Oxygen catalytic gel microspheres regulate mitochondrial energy metabolism and its tissue repair applications

Abstract

This invention discloses an oxygen-catalyzing gel microsphere for regulating mitochondrial energy metabolism and its application in tissue repair. The microsphere comprises a calcium alginate hydrogel framework and multiple separate micro-reaction chambers embedded within the calcium alginate hydrogel framework. Each micro-reaction chamber independently contains ozone oil and graphene. This invention constructs a near-infrared responsive oxygen-catalyzing gel microsphere, achieving structured regulation of local oxygen supply and effective remodeling of cellular metabolic states. The system utilizes a dual-emulsion-microfluidic electrospray strategy to construct a spatially separated multi-chamber structure. Under near-infrared stimulation, the Gr-mediated photothermal-electronic synergy promotes the in-situ, efficient, and relatively mild conversion of ozone to oxygen, providing new insights for the design of hypoxia-related bone regeneration and other tissue repair materials.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of oxygen-catalyzed gel microspheres in regulating mitochondrial energy metabolism and tissue repair. Background Technology

[0002] Lipid droplets, as a dynamic storage and allocation platform for intracellular lipids, play a fundamental role in the energy supply required for bone regeneration by continuously delivering fatty acid substrates to mitochondria, supporting β-oxidation and oxidative phosphorylation processes. This lipid mobilization-mitochondrial oxidation pathway provides a stable source of acetyl-CoA and adenosine triphosphate (ATP) for bone marrow mesenchymal stem cells (BMSCs) during high-energy-consuming activities such as proliferation, osteogenic differentiation, and related matrix synthesis. Simultaneously, the efficient utilization of fatty acids in lipid droplets helps maintain mitochondrial metabolic flux and redox homeostasis, avoiding metabolic stress caused by lipid accumulation. Therefore, the functional energy supply from lipid droplets to mitochondria is considered a key link in supporting mitochondrial energy metabolism during bone regeneration.

[0003] However, persistent hypoxia in bone defect areas directly interferes with the functional energy supply from lipid droplets to mitochondria. Since fatty acid β-oxidation and oxidative phosphorylation depend on oxygen as the final electron acceptor in the electron transport chain (ETC), insufficient oxygen limits the sustained oxidative utilization of fatty acids within mitochondria, making it difficult to effectively mobilize lipids stored in lipid droplets, leading to abnormal accumulation of lipid droplets within the cell. This process is accompanied by a decrease in the oxidized nicotinamide adenine dinucleotide / reduced nicotinamide adenine dinucleotide (NAD⁺ / NADH) ratio, reflecting limited mitochondrial oxidative phosphorylation flux and ATP production capacity, thus becoming a key limiting factor restricting energy metabolism and weakening osteogenic potential in the bone defect microenvironment. Therefore, restoring local oxygen supply to rebuild the effective energy supply from lipid droplets to mitochondria is a crucial prerequisite for promoting bone defect repair.

[0004] However, existing oxygen supply strategies still face significant limitations in achieving continuous, controllable, and efficient oxygen supply. Direct oxygen injection relies on external supply, resulting in large fluctuations in oxygen concentration and short duration of operation; the release process of chemical or biological oxygen release systems is limited by reaction kinetics, often making precise control difficult; while nano- or micro-sized oxygen-carrying platforms can delay oxygen release, their oxygen supply efficiency remains limited due to constraints on reaction interfaces and diffusion pathways. Furthermore, although catalytic reaction-based oxygen release systems possess the potential for continuous oxygen supply, the scale of the reaction interface, catalytic efficiency, and potential side reactions still restrict their application effectiveness. Therefore, there is an urgent need to achieve precise control of the oxygen supply process through novel structural designs. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing oxygen-catalyzed gel microspheres for regulating mitochondrial energy metabolism and their application in tissue repair.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide an oxygen catalytic gel microsphere, the microsphere comprising: Calcium alginate hydrogel framework; and Multiple separate microreaction chambers are embedded within the calcium alginate hydrogel framework. Each of the micro-reaction chambers independently contains ozone oil and graphene.

[0007] Furthermore, the microreaction chambers are spherical or near-spherical substructures, and multiple microreaction chambers are distributed in an array within the hydrogel framework.

[0008] Furthermore, the microspheres also contain platelet-derived growth factor BB.

[0009] The second aspect is to provide a method for preparing the aforementioned oxygen catalytic gel microspheres, comprising the following steps: Step 1: Dissolve polyvinyl alcohol in graphene dispersion to prepare inner aqueous phase W1; dissolve polylactic acid-glycolic acid copolymer in organic solvent and add ozone oil to prepare oil phase O; dissolve sodium alginate in water to prepare outer aqueous phase W2. Step 2: Under ice-water bath conditions, the inner aqueous phase W1 is added to the oil phase O in batches, and ultrasonic emulsification is performed after each addition to form a stable W1 / O nanoemulsion. Step 3: The W1 / O nanoemulsion is added to the external aqueous phase W2 in batches, and ultrasonic treatment is continued after each addition to obtain W1 / O / W2 dual nanoemulsion. Step four: The W1 / O / W2 dual nanoemulsion is added dropwise to a crosslinking solution containing calcium ions through a microfluidic electrospray system, causing sodium alginate to undergo in-situ ionic crosslinking and solidify to form the oxygen catalytic gel microspheres.

[0010] Further, in the inner aqueous phase W1, the concentration of graphene dispersion is 0.5~2 mg / mL, and the concentration of polyvinyl alcohol is 10~15 mg / mL; in the oil phase O, the organic solvent is dichloromethane, the concentration of polylactic acid-glycolic acid copolymer is 80~120 mg / mL, and the volume fraction of ozone oil is 2%~5%; in the outer aqueous phase W2, the mass fraction of sodium alginate is 0.5%~2%.

[0011] Furthermore, the external aqueous phase W2 also contains a bioactive factor, namely platelet-derived growth factor BB.

[0012] Further, the batch addition in step two refers to adding the inner aqueous phase W1 to the oil phase O in 4 to 8 portions of 0.1 to 0.3 mL each time; the batch addition in step three refers to adding the W1 / O nanoemulsion to the outer aqueous phase W2 in 4 to 8 portions of 0.1 to 0.3 mL each time.

[0013] Furthermore, the calcium-containing crosslinking solution is an aqueous solution of calcium chloride with a concentration of 1 mol / L.

[0014] Furthermore, the operating voltage of the microfluidic electrospray system is 10kV, and the propulsion speed is 5mL / h.

[0015] The third aspect is to provide the application of the aforementioned oxygen-catalyzing gel microspheres in the preparation of products that promote bone regeneration.

[0016] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention constructs a near-infrared responsive oxygen-catalyzing gel microsphere, achieving structured regulation of local oxygen supply and effective remodeling of cellular metabolic state. The system utilizes a dual-emulsion-microfluidic electrospray strategy to construct a spatially separated multi-compartment structure. Under near-infrared stimulation, it promotes the in-situ, efficient, and relatively mild conversion of ozone to oxygen through Gr-mediated photothermal-electronic synergy.

