Preparation and application of pomegranate bionic microgel / periosteum composite material

Abstract

The invention discloses preparation and application of a pomegranate bionic microgel / periosteum composite material, and belongs to the technical field of biomedical engineering. The composite material (PBC) is a'membrane-seed 'hierarchical system, the'membrane' component is composed of a poly-L-lactic acid (PLLA) stent modified by polydopamine (PDA), simulates a pomegranate outer membrane, and has the effects of oxidation resistance, protection and stabilization; according to the seed component, poly (3, 4-ethylenedioxythiophene)-poly (styrenesulfonic acid) (PEDOT: PSS) and methacrylated gelatin (GelMA) are combined to form conductive hydrogel microspheres, pomegranate seeds are simulated, electrical stimulation can be transmitted, and the redox cycle of PDA can be maintained. Reactive oxygen (ROS) is removed through PDA, the conductive microspheres promote catechol regeneration, mitochondrial metastasis between macrophages and osteoblasts is enhanced, the bone defect microenvironment is remarkably improved, and functional bone regeneration is promoted. The material is suitable for bone defect repair, and has excellent biocompatibility, mechanical property and long-acting antioxidant function.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to the preparation and application of a pomegranate-inspired biomimetic microgel / periosteum composite material. Background Technology

[0002] Bone defects caused by tumor resection, traumatic fractures, inflammation, and osteonecrosis pose significant clinical challenges. It is now well understood that osteogenic processes are not solely dependent on osteoblasts within the skeletal system, but rather the result of complex synergistic interactions among multiple biological systems. Increasing evidence highlights the close interaction between the immune system and the skeletal system, manifested in their sharing of various cytokines, receptors, signaling molecules, and transcription factors. Natural bone healing involves a series of phases: inflammation, repair, and remodeling. However, persistent inflammation and oxidative stress disrupt this process, creating a vicious cycle that further hinders bone regeneration. The dynamic and interdependent nature of the bone defect microenvironment limits the effectiveness of current treatments. This underscores the need for innovative strategies to break this pathological cycle and restore functional bone regeneration.

[0003] Polydopamine (PDA) has emerged as a promising material in bone tissue engineering in recent years due to its unique chemical structure and multifunctional biomimetic properties. Structurally similar to melanin and polyphenolic compounds, PDA exhibits excellent biocompatibility and low immunogenicity. A key advantage of PDA is its ability to spontaneously form uniform, viscous coatings on various material surfaces through self-polymerization, effectively improving the bioinertness of many traditional bone scaffolds. Notably, PDA contains catechol functional groups, which can scavenge reactive oxygen species (ROS) in inflammatory environments, thereby protecting osteoblasts from oxidative stress-induced damage. Although PDA has shown some effectiveness in bone defect repair, its catechol groups are prone to rapid oxidation under inflammatory conditions, limiting its long-term antioxidant function. After neutralizing ROS, the catechol groups are oxidized to quinone structures. These oxidized groups can be regenerated into reduced catechol forms by accepting electrons (from endogenous currents or biological reducing agents). This redox cycle mechanism is the basis for the sustained antioxidant activity of PDA. Enhancing this regenerative process—especially by introducing an endogenous electric field—may help maintain the antioxidant potential of PDAs in the long term. In this context, conductive hydrogels have attracted attention due to their electron transport capabilities, which could help PDAs achieve sustained ROS clearance. Compared to bulk scaffolds, microspheres offer additional advantages such as injectability, high surface area, and suitability for minimally invasive delivery. However, microspheres are prone to displacement after implantation, and soft tissue interference may hinder the osteogenic process. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a pomegranate-inspired biomimetic microgel / periosteum composite material and its application. The material (PBC) described in this invention is used for bone defect repair. This hierarchical system integrates two components—a "membrane" and "seeds"—which respectively mimic the outer membrane and internal seeds of a pomegranate.

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

[0006] This invention discloses a pomegranate-inspired bio-gel / periosteum composite material, characterized in that it comprises: Bionic periosteum layer: composed of poly-L-lactic acid (PLLA) modified with polydopamine (PDA); Conductive microgel layer: Dispersed on the surface of the periosteal layer, it is formed by a conductive hydrogel containing poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) (PEDOT:PSS) and methacrylamide gelatin (GelMA), with a microgel particle size of 160–170 µm.

