bone-targeting microsphere (A-SCH / OSA@EXO microsphere) loaded with CD301b

[0023] The present application has the following advantages and positive effects compared with the prior art: (1) Improve the synergy and reliability of treatment: the present application realizes the synergistic regulation of the whole bone repair process, fundamentally avoiding the complexity of multi-factor compounding; the beneficial effects of the present application are mainly brought by the technical feature of using CD301b + -EXO as the core functional component; CD301b +-EXO naturally inherits the "immune regulation-angiogenesis-osteogenic induction" triple biological function of its parent cells, which can simultaneously regulate the three key stages of inflammation resolution, vascularization and bone formation necessary for bone healing in the form of a single component; compared with the strategy in the prior art that multiple functionally single active factors (such as BMP-2 for osteogenesis, VEGF for angiogenesis, IL-4 for anti-inflammatory) must be physically mixed, the complexity of multiple factor matching, the uncontrollability of interaction between different factors and the difficulty in matching the release kinetics are completely avoided, and the synergy and reliability of the treatment are significantly improved; (2) Intelligent bone targeting delivery: The CD301b + loaded EXO of the application achieves active targeting enrichment of exosomes at bone defect sites by utilizing the specific binding of alendronate sodium and hydroxyapatite, avoiding systemic non-target organ clearance, improving local bioavailability, and actually achieving intelligent bone targeting delivery; (3) Controlled release and stability optimization: The CD301b + loaded EXO of the application is prepared by microfluidic technology to prepare microspheres, achieving uniform particle size and stable dispersion, and at the same time, utilizing the pH-responsive Schiff base bond formed by OSA and A-SCH to achieve intelligent controlled release of exosomes in an acidic microenvironment, prolonging the action time and maintaining biological activity, thereby significantly improving the local retention and function of exosomes; compared with simple sustained release, this environment-responsive release characteristic can better match the dynamic process of bone repair, further improving the repair efficiency; (4) Functional synergy enhancement: The CD301b + loaded EXO of the application is a microsphere three-dimensional structure that not only provides physical support, but also promotes bone defect repair, making CD301b + -EXO further amplified its multiple functions. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 CD301b - -EXO and CD301b + -EXO separation and transcriptomic analysis chart; wherein A is CD301b + macrophage subpopulation fluorescence activated cell sorting (FACS) flowchart; B is a flow cytometry analysis chart of CD301b + expression of RAW264.7 cells after 72 hours of induction with or without IL-4; C is a chart of CD301b- -EXO, CD301b + -Representative TEM images of EXO and M2-EXO; D is CD301b detected by NTA - -EXO, CD301b + -Particle size distribution of EXO and M2-EXO; E is Western blot analysis of RAW264.7 cells (Cells), CD301b - -EXO and CD301b + -Expression of exosome markers (CD63, CD81, CD9 and TSG101) in EXO; F is Western blot analysis of RAW264.7 cells (Cells), M2-EXO and CD301b + -Expression of exosome markers (CD63, CD81, CD9 and TSG101) in EXO; G is CD301b - -EXO and CD301b + -Volcano plot of differentially expressed genes from EXO transcriptome sequencing, where red data points represent up-regulated genes, blue data points represent down-regulated genes, and gray data points represent genes with no significant changes; H is CD301b - -EXO and CD301b + -EXO transcriptome sequencing, GO enrichment analysis chart of significantly up-regulated differentially expressed genes; Figure 2 -CD301b + -Comparison chart of the promoting effect of EXO on macrophage M2 polarization, angiogenesis and osteogenic differentiation in vitro; where A is in LPS-induced RAW264.7 cells, M2-EXO and CD301b + -Representative immunofluorescence images of iNOS (green, M1 marker) and CD206 (red, M2 marker) after EXO treatment, and nuclei were stained with DAPI (blue); B is a quantitative analysis chart of RAW264.7 cell iNOS and CD206 fluorescence intensity; C is the control group, M2-EXO and CD301b + -Representative fluorescence images of Matrigel HUVECs tube formation after EXO treatment; D is a quantitative analysis chart of vascular network parameters; E is the control group, M2-EXO and CD301b + -ALP staining chart of BMSCs after EXO osteogenic induction for 6 days; F is the control group, M2-EXO and CD301b +- Representative ARS staining of mineralized nodules in BMSCs after 12 days of EXO osteoinduction; G is the quantitative analysis of calcium deposition measured by spectrophotometric absorption at 562 nm after ARS dissolution; Figure 3 Figure for preparation and characterization of SCH / OSA microspheres, A-SCH / OSA microspheres and A-SCH / OSA@EXO microspheres; A is the Fourier transform infrared spectroscopy (FT-IR) spectrum of SA and OSA; B is the FT-IR spectrum of CH, SCH and A-SCH; C is the FT-IR spectrum of SCH / OSA microspheres, A-SCH / OSA microspheres and A-SCH / OSA@EXO microspheres; D is the nuclear magnetic resonance (NMR) 31 P spectrum; E is the scanning electron microscopy (SEM) morphology of SCH / OSA microspheres, A-SCH / OSA microspheres and A-SCH / OSA@EXO microspheres; F is the autofluorescence imaging of SCH / OSA microspheres, A-SCH / OSA microspheres and A-SCH / OSA@EXO microspheres under standard DAPI, FITC and TRITC filters; G is the injectability of SCH / OSA microspheres, A-SCH / OSA microspheres and A-SCH / OSA@EXO microspheres; H is the particle size distribution of SCH / OSA microspheres, A-SCH / OSA microspheres and A-SCH / OSA@EXO microspheres; I is the fluorescence imaging of SCH / OSA microspheres, A-SCH / OSA microspheres attached to the surface of hydroxyapatite (HAp) tablets; J is the quantitative analysis of fluorescence intensity corresponding to I; K is the SEM comparison of microspheres loaded with DIR-labeled exosomes (A-SCH / OSA@EXO DIR ) and blank microspheres (A-SCH / OSA) without loading exosomes under cryosections; L is the BSA release kinetics curve of A-SCH / OSA@BSA RBITC microspheres in weakly acidic (pH 5.0) or physiological conditions (pH 7.4) simulated body fluid; Figure 4Figure 1 is a graph of the cell compatibility of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; wherein A is a graph of live / dead cell staining (live cells appear green fluorescence, dead cells appear red fluorescence) after SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres were co-cultured with BMSCs, HUVECs, and RAW264.7 for 3 days; B is a graph of the proliferation activity of BMSCs after being co-cultured with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres for 1 day and 3 days, as detected by CCK-8; C is a graph of the proliferation activity of HUVECs after being co-cultured with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres for 1 day and 3 days, as detected by CCK-8; D is a graph of the proliferation activity of RAW264.7 after being co-cultured with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres for 1 day and 3 days, as detected by CCK-8; Figure 5 Figure 2 is a graph of the verification of the potential of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres in promoting macrophage polarization, angiogenesis, and osteogenic differentiation in vitro; wherein A is a graph of immunofluorescence staining of CD206 (red), iNOS (green), and nuclei (blue) in LPS-induced RAW264.7 cells after treatment with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; B is a graph of quantitative analysis of iNOS and CD206 fluorescence intensity; C is a representative graph of matrigel HUVECs tube formation after treatment with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; D is a quantitative analysis of blood vessel network parameters; E is alizarin red staining (ARS) of calcium deposition in BMSCs after treatment with a control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres for 14 days; F is a quantitative analysis of calcium deposition (absorbance at 562 nm after ARS dissolution); Figure 6The images show the bone repair effects of the control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres in a rat skull defect model. Specifically, A shows representative Micro-CT 3D and 2D reconstructed images of the skull defect areas in each group (n = 5) 10 weeks after implantation of the control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres, with red circles indicating new bone formation; B shows the quantitative statistical analysis of bone mineral density based on Micro-CT data for the control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; and C shows the quantitative statistical analysis of bone volume fraction for the control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres. Figure 7 The images show hematoxylin-eosin staining of major organs (heart, liver, spleen, kidney, and lung) 10 weeks after implantation of control group, SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO microspheres, respectively. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. All materials used in the embodiments of the present invention can be purchased commercially.