[0017] In vitro and in vivo experiments showed that this system could continuously improve hypoxia, alleviate abnormal lipid droplet accumulation, restore lipid droplet-mitochondrial interactions and mitochondrial oxidative phosphorylation function, enhance cellular energy metabolism, and further activate the integrin-mediated FAK-PI3K-AKT signaling pathway, promoting extracellular matrix deposition and remodeling, and enhancing osteogenic differentiation and vascular remodeling. In a rat bone defect model, the microspheres significantly promoted new bone formation, bone tissue maturation, and overall repair of the bone defect area, while exhibiting good in vivo biocompatibility.

[0018] In summary, the near-infrared responsive oxygen-catalyzing gel microspheres of the present invention provide a bone repair strategy based on the synergistic effect of structured oxygen supply and metabolic remodeling, offering new ideas for the design of hypoxia-related bone regeneration and other tissue repair materials. Attached Figure Description

[0019] Figure 1This document describes the construction and physicochemical characterization of the oxygen-catalyzing gel microspheres of this invention. A is a schematic diagram of the preparation of the oxygen-catalyzing gel microspheres; B) shows the Raman spectra of the Gr, W1 / O, and W1 / O+NIR systems; C is an optical microscope image of the microspheres; D is a confocal laser scanning microscope image of the multi-chamber structure inside the microspheres; E is a scanning electron microscope image of the overall morphology of the microspheres; F and G are high-magnification scanning electron microscope images of the spherical substructures inside the microspheres, where G is a pseudo-color annotation; H and J are the elemental surface scan results of the microspheres, corresponding to the distribution of O, Ca, and N elements, respectively; K is an infrared thermal image of different treatment groups under near-infrared irradiation; and L is the temperature change curve of each group during near-infrared irradiation.

[0020] Figure 2 This invention describes the NIR-responsive oxygen supply behavior of the multi-compartment oxygen catalytic microspheres prepared in this invention and their regulatory effect on BMSC metabolism and chemotaxis. A represents in vivo infrared thermography images of the defective area and the PBS control group under near-infrared irradiation after implantation with Alg-Ca@W1 / O / W2-Gr microspheres; B represents the quantitative analysis of local temperature changes in different treatment groups in (A); C represents the cyclic temperature response curves of Alg-Ca@W1 / O / W2-Gr microspheres under multiple rounds of NIR on / off conditions; D represents the changes in dissolved oxygen concentration in different systems during NIR irradiation; EG represents the electron spin resonance (ESR) spectra of relevant reactive oxygen species under different treatment conditions, corresponding to ·O2. - ·OH and 1 O2; H represents representative images of cell aggregation / recruitment behavior around microspheres; IL represents XPS fine spectral analysis of Gr after different treatments, where I and J are C1s spectra, and K and L are O1s spectra; M represents scanning electron microscope images of Gr and Gr+O3+NIR treated; N represents elemental surface scan images of the corresponding samples; O represents fluorescence staining images of lipid droplets (BODIPY, green) and mitochondria (Mito-Tracker, red) in BMSCs under different conditions, with cell nuclei labeled with Hoechst (blue).

[0021] Figure 3This study aimed to characterize the biocompatibility and osteogenic associations of the Alg-W1 / O / W2+NIR system. A shows live / dead cell fluorescence staining images of BMSCs under different material treatments; B shows fluorescence staining images of the cytoskeleton (F-actin) and nucleus (DAPI) of BMSCs; C shows the results of the material hemolysis experiment and corresponding sample photographs; D shows the proliferation of BMSCs under different treatments detected by the CCK-8 assay; E shows alkaline phosphatase (ALP) activity staining images; F shows Alizarin Red S (ARS) staining images, used to observe calcified nodule formation; G and H show immunofluorescence staining images of osteogenic-related proteins COL-1 (G) and OCN (H); I shows the quantitative analysis results of ARS staining; J and K show the quantitative analysis of COL-1 and OCN immunofluorescence intensity; LP shows the expression level of osteogenic-related genes detected by qRT-PCR; (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001.

[0022] Figure 4 The study aimed to characterize the angiogenic behavior of endothelial cells. A represents images from the lumen formation assay of HUVECs; B represents images from the scratch migration assay of HUVECs; C represents immunofluorescence staining images of CD31, F-actin, and DAPI; DF represents quantitative analysis of the number of nodes, branches, and total length in the lumen formation assay; G represents quantitative analysis of the healing rate in the scratch assay; and H represents quantitative analysis of CD31 fluorescence intensity. (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0023] Figure 5 Characterization of mitochondrial function in BMSCs using Alg-W1 / O / W2+ NIR; where A is a transmission electron microscope (TEM) image of BMSCs; B is a fluorescence image of total ROS detected by the DCFH-DA probe; C is a fluorescence image of mitochondrial ROS detected by the MitoSOX probe; D is a fluorescence image of mitochondrial membrane potential detected by the TMRE probe; E is a fluorescence image of mitochondrial membrane potential changes detected by the JC-1 double staining method; F is a fluorescence image of mitochondrial Ca²⁺ levels detected by the Fluo-4 AM probe; G is a fluorescence image of mitochondrial ATP levels detected by the pCMV-Mito-ATP probe; HN represents the quantitative analysis results of the corresponding fluorescence signals; O represents NAD. + / NADH ratio test results; (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0024] Figure 6This section presents transcriptomics and signaling pathway correlation analyses. A represents principal component analysis (PCA) plots; B represents a heatmap of gene expression clusters among samples; C represents the number of differentially expressed genes (DEGs); D represents a volcano plot of differentially expressed genes; E represents GO enrichment analysis results; F represents KEGG enrichment analysis results; GI represents gene set enrichment analysis (GSEA) results; J represents protein-protein interaction network (PPI) analysis results; L represents Western blot analysis of osteogenic-related proteins; M represents Western blot analysis of mitochondrial function-related proteins; and N represents Western blot analysis of FAK, PI3K, AKT, and their phosphorylated forms.

[0025] Figure 7 This invention provides a micro-CT evaluation of the multi-compartment oxygen catalytic microspheres prepared in this invention in promoting bone defect repair in vivo. A shows a schematic diagram of the animal experiment process, including rat bone defect model construction, material implantation, and near-infrared (NIR) intervention. B shows micro-CT three-dimensional reconstruction images, longitudinal sections, pseudo-color images, and cross-sectional images of the defect area in each group at 4 weeks post-operation. C shows micro-CT three-dimensional reconstruction images, longitudinal sections, pseudo-color images, and cross-sectional images of the defect area in each group at 8 weeks post-operation. DF shows quantitative analysis of bone repair-related parameters in the defect area of ​​different treatment groups at 4 and 8 weeks post-operation, including bone volume fraction (BV / TV), bone mineral density (BMD), and trabecular bone number (Tb.N). Data are expressed as mean ± SD.

[0026] Figure 8 The images show the histological and immunological characteristics of the bone defect area. Among them, A is the H&E staining image of the bone defect area at different time points; B is the Masson trichrome staining image; C is the immunohistochemical staining image of osteogenic-associated protein COL-1; and D is the immunofluorescence staining image of TOMM20, OCN, and CD31.