[0007] Furthermore, the PDA coating thickness of the biomimetic periosteum layer is 0.5–5µm, and the adhesion strength is ≥25 kPa.

[0008] Furthermore, the mass ratio of PEDOT:PSS in the conductive microgel is 0.05–0.2%; the conductivity of the conductive hydrogel is 0.08–0.12 mS / cm; and the compressive strength of the conductive microgel is 130–150 kPa.

[0009] This invention also discloses a method for preparing the pomegranate biomimetic microgel / periosteum composite material according to any one of the above claims, characterized in that it includes: (1) Preparation of PLLA / PDA membrane: PLLA particles were dissolved in a mixed solvent of dichloromethane (DCM) and N,N-dimethylformamide (DMF) to form a 10wt% suspension. After spin coating, the membrane was immersed in a mixed solution containing 95% PDA solution and 5% ethanol and soaked at 4°C for 24h. After washing, the membrane was obtained. (2) Preparation of GelMA prepolymer: Dissolve gelatin in PBS, stir at 50°C to dissolve, add methacrylic anhydride (MA) dropwise, react at 50°C for 2 h, dialyze and freeze dry to obtain the product; (3) GelMA / PEDOT:PSS conductive hydrogel microspheres were prepared by microfluidic technology: the dispersed phase was a GelMA / PEDOT:PSS prepolymer solution, the continuous phase was a 2:1 mixture of mineral oil and Span80, the flow rate of the continuous phase was 1µL / min, the flow rate of the dispersed phase was 2µL / min, and microspheres with a size of 160-170µm were obtained after UV curing. (4) The PLLA / PDA membrane from step (1) and the microspheres from step (3) are bonded together by adhesion to obtain a composite material.

[0010] Furthermore, in the mixed solvent of (1), the volume ratio of DCM to DMF is 9:1.

[0011] Furthermore, the final volume concentration of PEDOT:PSS in the conductive hydrogel microspheres in (3) is 0.1%.

[0012] Furthermore, in step (3), the GelMA / PEDOT:PSS prepolymer solution contains 1 wt% photoinitiator Irgacure2959.

[0013] The present invention also discloses the application of the pomegranate biomimetic microgel / periosteum composite material described in any of the above claims in the preparation of bone defect repair drugs.

[0014] The present invention also discloses the application of the pomegranate biomimetic microgel / periosteum composite material described in any of the above claims in the preparation of medical devices for bone defect repair.

[0015] Furthermore, the material enhances bone regeneration efficiency by promoting macrophage M2 polarization and mitochondrial transfer to osteoblasts.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] Inspired by the structure of pomegranate, this invention develops a biomimetic microgel / periosteum composite (PBC) biomaterial for bone defect repair. This hierarchical system integrates two components: a "membrane" and "seeds." The biomimetic periosteum is constructed using a polydopamine-modified poly-L-lactic acid (PLLA) scaffold, mimicking the seed coat and seed structure of a pomegranate, forming a new generation of guided bone regeneration membrane with both protective and osteogenic functions. The "seeds" are formed by combining the electrochemically stable conductive polymer poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) (PEDOT:PSS) with a methacrylamide gelatin (GelMA) matrix to form conductive hydrogel microspheres. The PDA-coated membrane effectively neutralizes reactive oxygen species (ROS) at the injury site through catechol groups, while simultaneously protecting, supporting, and stabilizing the internal microgel. The microgel, acting as a filler in the defect area, simulates the natural electrophysiological environment by transmitting electrical stimulation, thereby maintaining the catechol-quinone redox balance and achieving long-lasting antioxidant effects. The study further revealed that the PBC system can enhance mitochondrial transfer between macrophages and osteoblasts, providing new insights into the mechanism by which catechol groups promote osteoogenesis. Attached Figure Description

[0018] Figure 1Preparation and properties of PLLA and PLLA / PDA films. (A) Typical SEM image of PLLA film. (B) Typical SEM image of PLLA / PDA film. (C) FT-IR spectra of PLLA and PLLA / PDA films. (D) Stress-strain curves of PLLA and PLLA / PDA films. (E) Tensile strength of PLLA and PLLA / PDA films. (F) Contact angle of PLLA and PLLA / PDA films. (G) Adhesive strength of PLLA and PLLA / PDA films. (H) 3D morphology image of PLLA / PDA film structure. (I) Electro-response signal of PLLA / PDA film. *P<0.05, ***P<0.001.