[0026] Example 1 1.CD301b - -EXO and CD301b + -Identification and transcriptomic analysis of EXO 1.1 RAW264.7 Cell Culture Mouse macrophage cell line RAW264.7 was purchased from Wuhan Pronosei Biotechnology Co., Ltd., and routinely cultured in high-glucose DMEM complete medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin. It was incubated at 37℃ in a 5% (v / v) CO2 incubator. When the cell confluence reached 70%–80%, it was passaged using the following steps: ① Aspirate the original medium using a Pasteur tube or pipette; ② Gently wash the cells twice with sterile PBS; ③ Add fresh complete medium, gently pipette with a 1 mL pipette tip until the cells are completely detached, and collect the cell suspension; ④ Seed the cell suspension at a 1:5 ratio into new culture dishes for continued culture. For long-term culture, cells were passaged every 48 hours.

[0027] 1.2 CD301b - With CD301b + Macrophage sorting RAW264.7 macrophages were induced for 72 hours with 20 ng / mL IL-4. Cells were cultured in high-glucose DMEM complete medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin. After induction, cells were collected and single-cell suspensions were prepared. A surface marker staining was performed using the following antibodies to form an antibody mixture: APC-labeled anti-mouse F4 / 80 antibody (1:400 dilution) and PE-labeled anti-mouse CD301b antibody (1:200 dilution). The single-cell suspension and antibody mixture were incubated at room temperature in the dark for 30 minutes, centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed once more. Finally, the cells were resuspended in 500 μL PBS and filtered through a 40 μm cell sieve into flow cytometry tubes. Figure 1 A and Figure 1 As indicated by B in the data, CD301b was successfully sorted using a BD FACSAria II flow cytometer. - With CD301b + Macrophage subsets, quantitative analysis showed that CD301b was present in the IL-4-induced macrophage population. + The percentage of positive cells reached 40.7%.

[0028] 1.3 Preparation of macrophage-derived exosomes CD301b - With CD301b + Macrophages and IL-4 (20 ng / mL)-stimulated RAW264.7 cells were cultured for 72 hours in high-glucose DMEM medium containing 10% (v / v) exosome-free FBS and 1% (v / v) penicillin-streptomycin. Conditioned media from each cell type were collected and centrifuged separately using the following methods: 300–400 ×g for 10–15 minutes at 4°C to remove live cells, 2000–3000 ×g for 10–15 minutes to remove dead cells, and 10000–12000 ×g for 30–40 minutes to remove cell debris. The supernatant was then ultracentrifuged at 100000–120000 ×g for 70–90 minutes at 4°C–10°C to enrich exosomes. The resulting precipitate was resuspended in pre-chilled PBS and washed again by centrifugation at 100000–120000 ×g for 70–90 minutes. The final precipitate was resuspended in PBS, and the three groups of exosomes obtained were: CD301b - Macrophage-derived exosomes (CD301b) - -EXO), CD301b + Macrophage-derived exosomes (CD301b)+ The exosomes derived from M2 macrophages (M2-EXO) and M2-type macrophages (M2-EXO) were aliquoted and stored at -80°C for later use.

[0029] 1.4 EXO identification 1.4.1 Observation of Ultrastructure Take 10 μL of freshly extracted CD301b + -EXO, CD301b - -EXO and M2-EXO were dropped onto a clean sealing film surface. A 200-mesh copper mesh (carbon film side down) was gently placed on the droplet and allowed to absorb at room temperature for 2 minutes. Residual liquid was slowly absorbed from the edge of the mesh using filter paper. The sample was then transferred to a 2% (v / v) uranium acetate staining solution for negative staining for 2 minutes. Excess staining was removed again, and the sample was dried at room temperature for 10 minutes. After the sample was completely dry, the morphological characteristics of exosomes were observed using a transmission electron microscope (TEM) at an accelerating voltage of 80 kV. At least five different fields of view were observed for each sample to ensure representativeness of the results. Figure 1 As shown in C, CD301b + -EXO, CD301b - Both -EXO and M2-EXO exhibit a typical cup-shaped morphology and a clear bilayer lipid membrane structure.

[0030] 1.4.2 Particle Size Analysis Detection of CD301b using nanoparticle tracking analysis (NTA) + -EXO, CD301b - Particle size distribution of -EXO and M2-EXO. The three EXO suspensions were diluted to 1 mL with PBS, mixed, and equilibrated at room temperature for 2 minutes. Samples were injected into the detection cell at a constant flow rate (30 μL / min) using a microinjection pump. Three videos (60 seconds / video) were captured for each sample, and particle size distribution characteristics were analyzed using a NanoSight NS300 system. Figure 1 As shown in D, the NTA results indicate that CD301b + -EXO, CD301b - -EXO and M2-EXO have diameters ranging from 70 to 200 nm, with the main peak at approximately 100 nm.

[0031] 1.4.3 Surface Marker Detection To confirm that the extracted substance was EXO, Western blotting (WB) was used to detect the expression of EXO-specific surface markers CD9, CD63, CD81, and TSG101. Figure 1 E and Figure 1As shown in F, all three EXO strains stably express the EXO marker proteins CD9, CD63, CD81, and TSG101.