[0027] Figure 9 Images of hematoxylin-eosin (H&E) staining of major organs such as the heart, kidneys, liver, lungs and spleen in different treatment groups. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0029] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0030] Example 1: Preparation and physicochemical characterization of oxygen catalytic gel microspheres This embodiment provides a multi-compartment oxygen catalytic microsphere and its preparation method, which is as follows: The microspheres were prepared using a dual emulsion (W1 / O / W2) combined with microfluidic electrospray technology. The inner aqueous phase (W1) was prepared by dissolving 0.05 g of polyvinyl alcohol (PVA) in 4 mL of graphene (Gr, 1 mg / mL) dispersion. The oil phase (O) was prepared by dissolving 300 mg of polylactic-co-glycolic acid copolymer (PLGA) in 3 mL of dichloromethane (DCM) and adding 0.1 mL of ozone oil, mixing thoroughly. The outer aqueous phase (W2) was 4 mL of a 1 wt% sodium alginate (Alg) aqueous solution. To impart certain bioactivity to the microspheres, PDGF-BB was added to the outer aqueous phase W2 during the preparation process and mixed thoroughly. In preparation, 1 mL of the inner aqueous phase W1 was added in portions of 0.2 mL to 4 mL of the oil phase under ice-water bath conditions, with ultrasonic emulsification performed after each addition to form a stable W1 / O nanoemulsion. Subsequently, 1 mL of the above W1 / O nanoemulsion was taken and added in portions of 4 mL of the outer aqueous phase W2 in the same manner, with ultrasonic treatment continued after each addition, ultimately obtaining a W1 / O / W2 dual nanoemulsion. The obtained dual emulsion was then dropwise added to a crosslinking solution (1 mol / L) containing CaCl2 using a microfluidic electrospray system. 2+ In-situ ionic cross-linking of sodium alginate under induction occurs, forming structurally stable multi-compartment oxygen catalytic microspheres. After preparation, the microspheres are thoroughly washed and used for subsequent physicochemical characterization and biological experiments.

[0031] For simplicity, the naming emphasis varies depending on the scenario in this invention. In the material preparation and structural characterization section, the microspheres are named "Alg-Ca@W1 / O / W2-Gr", where "Alg-Ca" represents the calcium alginate hydrogel framework, "W1 / O / W2" indicates that the microspheres originate from the W1 / O / W2 dual emulsion precursor system, and "Gr" indicates that the system contains graphene components. This naming is mainly used to characterize the composition and origin of the microspheres, and does not indicate the existence of a complete W1 / O / W2 three-phase continuous structure in the final microspheres, nor does it strictly limit the absolute spatial distribution of each component in the microspheres. In the above naming, "W1 / O / W2" characterizes the precursor construction method; the final microspheres are essentially composite microsphere structures supported by a calcium alginate framework and forming multiple independent micro-compartments inside. These micro-compartments are formed by an oil phase encapsulating an inner aqueous phase and being isolated by the calcium alginate framework. In cell and animal experiments, simplified naming conventions, such as "Alg-W1 / O / W2," are used to facilitate comparison between groups. This primarily indicates whether the system contains the corresponding structural unit or functional component, highlighting the compositional differences between different experimental groups. This type of naming serves as an experimental group identifier and is not used to strictly define the precise spatial configuration of the microspheres.

[0032] 1.1 Morphological and structural characterization The Alg-Ca@W1 / O / W2-Gr microspheres prepared in Example 1 were observed for their overall morphology using an optical microscope. The results are as follows: Figure 1 As shown in C, the microspheres are regular spherical with clear boundaries.

[0033] The internal structure of the microspheres was observed using confocal laser scanning microscopy. The results are as follows: Figure 1 As shown in Figure D, a large number of fluorescently labeled oil droplets are uniformly distributed inside the calcium alginate (Alg-Ca) matrix, indicating that the internal reaction units formed by the dual emulsion were successfully embedded in the hydrogel framework, and the microspheres have a distinct multi-compartment spatial separation structure.

[0034] The microstructure of the microspheres was observed using scanning electron microscopy (SEM). Low-magnification SEM images (...) Figure 1 The high-magnification SEM images show that the surface of the dried microspheres is rough and wrinkled, which helps to increase the contact area between the material and the external environment; Figure 1 Figures F and G (where G is a pseudo-color annotation) show that the microspheres contain numerous relatively uniform, separated spherical substructures, corresponding to the W1 / O reaction units stably encapsulated by the Alg-Ca matrix. This indicates that multiple independent micro-reaction chambers are constructed within each microsphere. This spatially separated structure not only improves the local confinement of the ozone reaction system but also amplifies the effective reaction interface through the arrayed distribution of reaction units, thus providing a structural advantage for improving the conversion efficiency of O3 to O2.

[0035] 1.2 Characterization of Composition and Structural Stability Raman spectroscopy was used to characterize the graphene (Gr) in the microspheres. The results are as follows: Figure 1 As shown in Figure B, the Gr-containing microsphere system exhibits characteristic D and G peaks of Gr at ~1350 cm⁻¹ and ~1580 cm⁻¹, indicating that Gr has been successfully integrated into the microsphere system and the structure remains intact.

[0036] The elemental distribution of the microspheres was analyzed using energy dispersive spectroscopy (EDS). The results are as follows: Figure 1 As shown in Figure HJ, oxygen (O), calcium (Ca), and nitrogen (N) elements are all clearly distributed in the microspheres, indicating that the Alg-Ca ion crosslinking network has been successfully constructed and has good overall stability.

[0037] 1.3 Characterization of photothermal response performance Alg-Ca@W1 / O / W2-Gr microspheres, Alg@W1 / O / W2-Gr, Gr-free Alg@W1 / O / W2 microspheres, and a PBS control group were used. Temperature changes were recorded using an infrared thermal imager under near-infrared (NIR) irradiation. Results are as follows: Figure 1 As shown in Figure K, the temperature of the Gr-containing microsphere group increased rapidly over time, while the PBS group and the Gr-free microsphere group only showed a slight temperature increase. Quantitative analysis ( Figure 1 The results (L) show that after 10 min of NIR irradiation, the temperature rise of the Gr-containing microsphere system can reach 15.0–18.0 °C. This indicates that the introduction of Gr endows the system with good near-infrared response and photothermal conversion capability, which can provide the necessary energy input for subsequent in-situ catalytic decomposition of ozone.

[0038] Example 2: NIR-responsive oxygen supply behavior and its regulatory effect on BMSC metabolism and chemotaxis 2.1 To verify the photothermal response capability of the system under in vivo conditions, near-infrared (NIR) irradiation was applied to the defective area of ​​the implanted Alg-Ca@W1 / O / W2-Gr microspheres, and local temperature changes were monitored in real time using infrared thermal imaging. Figure 2 As shown in Figures A and B, after NIR irradiation, the temperature of the microsphere implantation area rapidly increased, rising from approximately 24.0°C to 42.8°C within 2 minutes, while the PBS group only showed a slight temperature increase. Quantitative analysis further indicated that the Alg-Ca@W1 / O / W2-Gr group maintained a significantly higher temperature level than the PBS group throughout the irradiation process, demonstrating that the system retains good near-infrared light response and photothermal conversion capabilities in vivo. Notably, the temperature rise was relatively stable, without any significant temperature spikes, suggesting good controllability and potential for in vivo application. Further cyclic irradiation experiments revealed that the system maintained stable and reversible temperature response behavior under multiple rounds of NIR on / off conditions. Figure 2 The presence of C indicates that the photothermal triggering process has good repeatability, laying the foundation for subsequent on-demand oxygen supply.