[0019] Figure 2 Preparation and characterization of GelMA and GelMA / PEDOT:PSS microhydrogels. (A) Exploring the effect of different concentrations of PEDOT:PSS on cell growth. (B) Effect of flow rate on the size of hydrogel microspheres. (C) Degradation of GelMA / PEDOT:PSS microspheres. (D) Swelling ratio of GelMA / PEDOT:PSS microspheres. (E) Typical images of GelMA and GelMA / PEDOT:PSS microspheres. (F) Size distribution of GelMA / PEDOT:PSS microspheres. (G) Zeta potential of GelMA and GelMA / PEDOT:PSS hydrogels. (H) Cyclic voltammetry curves of GelMA / PEDOT:PSS hydrogels. (I) Conductivity of GelMA and GelMA / PEDOT:PSS hydrogels. (J) Stress-strain curves of GelMA and GelMA / PEDOT:PSS hydrogels. (K) Compressive strength of GelMA and GelMA / PEDOT:PSS hydrogels. *P<0.05, **P<0.01.

[0020] Figure 3 Biocompatibility assessment of the PBC system: morphology of cells after 1, 3 and 5 days of growth on the microsphere surface.

[0021] Figure 4Study on the immunomodulatory effects of the PBC system. (A) iNOS mRNA expression level in each group. (B) TNF-α mRNA expression level in each group. (C) IL-10 mRNA expression level in each group. (D) Arg-1 mRNA expression level in each group. (E) Flow cytometry detection of CD86 and CD206 expression levels and quantitative analysis of flow cytometry results. (F) Immunofluorescence detection of CD86 (green) and CD206 (red) expression levels and quantitative analysis of immunofluorescence results. (G) Detection of intracellular ROS generation by the DCFH-DA probe and quantitative analysis of ROS generation results. n=3, *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 5 The PBC system promotes enhanced macrophage metabolism and mitochondrial translocation. (AB) Differentially expressed genes (DEGs) of RAW264.7 macrophages cultured under LPS+PBC and LPS+Gel conditions are shown in heatmaps and volcano plots, respectively. (C) Gene Ontology (GO) analysis of DEGs. (DE) Gene set enrichment analysis (GSEA) of overall differences between the LPS+PBC and LPS+Gel groups. (F) Oxygen consumption rate (OCR) curves of RAW264.7 cells cultured under LPS+PBC and LPS+Gel conditions. (G) Extracellular acidification rate (ECAR) curves of RAW264.7 cells cultured under LPS+PBC and LPS+Gel conditions. (H) ATP production quantified from OCR curves. (I) Basal respiration quantified from OCR curves. (J) Maximum respiratory capacity quantified from OCR curves. (K) Respiratory reserve capacity quantified from OCR curves. (L) Schematic diagram of mitochondrial translocation. (M) Number of mitochondria in the culture medium of macrophages in the LPS+PBC group and the LPS+Gel group. (N) Number of macrophage-derived mitochondria in MC3T3-E1 cells as detected by flow cytometry. (O) Detection of macrophage-derived mitochondria levels in MC3T3-E1 cells by immunofluorescence, and quantitative analysis of the immunofluorescence results. **P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 6Mitochondrial transfer promotes osteogenic differentiation. (A) Schematic diagram of conditioned culture of MC3T3-E1 cells. (B) Quantitative statistics of MC3T3-E1 cells after 6 h of migration. (C) Quantitative statistics of MC3T3-E1 cells after 12 h of migration. (D) In ​​vitro study on the effect of mitochondrial transfer on osteogenic differentiation capacity. (E) Oxygen consumption rate (OCR) curves of MC3T3-E1 cells co-cultured with RAW264.7 macrophages of different treatment groups. (F) ATP production quantified according to OCR curves. (G) Basal respiration quantified according to OCR curves. (H) Maximum respiratory capacity quantified according to OCR curves. **P<0.05, **P<0.01, ***P<0.001.