[0032] 1.5 CD301b - -EXO and CD301b + -EXO RNA sequencing analysis To identify CD301b - -EXO and CD301b + Differential expression of RNA transcripts among the EXO groups was determined by whole-genome transcriptional sequencing performed by Beijing Aovisen Gene Technology Co., Ltd. RNA was extracted using the TRIzol method and treated with RNase-free DNase I. RNA degradation and contamination were monitored on 1% (w / v) agarose gels. RNA was quantified using an Agilent 2100 Bioanalyzer, and its quality and integrity were assessed using a NanoDrop spectrophotometer. Sequencing libraries were then constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® according to the manufacturer's recommendations, and library fragments were purified using the AMPure XP system. Finally, PCR products were purified using the AMPure XP system, library quality was assessed on an Agilent Bioanalyzer 2100 system, and sequencing was performed using the Illumina NovaSeq 6000 platform.

[0033] The sequencing data were aligned to the reference genome sequence using STAR software. The number of reads aligned to each gene was counted using HTSeq. Gene expression levels were estimated using the number of comparable fragments per million kilobase transcripts. Differentially expressed genes (DEGs) were analyzed between the two conditions / groups using the DESeq R package. A corrected p-value (q-value) < 0.05 and |log2 (fold change)| ≥ 1 were set as the threshold for significant differential expression. Based on the Wallenius noncentrocentric hypergeometric distribution, gene ontology (GO) enrichment analysis of differentially expressed genes was performed using the GOseq R package. Simultaneously, the significance of enrichment of differentially expressed genes in the KEGG pathway was tested using KOBAS software. Figure 1 The G results show that, compared with CD301b - Compared to EXO, CD301b +-EXO revealed a significant differential gene expression profile, identifying 8,909 significantly upregulated genes and 2,469 significantly downregulated genes. For example... Figure 1 As shown in H, functional enrichment analysis of differentially expressed genes using Gene Ontology (GO) revealed that CD301b... + -EXO's functional genes are mainly enriched in three key pathways: "macrophage activation", "angiogenesis regulation" and "positive regulation of osteogenic differentiation", suggesting that it may play multiple roles in coordinating immune regulation, promoting angiogenesis and inducing bone regeneration.

[0034] Example 2 2.1 Immunofluorescence staining detection of CD301b + -Expression of RAW264.7 macrophage polarization-related proteins by EXO The immune microenvironment plays a crucial role in bone regeneration. To investigate CD301b... + -EXO's immunomodulatory function, causing RAW264.7 cells to be at a rate of 5 × 10⁻⁶ 5 RAW264.7 cells were seeded at a density of 1 cell / well onto sterile glass slides in 24-well plates. After cell adhesion, the cells were stimulated with LPS (100 ng / mL) for 12 hours to establish an inflammation model. Subsequently, M2-EXO (40 μg / mL) or CD301b was added. + -EXO (40 μg / mL), with cells cultured in conventional tissue culture plates as a blank control. After 2 days of incubation, cells were fixed with 4% (v / v) paraformaldehyde for 30 min, gently washed twice with PBS, permeabilized with 0.1% Triton X-100 for 20 min, washed with PBS, and blocked with 1% bovine serum albumin for 30 min. Cells were incubated overnight at 4°C with anti-iNOS (1:200) and anti-CD206 (1:200) primary antibodies, respectively. After elution with PBS, cells were incubated for 40 min with a mixture of fluorescent secondary antibodies Alexa Fluor 488 (1:200) and Alexa Fluor 568 phalloidin (1:40). Finally, the cells were mounted with a DAPI-containing antifluorescence quencher, and images were acquired and observed using a confocal fluorescence microscope. The expression levels of iNOS and CD206 were quantitatively analyzed using ImageJ software. Figure 2 A in and Figure 2 The B section shows that, compared to the M2-EXO group, CD301b + -EXO exhibits a stronger dual regulatory capacity: it not only significantly inhibits the expression of the M1 phenotypic marker inducible nitric oxide synthase (iNOS), but also enhances the fluorescence intensity of the M2 phenotypic marker CD206.

[0035] 2.2 Detection of CD301b in matrix gelation tube formation experiment + -EXO promotes angiogenesis in HUVECs To investigate CD301b + The regulatory effect of EXO on angiogenesis was evaluated in this study using a matrix gel in vitro tube formation assay. First, the matrix gel was placed in a 4°C freezer overnight to allow for complete liquefaction. Simultaneously, pipettes, pipette tips, and 48-well plates were pre-chilled to ensure the matrix gel remained liquid during the procedure. The matrix gel was diluted 2:1 with high-glucose DMEM basal medium, and 130 μL of the diluted matrix gel was added to each well of the pre-chilled 48-well plate. The plate was kept on ice to ensure uniform spreading, and then transferred to a 37°C incubator for 30 minutes to 1 hour to allow the matrix gel to completely solidify. HUVECs were routinely digested, centrifuged, and resuspended in high-glucose DMEM basal medium at 1.6 × 10⁻⁶ cells / well. 5 Inoculate the cells onto the solidified matrix gel at a density of cells / well, and simultaneously add 40 μg / mL of M2-EXO or CD301b. + -EXO was used to treat cells, with the untreated group serving as a control. After incubating the plates at 37°C in a 5% (v / v) CO2 incubator for 6 hours, the culture medium was carefully aspirated, and Calcein AM working solution was added for staining in the dark for 15 minutes. After washing with high-glucose DMEM medium, cell morphology was observed under a 10x fluorescence microscope, and three fields of view were randomly selected for photographic recording. Finally, ImageJ software with an angiogenesis analysis plugin was used to quantitatively analyze parameters such as the number of luminal junctions, the number of luminal grids, and the number of branch points to evaluate the effect of different treatment groups on the angiogenesis ability of hUVECs. Figure 2 As shown in C, CD301b + -EXO significantly enhances the complexity of the tubular networks formed by HUVECs. For example... Figure 2 Quantitative analysis of D in the study confirmed that key parameters of vascular structure, including the total number of connections, average vascular area, and average vascular length, were significantly improved.

[0036] 2.3. CD301b + Effects of EXO on osteogenic differentiation of BMSCs 2.3.1 ALP staining BMSCs were divided into 5 × 10 4 Cells were seeded at a density of 10 cells / well in 12-well plates. When cell confluence reached 80%, the medium was replaced with osteogenic induction medium (OIM). Cells were randomly divided into three groups: control group, 40 μg / mL M2-EXO group, and 40 μg / mL CD301b group. +- EXO group: Change culture medium and EXO every 3 days. After 5-6 days of induction, wash cells twice with PBS, fix with 4% (v / v) paraformaldehyde for 15 minutes, prepare working solution according to the BCIP / NBT alkaline phosphatase chromogenic kit instructions, stain at room temperature in the dark for 30 minutes, then stop the reaction with distilled water, and observe and photograph under an inverted microscope. Figure 2 As shown in E, CD301b + -EXO induced ALP staining intensity higher than M2-EXO, indicating that it has a stronger ability to initiate early osteogenic processes.