[0039] 2.2 To evaluate the oxygen supply behavior of different systems under near-infrared stimulation, samples from each treatment group were placed in a reaction system of the same volume, and the changes in dissolved oxygen concentration in the system were measured in real time using a dissolved oxygen analyzer under near-infrared irradiation. The detection groups included PBS, Alg-Ca-O3-Gr (Bulk), Alg-Ca@W1 / O / W2, and Alg-Ca@W1 / O / W2-Gr, used to compare the effects of different structural designs and functional components on oxygen supply kinetics.

[0040] The results are as follows Figure 2As shown in Figure D, the PBS group maintained a consistently low dissolved oxygen level, while the Gr-free Alg-Ca@W1 / O / W2 group only showed a limited increase in oxygen concentration. In contrast, the dissolved oxygen concentration of the Alg-Ca@W1 / O / W2-Gr group continuously increased with irradiation time, reaching its highest level at 10 min, demonstrating a more stable and sustained oxygen supply capacity. Notably, although the Bulk group rapidly released high levels of oxygen in the early stages, the release subsequently dropped significantly, exhibiting typical burst release characteristics. The multi-compartment Alg-Ca@W1 / O / W2-Gr system, however, avoided this phenomenon, showing a continuously increasing oxygen release trend. These results indicate that the spatially separated multi-compartment structure not only helps to delay reactant depletion but also amplifies the catalytic interface through arrayed micro-reaction units, thereby achieving a shift from "rapid burst release" to "continuous oxygen supply." This demonstrates that the introduction of Gr and the multi-compartment configuration play a crucial role in both the O3–O2 conversion efficiency and sustainability.

[0041] 2.3 To further elucidate the reaction characteristics of O3 to O2 conversion in this system, this invention employs electron spin resonance (ESR) to analyze the generation of reactive oxygen species during the reaction process. Singlet oxygen (¹O2) and superoxide radicals (·O2) in the O3+NIR and O3+Gr+NIR systems were detected respectively. - The results showed that, compared with the O3+NIR group, the ESR signals of singlet oxygen and superoxide radicals were significantly weakened in the O3+Gr+NIR group. Figure 2 The presence of Gr (as seen in the near-infrared irradiation) indicates that the introduction of Gr did not promote the enhancement of free radical chain reactions in the solution phase; instead, it inhibited the generation and accumulation of ROS related to oxygen supply. This result suggests that under near-infrared irradiation conditions, the pure O3 system tends to generate ¹O2 and ·O2, which can be captured by ESR, through solution phase decomposition and photochemical chain reactions. - And ·OH; however, after the introduction of Gr, O3 preferentially undergoes electron transfer-mediated decomposition reactions on its surface, shifting the reaction pathway from a radical-dominated process to a non-radical process dominated by O2 generation. In other words, Gr provides a surface electron transfer channel, prompting O3 to undergo rapid disproportionation and directional conversion into O2, thereby reducing the formation of reactive oxygen species related to oxygen content in the solution environment. This result indicates that Gr does not simply exist as a photothermal material in this system, but also participates in the regulation of the O3 decomposition pathway, making the reaction more inclined towards efficient oxygen supply rather than disordered oxidation. Consistent with the ESR results, XPS analysis further revealed the changes in the surface chemical state of Gr after the combined effects of O3 and NIR. The fine spectra of C1s and O1s of the treated samples were significantly altered ( Figure 2The NIR spectrum showed enhanced carbon-oxygen bond-related peaks and further detected ester bond-related signals. Combined with the comprehensive analysis of carbon skeleton oxidation characteristics, this suggests that a carbonate-like structure may have formed on the Gr surface during the reaction due to a strong oxidation process. This indicates that a strong oxidation reaction occurred on the Gr surface after O3 contacted and triggered by NIR, reflecting that O3 was indeed continuously activated and consumed on the Gr surface. Simultaneously, SEM and elemental surface scanning results showed changes in the surface morphology and oxygen distribution of the treated Gr. Figure 2 The results from M and N further demonstrate that Gr is not passively endothermic, but rather directly participates in the conversion of O3 as a catalytic interface during the reaction. Combined with ESR and XPS results, it can be concluded that Gr's role is to guide the preferential catalytic decomposition of O3 into O2 via a non-radical pathway along its surface, while simultaneously improving oxygen supply efficiency and reducing the accumulation of oxygen-related ROS. This provides a mechanistic basis for the system to achieve continuous, mild, and relatively safe oxygen supply.

[0042] 2.4 Improved local oxygen supply should ultimately be reflected in the regulation of the cellular microenvironment and cell behavior. The prepared microspheres were placed in designated areas of a cell culture plate, and BMSCs were subsequently seeded into the same culture system and co-cultured under standard conditions for a certain period. The distribution, migration trends, and aggregation behavior of cells around the material were observed and recorded in real time using bright-field optical microscopy to evaluate the material's recruitment effect on BMSCs. In vitro observations showed significant cell aggregation around the Alg-Ca@W1 / O / W2-Gr microspheres, suggesting that this system can create a local microenvironment conducive to BMSC adhesion and recruitment around the material. Figure 2 (H).

[0043] To observe the spatial distribution relationship between lipid droplets and mitochondria in BMSCs under different treatment conditions, cells were cultured under normal, hypoxic, and hypoxic + material treatment conditions. Lipid droplets were then labeled with BODIPY dye, mitochondria with MitoTracker, and cell nuclei with Hoechst. Fluorescence images were acquired using confocal laser scanning microscopy to analyze lipid droplet accumulation and changes in lipid droplet-mitochondrial spatial interactions. Figure 2As shown in Figure O, under normal culture conditions, lipid droplets and mitochondria maintained good spatial association within BMSCs. However, under hypoxic conditions, the lipid droplet signal was significantly enhanced, and the co-localization with mitochondria decreased, suggesting that the functional energy supply process from lipid droplets to mitochondria was inhibited. Notably, the addition of Alg-Ca@W1 / O / W2-Gr microspheres under hypoxic conditions significantly alleviated intracellular lipid droplet accumulation, and the spatial overlap between lipid droplets and mitochondria was restored. This indicates that the system can rebuild lipid droplet-mitochondrial interactions by improving local oxygen supply, thereby contributing to the restoration of mitochondrial oxidative metabolism. Combined with the aforementioned oxygen release results, it is evident that multi-compartment oxygen catalytic microspheres can convert exogenous near-infrared stimulation into a continuous local oxygen supply, further acting on cellular metabolic processes and alleviating lipid utilization impairment caused by hypoxia.

[0044] In summary, Alg-Ca@W1 / O / W2-Gr microspheres can achieve a stable and controllable photothermal-oxygen supply coupled response under near-infrared stimulation both in vivo and in vitro. Their multi-compartment structure effectively avoids the oxygen burst release phenomenon common in traditional homogeneous systems and significantly improves the sustainability and effectiveness of oxygen supply. Simultaneously, Gr-mediated photothermal-electron co-catalysis provides the reaction basis for the in-situ conversion of O3 to O2, enabling the system to further translate its structural advantages into metabolic regulatory effects. By improving local hypoxia, this system not only promotes the aggregation of BMSCs around the material but also restores lipid droplet-mitochondrial interactions damaged under hypoxic conditions, providing favorable conditions for subsequent mitochondrial energy metabolism reconstruction and osteogenic activity during bone repair.