[0024] Figure 7 In vivo macrophage polarization and bone regeneration. (A) Dual immunofluorescence staining of CD68 (macrophage) and CD86 (M1 type) in the defect area. (B) Dual immunofluorescence staining of CD68 (macrophage) and CD206 (M2 type) in the defect area. (C) Micro-CT image of the rat mandible at the implantation site. (D) Hematoxylin and eosin (H&E) staining and Masson's trichrome staining in the defect area. n=4 (sample size n=4 per group). (E) Heatmap of relative metabolite abundance between the Gel group and the PBC group based on LC-MS / MS analysis. (F) KEGG pathway enrichment of differentially expressed metabolites. **P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0026] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0027] I. Methods.

[0028] 1. Preparation of a pomegranate-inspired biomimetic microgel / periosteum composite system.

[0029] 1.1 Preparation of PLLA / PDA membrane. Poly-L-lactic acid (PLLA) particles (Aladdin, China) were dissolved in a mixed solvent of dichloromethane (DCM, Aladdin, China) and N,N-dimethylformamide (DMF, Aladdin, China) (DCM:DMF volume ratio 9:1) to prepare a 10 wt% PLLA particle suspension. The solution was then homogenized using a rotary homogenizer (KW-4A, Institute of Microelectronics, Chinese Academy of Sciences, China) to form a uniform membrane. After drying, the resulting film was carefully peeled off and cut to the desired size. For polydopamine (PDA) surface modification, a dopamine solution was prepared using Tris-buffered saline (10 mM, pH 8.5). The PLLA membrane was immersed in a mixed solution of 95% PDA solution and 5% ethanol (v / v) at 4 °C for 24 h to promote PDA grafting onto the fiber surface. Finally, the modified membrane was thoroughly rinsed five times with deionized water to remove any unbound residue.

[0030] 1.2 Preparation of GelMA. 20 g of gelatin was added to 200 mL of PBS solution and completely dissolved under magnetic stirring at 50 °C and 240 rpm. Under continuous stirring, 16 mL of methacrylic anhydride (MA) was added dropwise to the dissolved gelatin solution at a rate of 0.2 mL / min using a microinjection pump. The mixture was reacted for 2 h under magnetic stirring at 50 °C and 240 rpm. 50 mL of PBS solution preheated to 50 °C was added to the mixture, followed by magnetic stirring at 50 °C for 10 min. The reaction solution was then transferred to a dialysis bag and dialyzed against deionized water for 10 days at 40 °C with stirring at 500 rpm to remove salts and unreacted MA. After dialysis, 400 mL of deionized water was added to the dialysate, the mixture was heated to 40 °C, and stirred for 15 min. Finally, the solution was aliquoted into 5 mL centrifuge tubes and frozen at -80 °C for 2 days. The frozen sample was freeze-dried for 4 days to obtain a white, foamy GelMA prepolymer, which was then stored in a desiccator at room temperature for later use.

[0031] 1.3 Fabrication of the microfluidic device. This PDMS microfluidic chip features a flow focusing sheath (50 µm wide) for generating O / W droplets and a 100 µm wide meandering channel (75 µm deep, rectangular cross-section) for initial solvent evaporation and phase separation, as well as a dispersed / continuous phase inlet and a collection chamber. The fabrication process began with the preparation of an SU-8 mold, followed by pouring a degassed 10:1 PDMS / curing agent mixture onto the mold and curing at 80 °C for 40 min to form a 5 mm thick channel layer and a 1 mm thick substrate layer. After perforation, the two layers were plasma-bonded. For hydrophilic surface modification, the channel was treated with a 0.3 wt% PVA solution (flow rate 0.1 µL / min, treatment for 60 min) and then dried with nitrogen.