[0037] 2.3.2 Alizarin Red Staining BMSCs were divided into 5 × 10 4 Cells were seeded at a density of 10 cells / well in 12-well plates, and replaced with OIM when cell confluence reached 80%. Cells were randomly assigned to three groups: control, 40 μg / mL M2-EXO, and 40 μg / mL LCD301b. + - EXO group: Culture medium and EXO were changed every 3 days. After 14 days of induction, the nodules were washed with PBS, fixed with 4% (v / v) paraformaldehyde for 15 minutes, incubated with Alizarin Red S staining solution at room temperature for 15 minutes, washed with PBS, and observed under an inverted fluorescence microscope. The stained nodules were then dissolved with 10% (w / v) cetylpyridinium chloride (CPC), and the absorbance was measured at 562 nm using a multi-mode microplate reader for quantitative analysis. Figure 2 As shown in F, ARS staining results indicate that the formation of mineralized nodules gradually increased in each group, in the following order: control group <M2-EXO<CD301b + -EXO. (e.g.) Figure 2 The quantitative analysis of G further validated this trend.

[0038] Example 3 3. Preparation and characterization of microspheres 3.1 Preparation of microspheres 3.1.1 OSA Preparation Partially oxidized OSA was prepared by oxidation with sodium periodate: 4 g of SA was weighed into a beaker containing 20-25 mL of anhydrous ethanol and stirred thoroughly for 20-30 minutes. 1.98 g-2.40 g of sodium periodate was added to a beaker containing 20 mL of deionized water and stirred thoroughly for 20-30 minutes. Then, the dissolved sodium periodate was poured into 20 mL of SA in anhydrous ethanol and magnetically stirred for 6 hours. Finally, the solution was dialyzed against deionized water for three days (dialysis bag molecular weight cutoff: 3500 Da), with the water changed at least twice daily. The lyophilized liquid was then used as OSA. SA and OSA were characterized by FT-IR. Figure 3 As shown in A, the OSA sample was at 1725 cm⁻¹. -1 A weaker aldehyde characteristic peak appears at this location.

[0039] 3.1.2 Preparation of SCH Preparation of 5% (v / v) lactic acid: Weigh 40 mL of deionized water into a beaker using a graduated cylinder, add 4.25 mL of 85% lactic acid, and stir magnetically for 5 minutes. Weigh 0.5 g of chitosan (CH), add 40 mL of the well-mixed 5% (v / v) lactic acid aqueous solution, and stir magnetically for 4 hours. Then add 160 mL of methanol solution and continue stirring for 20-30 minutes to fully disperse the chitosan in the methanol solution. Add 150 mg of succinic anhydride and stir rapidly magnetically at room temperature for 24 hours. Then, while stirring, add 8M-10M NaOH dropwise to adjust the pH of the solution to 7.00-7.11. During this process, a precipitate will gradually form. Then, filter the liquid through filter paper. After filtration, transfer the precipitate on the filter paper to a 200 mL beaker, add 100 mL of deionized water, and stir magnetically at 800 rpm overnight until the precipitate dissolves. Finally, the solution was dialyzed with deionized water for three days (dialysis bag molecular weight cutoff: 3500 Da), with the water changed at least twice a day. The dialyzed liquid was lyophilized to obtain SCH. CH and SCH were characterized by FTIR. Figure 3 As shown in B, the amide I band (C=O stretching vibration, 1647 cm⁻¹) is clearly visible in the FT-IR spectrum. -1 ) and amide II band (NH bending vibration, 1554 cm -1 The characteristic peaks confirmed the successful acylation reaction. It is worth noting that, as... Figure 3 As shown in B, the A-SCH sample at 3530 cm⁻¹ -1 The presence of a significant NH stretching vibration peak further corroborates the formation of amide bonds.

[0040] 3.1.3 Synthesis of A-SCH Weigh 1.2 g of alendronate (ALE) and dissolve it in 30 mL–35 mL of 50% (v / v) glutaraldehyde solution. Stir the mixture at 45–50 °C for 12–16 hours. After the reaction, allow the mixture to stand for 20 minutes–1 hour. Wash the product with a large amount of cold acetone to obtain a brown precipitate. Dry the precipitate thoroughly in a ventilated environment to obtain aldehyde-activated ALE (ALE-CHO). Separately, under stirring, dissolve 250 mg of SCH in 150 mL of ultrapure water until completely dissolved. Then add 50 mg of ALE-CHO and stir continuously at 37 °C for 12 hours. Dialyze the resulting mixture to deionized water (molecular weight cutoff: 3500 Da) for 72 hours, then freeze-dry to obtain yellow porous A-SCH, which is stored at 4 °C. Nuclear magnetic resonance (NMR) was used to analyze the precipitate. 31 P-spectroscopy characterizes whether bisphosphonates have been grafted onto A-SCH. For example... Figure 3 As shown in D, both ALE and A-SCH exhibit characteristic chemical shifts at 17.6 ppm, directly proving that a covalent bond has formed between ALE and SCH.

[0041] 3.1.4 Preparation of A-SCH / OSA@EXO microspheres Monodisperse hydrogel microspheres were prepared using a microfluidic device. Aqueous phases (8–10 mg / mL SCH, 8–10 mg / mL LA-SCH, or containing 0.5 mg / mL CD301b) were used. + The oil phase (8–10 mg / mL A-SCH from EXO) was injected into the microfluidic chip at controlled flow rates of 2–4 μL / min and 40–80 μL / min, respectively. This caused the aqueous phase to form monodisperse spherical droplets under the shear force of the oil phase. The resulting emulsion droplets were collected in a 25–30 mg / mL OSA solution and crosslinked in a shaker at 4 °C for 12 hours. The microspheres were washed sequentially with anhydrous ethanol (to remove residual oil) and rinsed three times with deionized water. After centrifugation (3000–3500 rpm, 5–10 min), the purified microspheres (SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO) were stored at -20 °C to 80 °C for subsequent research.

[0042] 3.2 Testing of the physicochemical properties of the microspheres 3.2.1 FT-IR Detection of Microspheres Samples were prepared using the potassium bromide tableting method: lyophilized microspheres were mixed with potassium bromide at a ratio of 1:100 (w / w), thoroughly ground into a uniform and fine powder, then tableted. FT-IR was used for analysis of the chemical composition and functional group characteristics of the microspheres.Figure 3 As shown in C, all microsphere samples were at 1643 cm⁻¹. -1 The peaks at all locations showed obvious characteristic absorption peaks of imine bonds (-C=N-), confirming that intermolecular crosslinking was achieved through the Schiff base reaction.