[0045] Example 3: Comprehensive assessment of in vitro biocompatibility, osteogenic differentiation, angiogenesis, and mitochondrial function. Bone marrow mesenchymal stem cells (BMSCs) were extracted from 1-week-old Sprague-Dawley (SD) rats. The specific steps were as follows: First, the rats were euthanized, and then the leg muscle tissue was removed to separate the femur and tibia. Bone marrow contents were collected by centrifugation, and the cells were resuspended in complete culture medium for primary culture. Cells were cultured to passage 2 (P2) for subsequent experiments. HUVECs cell lines were purchased from IMMOCELL (Xiamen, Fujian, China) and cultured according to the supplier's recommended culture conditions for in vitro angiogenesis-related experiments. All cells were cultured in medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) in a 37°C, 5% CO2 incubator. Cell experiments were divided into the following four groups: Control group: Cells were cultured under standard culture conditions without any material treatment or near-infrared irradiation; NIR group: Cells were cultured under standard culture conditions and received only near-infrared (NIR) irradiation without any added materials; W1 / O / W2 group: Cells were not treated with microfluidic electrospraying or Ca2+. 2+ Cross-linked dual emulsion systems, lacking spatially fixed multi-compartment structures and light-controlled oxygen supply capabilities, are used to evaluate the effects of multiphase components themselves, rather than the structured system, on cell behavior; Alg-W1 / O / W2+NIR group: cells are fed with oxygen via microfluidic electrospraying and Ca... 2+ Cross-linked multi-compartment oxygen catalytic microspheres Alg-Ca@W1 / O / W2-Gr (hereinafter referred to as Alg-W1 / O / W2) were co-cultured. These microspheres were loaded with Gr and bioactive factors, and near-infrared irradiation was applied during the experiment to evaluate the comprehensive regulatory effect of the light-controlled oxygen supply system on cell behavior and metabolic function.

[0046] 3.1 In vitro biocompatibility assessment To evaluate Alg-W 1 / The biocompatibility of O / W2 was first analyzed by live / dead cell staining and cytoskeleton staining. Figure 3 (A, B). The results showed that after 1, 3, and 5 days of culture, the BMSCs in the Alg-W1 / O / W2+NIR group had higher survival rates, better morphology, and superior cell density and spreading compared to other groups. F-actin staining of the cytoskeleton showed that they exhibited regular and abundant fibrous structures, indicating good cell adhesion and spreading ability. Furthermore, the hemolysis experiment results ( Figure 3 The results (C) showed that the hemolysis rate in the Alg-W1 / O / W2+NIR group was less than 5% (0.69% ± 0.64%), far lower than the positive control (100%), indicating excellent blood compatibility. Further analysis using the CCK-8 assay (…) further confirmed this. Figure 3 Cell proliferation was detected by D) and the results showed no significant difference in cell activity among the groups at days 1, 3, and 5, indicating that Alg-W1 / O / W2+NIR has no significant cytotoxicity and good biocompatibility. In conclusion, Alg-W1 / O / W2 microspheres have good cell compatibility and blood compatibility.

[0047] 3.2 Assessment of in vitro osteogenic differentiation capacity To systematically evaluate the effects of Alg-W1 / O / W2+NIR treatment on osteogenic differentiation of bone mesenchymal stem cells (BMSCs), further analysis of osteogenic-related staining and biomarker expression was conducted. Alkaline phosphatase (ALP) activity staining results (…) Figure 3 (E) shows that Alg-W 1 / The ALP staining intensity was significantly enhanced in the O / W2+NIR group, indicating a markedly increased level of early osteogenic differentiation. Alizarin Red S (ARS) staining results ( Figure 3 The results of the quantitative analysis (F) further indicate that this group of mineralized nodules is the most abundant, and its quantitative analysis results (F) Figure 3 The concentration of α1 in the control group (I) was significantly higher than that in other treatment groups (control: 0.21 ± 0.03, NIR: 0.22 ± 0.06, W1 / O / W2: 0.49 ± 0.03, Alg-W1 / O / W2+ NIR: 0.55 ± 0.01), indicating that it had the most significant promoting effect on the mineralization process. Immunofluorescence staining results further confirmed the above trend. Figure 3 As shown in G and H, the protein expression levels of collagen I (COL-1) and osteocalcin (OCN) were significantly increased in the Alg-W1 / O / W2+NIR group. Corresponding quantitative analysis of fluorescence intensity (…) Figure 3 The results (J, K) showed that this group had significantly higher levels of extracellular matrix secretion and osteogenic differentiation compared to the control group and other treatment groups, suggesting that this system can effectively promote extracellular matrix secretion and late-stage osteogenic differentiation maturation. To further elucidate its molecular mechanism, qRT-PCR was used to detect the expression of osteogenic-related genes. Figure 3 As shown in LP, Alg-W 1 / In the O / W2+NIR group, RUNX2 (control: 1.03 ± 0.29, NIR: 1.39 ± 0.23, W 1 / O / W2: 1.66 ± 0.20, Alg-W 1 / O / W2+NIR: 2.69 ± 0.21), BMP-2 (control: 1.12 ± 0.58, NIR: 3.54 ± 1.23, W 1 / O / W2: 9.74 ± 1.57, Alg-W 1 / O / W2+NIR: 13.54 ± 2.36), SP7 (control: 1.04 ± 0.33, NIR: 1.39 ± 0.48, W 1 / O / W2: 0.98 ± 0.28, Alg-W 1 / O / W2+NIR: 2.67 ± 0.21), ALPL (control: 1.01 ± 0.16, NIR: 0.94 ± 0.39, W 1 / O / W2: 0.41 ± 0.12, Alg-W 1 / O / W2+ NIR: 2.85 ± 0.11) and SPP1 (control: 1.00 ± 0.04, NIR: 2.19 ± 0.38, W 1 / O / W2: 2.56 ± 0.17, Alg-W 1 / The expression levels of O / W2+NIR (4.61 ± 0.52) were significantly upregulated, indicating that this treatment can synergistically activate key transcription factors and signaling molecule networks in osteogenic differentiation.

[0048] In summary, oxygen-loaded hydrogel microspheres, under near-infrared irradiation-triggered photothermal effects, significantly promote the proliferation, adhesion, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Their superior osteogenic promoting effect stems primarily from two synergistic effects: firstly, the continuous release of oxygen by the oxygen-loaded hydrogel microspheres effectively alleviates local hypoxia, restores mitochondrial aerobic metabolism, and enhances cell activity; secondly, the loaded bioactive factors such as PDGF-BB promote the recruitment and activation of BMSCs, thereby synergistically constructing a local microenvironment conducive to osteogenic differentiation.