[0032] 1.4 Preparation of GelMA / PEDOT microspheres. A 7.5 wt% aqueous solution of GelMA hydrogel prepolymer was prepared by ultrasonic dispersion, with 1 wt% of photoinitiator Irgacure 2959 (BASF, Germany) added to the solution. The PEDOT:PSS solution (Aladdin, China) was filtered (0.22 µm filter membrane) to remove large particle aggregates, and the concentration of the filtered solution was determined to be 0.9 wt% by drying and weighing. The well-dispersed PEDOT:PSS solution was added to the GelMA solution at 0.1%, 0.2%, 0.3%, and 0.4% of the final volume, respectively. Undissolved particles in the prepolymer solution were ultrasonically dispersed again at 4 °C (KQ-100DB, Kunshan, China) for 30 min. The GelMA / PEDOT hydrogel prepolymer solution was used as the dispersed phase, and mineral oil and Span80 were formulated at a volume ratio of 2:1 as the continuous phase. The continuous phase and dispersed phase were connected to the chip inlet, respectively, and slowly injected into the channel using a syringe pump (LSP02-2A, Lange constant flow pump, China) at a continuous phase flow rate of 1 µL / min and a dispersed phase flow rate of 2 µL / min. Microdroplets with a particle size between 200 µm and 250 µm were cured by UV light and washed with ethanol and acetone. The washed microspheres were then lyophilized. The prepared microspheres and membrane were then implanted as a single unit into the defect site of rats via adhesion.

[0033] 2. Characterization of the pomegranate-inspired biomimetic microgel / periosteum composite system.

[0034] 2.1 Characterization of PLLA / PDA films. The surface morphology of the PLLA / PDA films was observed using a stereomicroscope with a digital camera (MC-D500U, Phoenix Optics, China), an atomic force microscope (Bruker, DimensionICON), and a scanning electron microscope (SEM, S-4800, Hitachi, Japan). Characteristic chemical groups of the films were analyzed using Fourier transform infrared spectroscopy (FTIR, ALPHA II, Bruker, Germany). To investigate the mechanical properties of the films (size: 24mm × 20mm × 0.07mm), a micro-force testing system was used. The applied strain rate was 0.1mm / s, and the test continued until the film fractured. The contact angles of the PLLA and PLLA / PDA films were measured using an interfacial tensiometer (DSA30, Kruss, Germany). Adhesion tests were performed at room temperature using a universal testing machine (WD-5A, Guangzhou Experimental Instrument Factory, China), with the tested film measuring 20mm × 20mm square. Additionally, a piece of bone was adhered to a plastic plate. First, the membrane was adhered to a moist bone surface under a pressure of 170 kPa for 2 minutes. Then, the ends of the membrane and the plastic plate were clamped and stretched upwards at a speed of 1 mm / min. At the same time, the adhesion force was determined using an overlap shear test, and the bond strength was calculated by dividing the maximum stress by the initial adhesion area.