[0043] 3.2.2 Morphological observation of microspheres The prepared SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO microsphere samples were resuspended in deionized water. A small amount of the dispersed microsphere solution was dropped onto a circular glass slide, air-dried at room temperature, and then sputtered with gold. The surface morphology and structural characteristics of the microspheres were then observed using a scanning electron microscope (SEM). Figure 3 The E results showed that the prepared microspheres had smooth surfaces, regular morphology, and uniform particle size distribution.

[0044] 3.2.3 Detection of microsphere particle size distribution and fluorescence properties Three groups of microsphere samples—SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO—were resuspended in deionized water. Small amounts of the dispersion were then added dropwise to 24-well plates, and their fluorescence characteristics were observed using an inverted fluorescence microscope. Three standard filters—DAPI, FITC, and TRITC—were used sequentially to record microsphere images under different fluorescence channels. NanoMeasure software was used to perform particle size statistical analysis on ≥200 microspheres in each group, and Origin software was used to plot the microsphere size distribution. Fluorescence microscopy confirmed that these microspheres were regularly spherical and possessed autofluorescence properties; for example... Figure 3 As shown in Figure F, under DAPI, FITC, and TRITC filters, it emits blue, green, and red fluorescence, respectively. It is worth noting that, as... Figure 3 As shown in Figure G, all microspheres can be successfully injected through a 32G fine-bore injection needle (110μm inner diameter) without clogging, and the structure remains intact after injection.

[0045] like Figure 3 As shown in Figure H, particle size statistical analysis using Nano Measure software revealed that the average diameters of the three microspheres—SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO—were 59 ± 9.5 μm, 53 ± 10.4 μm, and 53 ± 7.4 μm, respectively.

[0046] 3.2.4 In vitro affinity experiment with hydroxyapatite to verify the targeting ability of A-SCH / OSA microspheres The in vitro binding capacity of microspheres was evaluated using a hydroxyapatite (HAp) affinity assay. The procedure was briefly described as follows: One HAp disc (11 ± 1 mm in diameter, 3 ± 0.5 mm in thickness) was placed in each well of a 24-well plate. Equal volumes of prepared SCH microspheres (untargeted) and A-SCH microspheres (targeted) were dispersed separately in 0.5 mL of 0.9% (w / v) NaCl solution and added to the wells containing the HAp discs (three parallel wells per sample). The 24-well plate was incubated on a shaker. At 4, 16, and 24 hours, the HAp discs were removed from the wells and gently rinsed with 0.9% (w / v) NaCl solution to remove unbound microspheres. Subsequently, the microspheres adsorbed on the HAp discs were observed and images were acquired using an inverted fluorescence microscope. Finally, the images were analyzed quantitatively using ImageJ software. The bone-binding properties of A-SCH / OSA microspheres were systematically evaluated using a fluorescence tracer method. The experimental group (targeted A-SCH / OSA microspheres) and the control group (non-targeted SCH / OSA microspheres) were co-incubated with HAp tablets for 4 hours, 14 hours, and 24 hours, respectively. Observation under an inverted fluorescence microscope showed that... Figure 3 As shown in Figure I, A-SCH / OSA microspheres exhibit time-dependent fluorescence enrichment on the HAp surface, with significantly higher fluorescence intensity than the control group. Figure 4 Further quantitative verification using fluorescence analysis showed that the HAP binding capacity of A-SCH / OSA microspheres at 4 hours, 14 hours, and 24 hours was 4.5 times, 10 times, and 16 times that of the SCH / OSA microsphere group, respectively. P <0.001).

[0047] 3.2.5 Encapsulation Verification Experiment of EXO in Microspheres To verify CD301b + EXO was successfully encapsulated within A-SCH / OSA@EXO composite microspheres. First, exosomes were labeled with DIR near-infrared fluorescent dye. The specific steps were as follows: DIR dye working solution was added to the EXO suspension to adjust the final concentration to 10 μM. After vortexing and mixing, the mixture was incubated at 37°C in the dark for 30 minutes. After incubation, the mixture was diluted with PBS buffer and purified by ultracentrifugation (100,000–120,000 × g, 70–90 minutes) to remove unbound free dye. The precipitate was collected and resuspended in 200 μL of PBS to obtain DIR-labeled CD301b. + -EXO (EXO) DIR (For backup). Microfluidic technology is used to deliver EXO... DIRA-SCH / OSA@EXO was prepared by encapsulating it into A-SCH / OSA microspheres. DIR Microspheres were then sliced ​​into 5.0 μm thick samples using cryosectioning. CD301b was observed using SEM. + The distribution of -EXO within the microspheres. The results are shown in Figure 3, K, for A-SCH / OSA@EXO. DIR The microspheres have relatively rough cross-sections, forming granular aggregates, while the cross-sections of the A-SCH / OSA microsphere group are smoother. These experiments demonstrate from multiple perspectives that EXO was successfully encapsulated within the microspheres.

[0048] 3.2.6 EXO sustained-release experiment within microspheres To track CD301b in A-SCH / OSA@EXO microspheres + - The release characteristics of EXO were studied using rhodamine B isothiocyanate-labeled bovine serum albumin (BSA). RBITC As a tracer molecule to replace CD301b + -EXO, preparation of A-SCH / OSA@BSA RBITC Microspheres. Aliquots of microspheres were placed into dialysis bags (molecular weight cutoff: 3500 Da) containing 2 mL of simulated body fluid (SBF) at pH 7.4 or pH 5. The dialysis system was immersed in 45 mL of release buffer at the same pH and continuously agitated (1000 rpm / min, 37°C). At predetermined time intervals (days 1, 3, 5, 7, 9, 11, 13, and 15), 1 mL of external release buffer was collected, and the same volume of fresh buffer was added. The collected buffer was quantified using a multi-plate reader (excitation wavelength: 560 nm, emission wavelength: 600 nm). Based on a pre-established BSA... RBITC Standard curve to determine the BSA released by microspheres RBITC Concentration, and denoted as C. n Then calculate BSA using the following formula (1). RBITC The cumulative release amount.

[0049] Cumulative release amount = (1) : is the sum of the concentrations of all previous samples.

[0050] Example 4 4. Biocompatibility testing of microspheres 4.1 Live and Dead Staining BMSCs (2×10 3 (number / well), HUVECs (3×10)3 Cells per well) and RAW264.7 cells (5 × 10⁻⁶ cells / well) 3 Cells were seeded in the lower chamber of 24-well Transwell plates (8 μm pores) and cultured. After cell adhesion, three types of sterile microspheres (SCH / O, A-SCH / O, and A-SCH / O@EXO, 0.5 mg / well) were placed in the upper chamber of the Transwell plate. On day 3, live / dead cell staining was performed using the Calcein-AM / PI kit. 2 μL of PI and 0.5 μL of Calcein AM were added to 1 mL of culture medium to prepare the staining working solution. The 24-well plate medium was discarded, and the cells in the lower chamber were gently washed twice with PBS. 300 μL of the staining working solution was added, and the plates were incubated at 37°C in the dark for 30 minutes. Images were then observed and acquired using an inverted fluorescence microscope. Calcein AM labeled live cells (green fluorescence), and PI labeled dead cells (red fluorescence). Figure 4 The results of live / dead staining showed that after BMSCs, HUVECs and RAW264.7 cells were co-cultured with microspheres for 3 days, the proportion of live cells (green fluorescence) exceeded 95%, with only sporadic red fluorescence signals (dead cells).