[0049] 3.3 In vitro angiogenesis capacity assessment To evaluate the effect of Alg-W1 / O / W2+NIR on the angiogenesis ability of endothelial cells under hypoxic conditions, a systematic analysis was performed using methods such as lumen formation assay, scratch assay, and CD31 immunofluorescence staining. Figure 4 As shown in Figure A, in the lumen formation experiment of HUVECs, the Alg-W1 / O / W2+NIR group significantly promoted the formation of more branched vascular-like structures. Compared with the control group (0.56±0.14, 192.30±41.63, 22.52±2.47), the number of branch nodes in the Alg-W1 / O / W2+NIR group (1.98±0.27) was significantly higher. Figure 4 (D), number of branches (420.00±71.92) Figure 4 (E) and total length (56.78±4.17) Figure 4 The levels of both α and β (F) were significantly increased, suggesting that Alg-W1 / O / W2+NIR has excellent pro-angiogenic ability. Furthermore, the scratch assay (… Figure 4 (B) shows that Alg-W 1 / The scratch healing rate of the O / W2+NIR group (32.50±1.57) within 12 hours ( Figure 4 The concentration of CD31 in the medium-sized cells (CSCs) was significantly higher than in other groups (control: 19.31±2.19, NIR: 19.27±4.34, W1 / O / W2: 25.61±1.05), suggesting that this material helps promote endothelial cell migration, thereby accelerating vascular endothelial repair. Further analysis using CD31 immunofluorescence staining (CSCs) further confirmed this effect. Figure 4 The results of the detection of vascular endothelial-specific markers by (C) showed that Alg-W1 / O / W2+NIR treatment significantly enhanced the expression of CD31, with the average fluorescence intensity (C) being significantly higher than that of CD31. Figure 4 The highest concentration of H1N1 in H1N1 reflects its potential in promoting endothelial cell vascularization.

[0050] In summary, Alg-W1 / O / W2 microspheres release oxygen under photothermal action, which can significantly promote endothelial cell migration, lumen formation and CD31 expression under hypoxic conditions, demonstrating good angiogenic capacity and providing strong support for the formation of new blood vessels during bone repair, thereby improving the bone tissue microenvironment and further supporting the osteogenic process.

[0051] 3.4 In vitro assessment of mitochondrial function As the core site of fatty acid oxidation and oxidative phosphorylation, the structural and functional integrity of mitochondria directly determines the energy metabolism status and osteogenic potential of cells under hypoxic conditions. The above results indicate that Alg-W1 / O / W2+NIR can improve local oxygen supply and rebuild lipid droplet-mitochondrial interactions. Therefore, it is necessary to further systematically evaluate the regulatory effects of this system on mitochondrial structural integrity, redox homeostasis, and energy metabolism function in BMSCs at the subcellular level.

[0052] To investigate the regulatory role of Alg-W1 / O / W2+NIR on mitochondrial function in BMSCs under hypoxic conditions, the ultrastructure of mitochondria was first observed using transmission electron microscopy. Figure 5 (A). The results showed that BMSCs in the Control and NIR groups exhibited significant mitochondrial swelling and cristae breakage, while the W1 / O / W2 group showed slight improvement. In contrast, the Alg-W1 / O / W2+NIR group showed mostly intact mitochondrial morphology and clear cristae structure, suggesting that Alg-W1 / O / W2+NIR can effectively alleviate hypoxia-induced mitochondrial structural damage. Further analysis using DCFH-DA staining revealed intracellular ROS levels (…). Figure 5The results showed that the Alg-W1 / O / W2+NIR group had the lowest ROS fluorescence intensity (9.20±0.49), significantly lower than other groups (control: 21.74±3.93, NIR: 21.38±4.04, W1 / O / W2: 13.13±1.76), indicating that it had a better ability to alleviate oxidative stress. MitoSOX staining was then used to detect mitochondrial-specific ROS (…). Figure 5 In the control group (C, I), the fluorescence intensity of the Alg-W1 / O / W2+NIR group was significantly reduced (2.84±0.37), further confirming its significant advantage in inhibiting excessive accumulation of mitochondrial ROS (control: 6.31±1.78, NIR: 7.12±0.67, W1 / O / W2: 4.21±0.51). TMRE staining was used to evaluate mitochondrial membrane potential (ΔΨm) ( Figure 5 The highest fluorescence intensity (19.01±1.67) was observed in the Alg-W1 / O / W2+NIR group (D and J), indicating that it significantly improved the hypoxia-induced decrease in membrane potential. Subsequent JC-1 staining was used to verify the changes in mitochondrial membrane potential. Figure 5 In the E, K, and L groups, the fluorescence of red aggregates in the Alg-W1 / O / W2+NIR group was significantly increased (22.47±4.41), while the fluorescence of green monomers was decreased (1.71±0.69), indicating that this system helps restore mitochondrial polarization and maintain its functional stability. Furthermore, Fluo-4 AM staining detected mitochondrial Ca2+. 2+ level( Figure 5 The fluorescence intensity of the Alg-W1 / O / W2+NIR group was significantly increased (control: 2.724±0.14, NIR: 2.38±0.04, W1 / O / W2: 3.49±0.29, Alg-W1 / O / W2+NIR: 7.85±2.08) as shown in the F and M results, suggesting that it can improve mitochondrial calcium homeostasis disorder under hypoxic conditions. Further analysis using the pCMV-Mito-ATP probe detected mitochondrial ATP production levels (…). Figure 5 The highest fluorescence intensity (2.75±0.23) was observed in the Alg-W1 / O / W2+NIR group (G, N), indicating that it significantly enhanced mitochondrial energy metabolism. The NAD⁺ / NADH ratio was also measured. Figure 5 The results showed that the NIR in the Alg-W1 / O / W2+ group was significantly increased (control: 0.19±0.02, NIR: 0.099±0.064, W1 / O / W2: 0.398±0.032, Alg-W1 / O / W2+ NIR: 1.384±0.065), further reflecting that it promoted the recovery of mitochondrial oxidative phosphorylation.

[0053] In summary, this system not only effectively alleviates mitochondrial structural damage and oxidative stress, but also maintains membrane potential, calcium ion dynamics, and ATP production capacity, providing a metabolic basis for the efficient oxidation and utilization of lipid droplet-derived fatty acids. This precise regulation of mitochondrial function provides a crucial guarantee for the smooth progress of high-energy-consuming metabolic activities during subsequent osteogenic differentiation and bone tissue regeneration.

[0054] Example 4: RNA sequencing and Western blotting analysis of the molecular mechanism of bone regeneration The aforementioned in vitro experiments confirmed that Alg-W1 / O / W2+NIR significantly improved mitochondrial function and energy metabolism in bone mesenchymal stem cells (BMSCs) under hypoxic conditions. Bone regeneration essentially depends on the synergistic effects of extracellular matrix (ECM) remodeling, cell-matrix signal transduction, and osteogenic-angiogenic coupling regulation. Therefore, to systematically elucidate the molecular network and key signaling pathways of Alg-W1 / O / W2+NIR-mediated bone regeneration at the overall transcriptional level, this embodiment further performed RNA sequencing analysis on bone tissues from the treated and control groups. To further explore the molecular mechanism of Alg-W1 / O / W2+NIR for bone regeneration, transcriptome sequencing (RNA-seq) analysis was performed on bone tissues from the Alg-W1 / O / W2+NIR treated and control groups.

[0055] Principal component analysis (PCA) showed a clear separation between the two groups at the transcriptional level. Figure 6 The sample correlation heatmap also confirmed good reproducibility between experiments (A). Figure 6 (B) Differential gene statistics ( Figure 6 The results (C) show that after Alg-W1 / O / W2+NIR treatment, a total of 3563 differentially expressed genes (DEGs) were identified, of which 2589 were upregulated and 974 were downregulated. The volcano plot further illustrates the significantly upregulated and downregulated genes. Figure 6 D). GO functional enrichment analysis ( Figure 6 The results showed that genes significantly upregulated by Alg-W1 / O / W2+ NIR were mainly enriched in biological processes closely related to bone differentiation and cell-matrix interactions, such as extracellular matrix organization, cell adhesion, and integrin binding. Furthermore, processes such as angiogenesis and blood vessel remodeling were also significantly enriched, suggesting that Alg-W1 / O / W2+ NIR not only promotes osteogenic differentiation by enhancing ECM structure and signaling function, but also remodels the microenvironment supporting angiogenesis, providing the necessary oxygen supply for bone regeneration under hypoxic conditions.