[0051] 4.2 CCK-8 test BMSCs (2×10 3 (number / well), HUVECs (3×10) 3 Cells per well) and RAW264.7 cells (5 × 10⁻⁶ cells / well) 3 Cells were seeded in the lower chamber of 24-well Transwell chambers (8 μm pores) and cultured. After cell adhesion, three types of sterile microspheres (SCH / O, A-SCH / O, and A-SCH / O@EXO, 0.5 mg / well) were placed in the upper chamber of the Transwell chamber. After 1 and 3 days of culture, CCK-8 working solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance of each well was then measured at 450 nm. The results showed that the proliferation of all cell types was time-dependent. Notably, as shown in Figure 4, B... Figure 4 C in Figure 5 As shown in D, on day 3, both A-SCH / OSA and A-SCH / OSA@EXO microspheres significantly promoted the proliferation of RAW264.7 cells, while no significant difference was observed in the proliferation levels of BMSCs and HUVECs among the groups.

[0052] Example 5 5. Validation of the potential of A-SCH / OSA@EXO microspheres to promote macrophage polarization, angiogenesis, and osteogenic differentiation in vitro. 5.1 Effects of microspheres on RAW264.7 macrophage polarization RAW264.7 cells were cultured at a rate of 5 × 10⁻⁶. 5 Cells were seeded overnight on sterile slides in 24-well plates at a density of cells / well and induced to polarize to the M1 phenotype with 100 ng / mL lipopolysaccharide (LPS) for 12 hours. Then, various microspheres (SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO, at a concentration of 0.5 mg / well) were placed in the Transwell chambers of each well. Untreated cells served as a control. After 48 hours of culture, cells were fixed with 4% (v / v) paraformaldehyde, permeated with 0.1% Triton X-100 for 30 minutes, and blocked with 10% goat serum to prevent nonspecific staining. Cells were then incubated overnight at 4°C with primary antibodies: anti-iNOS (1:200) or anti-CD206 (1:200). After elution, the cells were incubated with either Alexa Fluor 488-labeled goat anti-rabbit IgG (1:400) or Alexa Fluor 568-labeled goat anti-rabbit IgG (1:400) and DAPI for nuclear labeling. Images were acquired using CLSM and analyzed using ImageJ software. Figure 5 Immunofluorescence analysis showed that LPS stimulation significantly induced intracellular high expression of the M1 biomarker CD86, while all microsphere groups effectively inhibited CD86 expression, with the A-SCH / OSA@EXO group showing the most significant effect. Notably, compared to the low expression of the M2 biomarker CD206 in the LPS group, all three microspheres induced specific high expression of CD206 in the perinuclear region, with the A-SCH / OSA@EXO group exhibiting the most significant fluorescence enhancement effect. Figure 5 The results of quantitative fluorescence analysis of B in the image were completely consistent with the image observation.

[0053] 5.2 Effects of microspheres on angiogenesis in HUVECs cells The effect of microspheres on the in vitro lumen-forming ability of HUVECs was evaluated using a Transwell co-culture system. The simplified procedure was as follows: 250 μL of liquid matrix gel was added to the lower chamber of a pre-cooled 24-well Transwell plate and allowed to solidify completely at 37 °C for 30 minutes. HUVECs were then routinely digested, centrifuged, and cultured at 3 × 10⁻⁶ microspheres. 5The density of the pores was resuspended in serum-free medium and evenly seeded on the surface of the solidified Matrigel. Subsequently, microspheres (SCH / OSA, A-SCH / OSA or A-SCH / OSA@EXO, with the amount of microspheres being 0.5 mg / well respectively) suspended in serum-free DMEM were added to the upper chamber of Transwell and co-cultured with HUVECs in the lower chamber for 4 - 6 hours. After the culture, the cells were stained with calcein-AM and the lumen structure was observed by an inverted fluorescence microscope. Quantitative analysis was performed using AngioTool software.

[0054] As Figure 5 The results in C showed that compared with SCH / OSA microspheres, the A-SCH / OSA group significantly enhanced the tube formation ability of HUVECs. Notably, as shown in D of Figure 5 , the A-SCH / OSA@EXO microsphere group exhibited the most superior angiogenesis-promoting performance, characterized by a dense and branched tubular network, with a significant upregulation of the total number of connection points, the percentage of vascular area and the average vascular length compared with other experimental groups.

[0055] 5.3 Effect of microspheres on the osteogenic differentiation ability of BMSCs BMSCs were seeded in 12-well plates at a density of 5×10 4 cells / well. When the cell confluence reached 80%, the medium was changed to osteogenic induction medium. The wells were randomly divided into four groups: (1) OIM as the control group; (2) SCH / OSA microsphere group; (3) A-SCH / OSA microsphere group; (4) A-SCH / OSA@EXO. After 12 - 14 days of induction, alizarin red S (ARS) staining was used to evaluate mineralization. The cells were fixed and then incubated with ARS solution for 15 minutes. The stained samples were observed under an inverted fluorescence microscope. To quantify calcium deposition, the ARS-stained nodules were dissolved in 10% (w / v) cetylpyridinium chloride and the absorbance was measured at 562 nm using a multifunctional microplate reader. As shown in the alizarin red staining results in E of Figure 5 , compared with the control group, each microsphere group significantly promoted the formation of mineralized nodules in BMSCs. And the staining area and intensity showed a gradient enhancement trend: SCH / OSA < A-SCH / OSA < A-SCH / OSA@EXO. Notably, the A-SCH / OSA@EXO group had the darkest staining, and dense dark red calcium deposition patches were visible around the microspheres. As shown in the quantitative analysis in F of Figure 6 , the amount of calcium salt deposition in the A-SCH / OSA@EXO group was significantly higher than that in the other groups.

[0056] Example 6 6. Effect of A-SCH / OSA@EXO microspheres on the repair of rat calvarial defects 6.1 Animal Purchase and Care Twenty-four SPF-grade female SD rats, 6-8 weeks old and weighing 260-300 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Certificate No.: 210726220102060432). The SD rats were fed under a 12-hour light / dark cycle, with unrestricted water and food, and the temperature was maintained at 23 ± 2℃. All animals were housed in the experimental animal facility for at least one week prior to the experiment to acclimatize. All animal studies were approved by the Animal Ethics Committee of the Affiliated Hospital of Guangdong Medical University (Approval No.: AHGDMU-LAC-B-202406-0039) and conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals".

[0057] 6.2 Rat skull defect modeling and microsphere implantation ① Material preparation Microspheres were prepared using a microfluidic method. The microspheres were immersed in a 75% (v / v) ethanol solution for 5 min, and then repeatedly washed with excess PBS to remove residual ethanol before use. ② Surgical procedure SD rats were fasted for 12 hours prior to surgery and then anesthetized with intraperitoneal injection of afodin (300 mg / kg body weight). The rats were placed prone on the operating table, and skin preparation, routine disinfection, and draping were performed. An incision was made along the midline of the skull, the skin was cut open, and the skin and subcutaneous tissue were dissected layer by layer to reach the periosteum, clearly exposing the flattest part of the midline of the parietal bone. A circular defect with a diameter of 5.0 mm and a thickness of 1.0 mm was drilled using a trephine. Care was taken during drilling to avoid damaging the dura mater and brain tissue. During the operation, physiological saline was dripped into the surgical area using a syringe to prevent tissue damage due to overheating of the trephine. Twenty-four rats were randomly divided into four groups: A: 6 rats served as a blank control, without implantation of any material; B: 6 rats implanted with SCH / OSA microspheres; C: 6 rats implanted with A-SCH / OSA microspheres; D: 6 rats implanted with A-SCH / OSA@EXO microspheres. After establishing the rat skull defect model, sterilized microspheres were immediately implanted into the bone defects of the rats according to the above-mentioned grouping to fill the defect area. After confirming proper placement of the microspheres and the absence of active bleeding, the incision was sutured layer by layer. After closing the surgical incision, it was disinfected with povidone-iodine, and the rats were intramuscularly injected with 200,000 units of penicillin sodium. The rats were placed in a warm, dry place and allowed free access to water for 2 hours after fully waking up. They were allowed free access to food on the first day after surgery. Ten weeks post-surgery, rat skull, heart, liver, spleen, lung, and kidney tissues were collected, fixed in 4% (v / v) paraformaldehyde solution for 48 hours, and then transferred to 75% (v / v) ethanol for subsequent experiments.

[0058] 6.3 Micro-CT Analysis The fixed rat skull specimen was removed and scanned using Micro-CT with the following parameters: slice thickness: 48 μm, slice interval: 48 μm, pixel size: 48 μm, and mode: high voltage. 3D reconstruction of the defect area was performed using CT Analyser Version 1.13 software, and parameters such as total tissue volume (TV) / bone volume (BV) and bone mineral density (BMD) of the defect area were analyzed. Figure 6 As shown in Figure A, the blank control group showed only a small amount of new bone tissue at the periphery of the defect area, while the central area still exhibited significant bone defects, indicating that bone defects are difficult to heal spontaneously without external intervention. All experimental groups showed varying degrees of bone repair effects. While the SCH / OSA group showed a significant increase in bone mineral density (BMD) compared to the control group, its bone volume fraction (BV / TV) only showed a non-significant increase. Notably, the A-SCH / OSA group and the A-SCH / OSA@EXO group showed significantly improved repair efficacy: [Example data would be inserted here]. Figure 6 B in Figure 7 Quantitative analysis of C showed that the BMD and BV / TV values ​​of both groups were significantly higher than those of the SCH / OSA group, and the A-SCH / OSA@EXO microsphere group showed the best repair effect, with BMD reaching 0.36±0.07 g / cm³ (an increase of 23.5% compared with the A-SCH / OSA group) and BV / TV reaching 31.5±3.5% (an increase of 13.5% compared with the A-SCH / OSA group), demonstrating a significant osteogenic promoting effect.

[0059] 6.4 Specimen decalcification and paraffin embedding Rat skull, heart, liver, spleen, lung, and kidney tissues were placed in an automated tissue dehydrator and sequentially immersed in ethanol solutions with volume fractions of 75.0%–30 min, 75.0%–1 h, 85.0%–3 h 30 min, 85.0%–1 h, 95.0%–1 h, 95.0%–4 h, 100.0%–30 min, and 100.0%–30 min for gradient dehydration. The samples were then immersed in xylene II (100% xylene) for 17 min, xylene II for 17 min, wax for 20 min, and wax for 1 h 30 min. The samples were then embedded in an embedding machine.

[0060] 6.5 HE staining Sectioning: Paraffin-embedded visceral tissue blocks were transversely sectioned into 5.0 μm thicknesses, unfolded in a 42℃ water bath, and mounted onto glass slides. The slides were then baked in a 60℃ incubator for 2 hours. Dewaxing and hydration: Sections were dewaxed by immersing each section in xylene I and xylene II for 15 minutes each, followed by rehydration in 100.0%, 100.0%, 95.0%, 85.0%, and 75.0% ethanol solutions and deionized water for 5 minutes each. Cell nuclei were stained with hematoxylin for 10 minutes, washed with deionized water for 5 minutes, differentiated with 1% hydrochloric acid alcohol for 3 seconds, and rinsed with running water for 10 minutes. Cytoplasm was stained with eosin for 30 minutes. The tissues were immersed for 5 seconds in 75.0%, 85.0%, 95.0%, and 95.0% ethanol solutions (v / v), 2 minutes each in 100.0% ethanol I and 100.0% ethanol II, and 2 minutes each in xylene I and xylene II. The tissues were then mounted with neutral resin and photographed under an optical microscope. Histological examinations were performed on major organs (including the heart, lungs, liver, spleen, and kidneys). ​ As shown, H&E staining revealed no significant histological changes or inflammatory lesions in all groups, confirming the excellent biocompatibility of the various microspheres.

[0061] Based on the above examples, the following conclusions can be drawn: (1) The experimental system can stably separate and enrich functional CD301b. + -EXO, whose morphology, particle size and marker proteins conform to the characteristics of exosomes, and has a clear expression profile related to immune / angiogenic / osteogenic functions (transcriptomics evidence).

[0062] (2) CD301b + -EXO promotes macrophage polarization toward reparative M2, enhances tube formation of human umbilical vein endothelial cells (HUVECs), and significantly promotes early (ALP) and late (mineralized nodule) osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs).

[0063] (3) The constructed payload CD301b + EXO's bone-targeting microspheres (A-SCH / OSA@EXO microspheres) have uniform particle size, good dispersibility and bone affinity (can specifically bind hydroxyapatite), and exhibit pH responsiveness—accelerating the release of exosomes in acidic (inflammatory) environments, thereby achieving targeted and environment-triggered release at damaged sites.