[0056] KEGG pathway analysis further revealed significant activation of Focal adhesion, PI3K-AKT, and calcium signaling pathways. Figure 6 The results suggest that Alg-W1 / O / W2+NIR may synergistically regulate osteogenic differentiation of BMSCs by reshaping ECM-cell interactions and activating key signaling pathways. GSEA analysis further validated the significant enrichment of bone repair-related gene sets, such as "extracellular matrix" (GO:0031012) and "BMP binding" (GO:0036122), in the Alg-W1 / O / W2+NIR group. Figure 6 In addition to G and H, the oxidative phosphorylation pathway is also significantly activated. Figure 6 The results suggest that Alg-W1 / O / W2+ NIR plays a crucial regulatory role in mitochondrial energy metabolism at the transcriptional level. Furthermore, protein-protein interaction network (PPI) analysis... Figure 6 The study revealed a close interaction between proteins related to ECM remodeling, mitochondrial metabolism, and bone differentiation, further emphasizing the potential molecular basis for Alg-W1 / O / W2+NIR promoting bone regeneration by regulating the ECM-mitochondrial metabolic coupling mechanism.

[0057] To validate the transcriptome results, the expression levels of key proteins related to bone differentiation, mitochondrial function, and signaling pathways were further examined. Western blot results showed that Alg-W1 / O / W2+NIR significantly upregulated the expression of bone formation markers COL-1, OPN, and RUNX2. Figure 6 The presence of L (a marker) suggests that it effectively promotes osteogenic differentiation of BMSCs. Simultaneously, key mitochondrial metabolism-related proteins SDHA, ATP5A, and TFAM were significantly upregulated (…). Figure 6 The presence of M further validated its ability to improve mitochondrial energy metabolism. Notably, the FAK-PI3K-AKT signaling axis, which is closely related to ECM sensing and angiogenesis-osteogenic regulation, was significantly activated (M). Figure 6 The phosphorylation levels of FAK, PI3K, and AKT (p-FAK, p-PI3K, p-AKT) were significantly increased, suggesting that Alg-W1 / O / W2+NIR may mediate downstream signal transduction by activating the FAK-PI3K-AKT pathway, and synergistically regulate osteogenic differentiation, angiogenesis, and mitochondrial metabolic function.

[0058] In summary, RNA-seq results reveal, at the systemic level, that Alg-W1 / O / W2+NIR constructs a multidimensional coupled regulatory network of osteogenic-angiogenic-energy metabolism by remodeling the ECM microenvironment, activating the FAK-PI3K-AKT signaling pathway, and synergistically enhancing mitochondrial oxidative phosphorylation. This mechanism not only explains the molecular basis of the aforementioned in vitro improvement in mitochondrial function but also elucidates the synergistic effects of local oxygen supply, matrix mechanical signals, and cellular metabolic reprogramming in bone regeneration, providing important theoretical support for achieving integrated bone-vascular regeneration.

[0059] Example 5: Comprehensive micro-CT and histological analysis of promoting bone defect repair in vivo In the process of bone defect repair in vivo, simple material filling or physical stimulation often fails to achieve sustained, structurally intact, and highly mature bone regeneration. The fundamental reasons lie in the hypoxic microenvironment, limited energy metabolism, and the temporal imbalance of osteogenic-angiogenic processes. Therefore, this invention combines micro-CT imaging and multi-level histological analysis to systematically evaluate the effect of Alg-W1 / O / W2+NIR on promoting bone defect repair in vivo. The animal experimental procedure is as follows: Figure 7 As shown in Figure A.

[0060] All animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University Affiliated Tongren Hospital (Approval No.: A2025-012-01), strictly following national and institutional guidelines regarding the care and use of laboratory animals. Six- to eight-week-old male SD rats were randomly divided into four groups (control group, NIR group, W1 / O / W2 group, and Alg-W1 / O / W2+NIR group), with 10 rats in each group. During the experiment, the rats underwent surgery under anesthesia (2% sodium pentobarbital, 40 mg / kg, intraperitoneal injection). Under aseptic conditions, a standard cylindrical bone defect with a diameter of 3 mm and a depth of 3 mm was prepared on the distal lateral femur of the rats using a miniature electric drill, and the corresponding material was implanted for treatment. All rats were injected with antibiotics for 3 days post-surgery to prevent infection. During this period, they were kept in routine feeding conditions, and their post-operative behavior, diet, and general condition were monitored to ensure animal welfare. Samples were collected at predetermined time points post-surgery for subsequent analysis. The grouping of animal experiments was the same as that of cell experiments.

[0061] Femoral specimens from each group were harvested at 4 and 8 weeks post-surgery and fixed in tissue fixative for 3 days. The specimens were then scanned using a Micro-CT system, and three-dimensional reconstruction analysis of the defect areas was performed. Based on the defined regions of interest (ROIs), the bone volume / tissue volume (BV / TV), bone mineral density (BMD), and trabecular number (Tb.N) were quantitatively analyzed to evaluate the promoting effect of different treatment groups on bone defect repair.

[0062] To assess tissue repair and osteogenic changes in the bone defect area, femoral specimens from each group were collected postoperatively for histological and immunological examinations. After fixation, decalcification, and paraffin embedding, 5μm thick serial sections were prepared from the specimens. Hematoxylin-eosin (H&E) staining was used to observe new bone formation and morphological changes in the defect area, while Masson's trichrome staining was used to assess collagen deposition and bone matrix remodeling. Collagen I (COL I; Shanghai Epizyme) expression was detected by immunohistochemistry, and images were acquired and analyzed after DAB staining.

[0063] For immunofluorescence staining, dual immunofluorescence staining with osteocalcin (OCN, Cell Signaling Technology) and CD31 (Shanghai Epizyme) was used to assess osteogenic and angiogenesis-related changes. TOMM20 antibody (Abcam, USA) was used to detect mitochondrial-related signals, and perilipin 2 antibody (PLIN2, Proteintech) was used to detect the expression level of lipid droplets-related proteins in the defect area. All immunofluorescent sections were counterstained with DAPI in the cell nuclei. After image acquisition, ImageJ software was used for quantitative analysis of the staining signal intensity of each group. To further assess the in vivo biocompatibility of the materials, major organs, including the heart, liver, spleen, lungs, and kidneys, were collected simultaneously during tissue sampling. After fixation, paraffin embedding, and sectioning, H&E staining was performed to observe the morphology of each organ tissue and the presence of obvious inflammatory infiltration or pathological damage.

[0064] All experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPadPrism 10.0 software (GraphPad Software, USA). Two-tailed unpaired t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups, with Bonferroni post-hoc tests performed. A p-value less than 0.05 was considered statistically significant (p < 0.05). All experiments were independently repeated at least three times to ensure the reliability of the results.