[0064] (4) Consistent in vitro and in vivo evidence: CD301b +-EXO loaded into bone-targeting microspheres (A-SCH / OSA@EXO) showed significantly better synergistic therapeutic effects than the control / single-component approach in terms of immune regulation (increasing CD206 and decreasing CD86), angiogenesis, and osteogenic effects. Ultimately, it achieved optimal bone defect repair in a rat skull defect model (highest BMD and BV / TV on Micro-CT, and histological findings showed a more dense and orderly bone matrix).

[0065] (5) Initial safety is good: No evidence of systemic toxicity was found during the dosing regimen and observation period of this study.

[0066] In summary, functional immune repair exosomes (CD301b) + The combination of -EXO with a controlled-release, bone-targeted microsphere delivery system is an effective multimodal bone repair strategy. By modulating the local immune microenvironment and simultaneously promoting angiogenesis and osteogenic differentiation, it significantly improves the repair effect of bone defects, with clear mechanistic support and potential clinical translational value.

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A CD301b + - method for the preparation of EXO, characterized in that, comprising the steps of: (1), CD301b + Preparation of macrophages: RAW264.7 macrophages were cultured in medium after stimulation by interleukin-4, and cell suspension was prepared by collecting the cells. The cell suspension was mixed with the antibody mixture at room temperature in the dark, centrifuged to discard the supernatant, washed repeatedly, resuspended, filtered, and sorted to obtain CD301b + macrophages; (2), CD301b + - Preparation of EXO: the CD301b + After culturing the macrophages in the medium after interleukin-4 stimulation, differential centrifugation was sequentially performed in a low-temperature environment, and then the supernatant was taken. The exosomes were enriched by first ultracentrifugation, the obtained precipitate was resuspended after precooling, and second ultracentrifugation was performed. The obtained precipitate was resuspended to prepare CD301b + - EXO, i.e. CD301b + exosomes derived from CD301b The mass concentration of interleukin-4 in step (1) and step (2) is 20 ng / mL; the culture medium is high-glucose DMEM complete medium containing 10% exosome-free FBS and 1% penicillin-streptomycin by volume percentage; The specific steps of sequentially performing differential centrifugation in the low-temperature environment in step (2) are as follows: first, remove living cells by centrifugation at 300-400xg for 10-15 minutes at 4-10℃, then remove dead cells by centrifugation at 2000-3000xg for 10-15 minutes, and then remove cell debris by centrifugation at 10000-12000xg for 30-40 minutes, and collect the supernatant.

2. The CD301b of claim 1 + - a method for the preparation of EXO, characterized in that, The antibody mixture in step (1) is a mixture of APC-labeled anti-mouse F4 / 80 antibody diluted 400 times and PE-labeled anti-mouse CD301b antibody diluted 200 times.

3. The CD301b of claim 1 + - a method for the preparation of EXO, characterized in that, The specific conditions of the first ultracentrifugation and the second ultracentrifugation in step (2) are 4-10℃, 100000-120000xg ultracentrifugation for 70-90 minutes.

4. A CD301b + - EXO, characterized in that, Prepared according to the preparation method of any one of claims 1-3.

5. A CD301b + - method for the preparation of bone-targeting microspheres of EXO, characterized in that, comprising the steps of: (a) Preparation of oxidized sodium alginate: sodium periodate aqueous solution is poured into a sodium alginate anhydrous ethanol solution and magnetically stirred, dialyzed, and freeze-dried to obtain oxidized sodium alginate. The mass concentration of the sodium periodate aqueous solution is 0.099-0.12 g / mL, the mass concentration of the sodium alginate anhydrous ethanol solution is 0.2-0.16 g / mL, and the volume ratio of the sodium periodate aqueous solution to the sodium alginate anhydrous ethanol solution is 1:1-1.

5. (b) Preparation of succinylated chitosan: chitosan, methanol solution, and succinic anhydride are sequentially added to a lactic acid aqueous solution, which is then uniformly magnetically stirred at room temperature. NaOH is then added dropwise until the pH of the solution is 7.00-7.

11. The precipitate is collected by filtration, dissolved in deionized water, dialyzed, and freeze-dried to obtain succinylated chitosan. The mass-volume ratio of chitosan, lactic acid aqueous solution, methanol solution, and succinic anhydride is 0.5 g:44.25 mL:160 mL:150 mg. (c) Synthesis of alendronate grafted succinylated chitosan: aldehyde-activated alendronate and succinylated chitosan are stirred in pure water to obtain a mixture, which is dialyzed and freeze-dried to obtain alendronate grafted succinylated chitosan. The mass ratio of aldehyde-activated alendronate to succinylated chitosan is 50 mg:250 mg. (d), CD301b-loaded + Preparation of bone-targeting microspheres of EXO: the water phase and the oil phase are injected into a microfluidic device at different flow rates to form monodisperse spherical droplets, which are collected in an oxidized sodium alginate solution, crosslinked at low temperature, sequentially washed with anhydrous ethanol and deionized water, centrifuged, and prepared to obtain CD301b-loaded + Bone-targeting microspheres of EXO, wherein the flow rate of the water phase is 2-4 μL / min, the flow rate of the oil phase is 40-80 μL / min, and the mass concentration of the oxidized sodium alginate solution is 25-30 mg / mL; the water phase is a CD301b-containing + Alendronate-grafted succinylated chitosan aqueous solution of EXO; the oil phase is mineral oil.

6. The loaded CD301b of claim 5 + - A method for preparing bone-targeting microspheres of EXO, characterized in that, The lactic acid aqueous solution in step (b) is a 5% lactic acid aqueous solution by volume percentage, and the concentration of NaOH is 8-10 M.

7. The loaded CD301b of claim 5 + - A method for preparing bone-targeting microspheres of EXO, characterized by, The aldehyde-activated alendronate in step (c) is prepared by reacting alendronate with 50% glutaraldehyde by volume percentage at a mass-volume ratio of 1.2 g:30-35 mL at 45-50℃ for 12-16 hours.

8. The load CD301b according to claim 5 + - A method for preparing bone-targeting microspheres of EXO, characterized by, The aqueous phase in step (d) contains 0.5 mg / mL CD301b + - An aqueous solution of alendronate-grafted succinylated chitosan EXO, wherein the concentration of alendronate-grafted succinylated chitosan is 10~8 mg / mL; the oil phase is mineral oil containing 5% Span 80 by volume; the temperature of low-temperature crosslinking is 4~8℃, the time of low-temperature crosslinking is 12~24 hours, the speed of centrifugation is 3000~3500 rpm, and the time of centrifugation is 5~10 minutes.

9. A CD301b + -EXO-loaded bone-targeting microspheres, characterized in that, Prepared according to the preparation method of any one of claims 6-8.

10. A loaded CD301b according to claim 9 + - Use of bone-targeted microspheres of EXO in the field of bone defect repair.

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