[0065] The results showed that at 4 weeks, the defect areas in the control group and the NIR-only group were still mainly composed of large cavities and sparse bone tissue, indicating limited spontaneous repair capacity. The W1 / O / W2 group showed some degree of new bone formation, but overall filling was still insufficient. In contrast, the Alg-W1 / O / W2+NIR group showed significant new bone ingrowth and defect filling at 4 weeks, and by 8 weeks, the defect area was basically covered by continuous and dense new bone tissue. Figure 7 (Figures B and C). Combined with pseudo-color images, it can be observed more intuitively that the bone tissue distribution in the defect area of ​​the Alg-W1 / O / W2+NIR group is more uniform, and the trabecular bone network is more complete, suggesting that this system not only promotes bone mass increase but also improves the overall bone structure reconstruction of the defect area. Micro-CT quantitative analysis further confirmed the above observations. Compared with the control group and the NIR group, the bone volume fraction (BV / TV) of the Alg-W1 / O / W2+NIR group was significantly increased at 4 and 8 weeks postoperatively. Figure 7 In the middle D, bone mineral density (BMD) also increased significantly. Figure 7 (E), while the number of small beams (Tb.N) significantly increased ( Figure 7 The results (F) indicate that the system can continuously promote bone tissue formation and improve the quality of bone microstructure. Notably, compared with the W1 / O / W2 system that has not been further assembled into cross-linked microspheres, the Alg-W1 / O / W2+NIR group showed a more significant repair advantage in all indicators, indicating that the structured delivery system after cross-linking into microspheres can more effectively construct a local oxygen supply microenvironment under near-infrared triggering, thereby promoting bone defect repair.

[0066] To further verify the quality of bone regeneration at the histological level, H&E and Masson staining analyses were performed on samples at 4 and 8 weeks post-surgery. H&E staining results showed that the defect areas in the control and NIR groups were mainly filled with fibrous tissue and a small amount of immature bone tissue, with sparse and poorly continuous trabecular bone structure. A certain amount of new bone formation was observed in the W1 / O / W2 group, but significant unrepaired areas remained in the defect area. In contrast, the Alg-W1 / O / W2+NIR group showed significant new bone formation at 4 weeks, and by 8 weeks, the defect area was further filled with a more complete and regularly arranged trabecular bone network. Figure 8 Masson staining results further showed that the Alg-W1 / O / W2+NIR group had richer collagen matrix deposition in the defect area, with a wider and more continuous blue-stained area, suggesting that it can effectively promote bone matrix deposition and tissue maturation. Figure 8 (B)

[0067] Immunohistochemical and immunofluorescence analyses further supported these results at the molecular and cellular levels. Immunohistochemical results showed that the Alg-W1 / O / W2+NIR group exhibited stronger expression signals of the osteogenic protein COL-1 at both 4 and 8 weeks post-surgery. Figure 8 The results (C) suggest that this system can continuously activate the osteogenic process in vivo. Immunofluorescence results showed that the fluorescence signals of mitochondrial marker protein TOMM20, vascular endothelial marker CD31, and osteogenic marker protein OCN were significantly enhanced in the Alg-W1 / O / W2+NIR group. Figure 8 (D) further indicates that while promoting osteogenicity, this system is accompanied by a more active mitochondrial metabolic state, more favorable angiogenesis, and enhanced bone formation activity.

[0068] In terms of biosafety assessment, H&E staining analysis was performed on major organs including the heart, liver, spleen, lungs, and kidneys. Figure 9 The images show H&E staining of major organs such as the heart, kidneys, liver, lungs and spleen in different treatment groups. The tissue structures of the major organs in each group are intact, and no obvious inflammatory infiltration, necrosis or other pathological damage is observed, indicating that the microsphere system has good tissue compatibility and system safety in in vivo application.

[0069] In summary, the results of micro-CT, histological, and immunological analyses corroborate each other at the macroscopic, tissue, and molecular levels, systematically demonstrating that Alg-W1 / O / W2+NIR can significantly promote bone defect repair in vivo. This repair effect is not only manifested in increased bone mass but also in enhanced bone tissue continuity in the defect area, maturation of trabecular bone structure, and overall improvement in bone matrix deposition and bone-vascular coupling. Combined with the aforementioned mechanistic studies, this high-quality bone regeneration likely originates from the near-infrared responsive oxygen-supplying microenvironment constructed in vivo by the microspheres. This microenvironment effectively repairs hypoxic bone defects by alleviating local hypoxia, reducing abnormal lipid droplet accumulation, improving mitochondrial energy metabolism, and promoting osteogenic-related microenvironment remodeling.

[0070] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. An oxygen-catalyzing gel microsphere, characterized in that, The microspheres include: Calcium alginate hydrogel framework; and Multiple separate microreaction chambers are embedded within the calcium alginate hydrogel framework. Each of the micro-reaction chambers independently contains ozone oil and graphene.

2. The oxygen catalytic gel microspheres according to claim 1, characterized in that, The microreaction chambers are spherical or near-spherical substructures, and multiple microreaction chambers are distributed in an array within the hydrogel framework.

3. The oxygen catalytic gel microspheres according to claim 1, characterized in that, The microspheres also contain platelet-derived growth factor BB.

4. The method for preparing oxygen catalytic gel microspheres according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Dissolve polyvinyl alcohol in graphene dispersion to prepare inner aqueous phase W1; dissolve polylactic acid-glycolic acid copolymer in organic solvent and add ozone oil to prepare oil phase O; dissolve sodium alginate in water to prepare outer aqueous phase W2. Step 2: Under ice-water bath conditions, the inner aqueous phase W1 is added to the oil phase O in batches, and ultrasonic emulsification is performed after each addition to form a stable W1 / O nanoemulsion. Step 3: The W1 / O nanoemulsion is added to the external aqueous phase W2 in batches, and ultrasonic treatment is continued after each addition to obtain W1 / O / W2 dual nanoemulsion. Step four: The W1 / O / W2 dual nanoemulsion is added dropwise to a crosslinking solution containing calcium ions through a microfluidic electrospray system, causing sodium alginate to undergo in-situ ionic crosslinking and solidify to form the oxygen catalytic gel microspheres.

5. The preparation method according to claim 4, characterized in that, In the inner aqueous phase W1, the concentration of graphene dispersion is 0.5~2 mg / mL, and the concentration of polyvinyl alcohol is 10~15 mg / mL; in the oil phase O, the organic solvent is dichloromethane, the concentration of polylactic acid-glycolic acid copolymer is 80~120 mg / mL, and the volume fraction of ozone oil is 2%~5%; in the outer aqueous phase W2, the mass fraction of sodium alginate is 0.5%~2%.

6. The preparation method according to claim 4, characterized in that, The external aqueous phase W2 also contains a bioactive factor, namely platelet-derived growth factor BB.

7. The preparation method according to claim 4, characterized in that, The batch addition in step two refers to adding the inner aqueous phase W1 to the oil phase O in 4 to 8 portions of 0.1 to 0.3 mL each time; the batch addition in step three refers to adding the W1 / O nanoemulsion to the outer aqueous phase W2 in 4 to 8 portions of 0.1 to 0.3 mL each time.

8. The preparation method according to claim 4, characterized in that, The calcium-containing crosslinking solution is an aqueous solution of calcium chloride with a concentration of 1 mol / L.

9. The preparation method according to claim 4, characterized in that, The microfluidic electrospray system operates at a voltage of 10kV and has a propulsion speed of 5mL / h.

10. The use of the oxygen catalytic gel microspheres as described in any one of claims 1-3 in the preparation of products that promote bone regeneration.

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