[0035] 2.2 Characterization of GelMA / PEDOT:PSS hydrogel microspheres. The morphology of the GelMA / PEDOT microspheres was observed using a stereomicroscope with a digital camera (MC-D500U, Phoenix Optics, China) and a scanning electron microscope (SEM, S-4800, Hitachi, Japan). The dimensions of the GelMA / PEDOT microspheres were measured using ImageJ software (version 1.53v, NIH, USA). Four images were used for measurement, with 50 microspheres measured per image. To obtain the mechanical properties of the microspheres, a compression test was performed on 60 µL cylindrical gels cross-linked in a cylindrical mold (6.0 mm diameter × 2.0 mm height). The gel was tested after swelling in PBS at 37 °C for 16 h to reach the maximum swelling ratio. The hydrogel was compressed to 70% strain at a rate of 1 mm / min in a PBS bath at room temperature. The zeta potentials of GelMA hydrogel and GelMA / PEDOT hydrogel were measured using a zeta potentiometer (ZS90, Malvern, UK). Electrochemical impedance spectroscopy (EIS) was performed at room temperature using an electrochemical workstation (Austin, TX, USA). This setup consisted of two 12mm × 12mm glass slides coated with a 100nm gold layer (Sigma-Aldrich). Electrodes were connected by silver-plated copper wire and covered with epoxy resin to prevent oxidation of the silver layer. A cylindrical hydrogel (60µL) was swollen in deionized water at 37°C for 16h. After fixation, data were measured and recorded at ±10mV AC amplitude within a frequency range of 0.1 to 100Hz. The conductivity of hydrogels at different concentrations was studied using a Fluke F17B+ digital multimeter. Conductivity was calculated using the formula: σ = L / (R × S), where L is the hydrogel height, R is the hydrogel resistance, and S is the hydrogel contact area. The hydrogel resistance was measured three times and the average value was taken. To determine the degradation rate of the microspheres, the lyophilized microspheres were placed in pre-weighed centrifuge tubes containing PBS solution. After swelling equilibrium was reached, the supernatant was removed by centrifugation, and the weight of the centrifuge tube containing the microspheres was recorded as W0. The microspheres were then placed in PBS solution containing 0.1 µM I collagenase. At specified time points, the supernatant was discarded by centrifugation, and the weight W of the degraded microspheres was measured. t The formula for calculating the degradation rate (DR) is: (W0 – W tThe swelling ratio (SR) was calculated as (W_wet – W_dry) / W_dry × 100%. For the swelling ratio of the microspheres, the microsphere samples were lyophilized, and the dry weight (W_dry) was recorded. The samples were then immersed in PBS and placed in an incubator. Samples were removed and weighed at different time points (0, 4, 8, 12, and 16 h). The swelling ratio (SR) was calculated using the formula: (W_wet – W_dry) / W_dry × 100%. After co-culturing cells with microspheres for 1, 3, and 5 days, cells were stained using a calcein-AM / PI staining kit (C2015S; Beyotime, China) or a phalloidin / DAPI staining kit (CA1620-300T; Solarbio, China), and observed using a confocal laser scanning microscope (Multizoom AZ-C2+; Nikon, Japan) or a fluorescence microscope (Axioscope 5; Carl Zeiss, Germany).

[0036] 3. In vitro induction of macrophage polarization.

[0037] 3.1 Macrophage Culture. RAW264.7 cell lines were cultured at 37°C using RAW 264.7 cell-specific medium (TCM-C766, Hycyte, China). RAW 264.7 cells were treated with 500 ng / mL lipopolysaccharide (LPS) for 3 h. Subsequently, inflammatory cytokine levels, macrophage polarization, and oxidative stress status were assessed.

[0038] 3.2 ROS detection. Cells were grouped as described above, with cells treated with H2O2 (1×10⁻⁶ cells)... 4 cells / cm 2 Cells were inoculated and cultured for 24 hours. Intracellular ROS were stained using a reactive oxygen species (ROS) detection kit (Beyotime). After staining, images were acquired using a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed using ImageJ software.

[0039] 3.3 RT-qPCR analysis of macrophage polarization. Total RNA was extracted from RAW264.7 cells after 3 days of culture. RT-qPCR analysis was performed to detect the expression of TNF-α, ARG-1, iNOS, and IL-10. Through 2... −ΔΔCt The relative expression levels of the target gene were standardized relative to the internal reference gene and expressed as mean ± standard deviation (mean ± SD). The primer sequences used for RT-qPCR are shown in Table 1.

[0040] Table 1. Gene and primer sequences.

[0041] 3.4 Immunofluorescence staining. The effect of the PBC system on macrophage polarization was investigated using immunofluorescence staining. Cells were fixed with 4% paraformaldehyde for 30 min and blocked with 5% bovine serum albumin for 2 h at room temperature. Samples were incubated overnight at 4°C with CD86 primary antibody (1:1000, 13395-1-AP, Proteintech, China) and CD206 primary antibody (1:1000, 18704-1-AP, Proteintech, China). Macrophages incubated with CD86 were subsequently incubated for 1 h with goat anti-rabbit IgG, Dylight 488 (A23220, Abbkine, China), while macrophages incubated with CD206 were incubated for 1 h with goat anti-rabbit IgG, Dylight 649 (A23620, Abbkine, China). Cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI, Solarbio, China) for 5 min. After staining, cells were observed under a laser confocal microscope, and fluorescence intensity was quantified using ImageJ software.

[0042] 3.5 Flow cytometry. Cells were collected, labeled with antibodies against CD11b (101205, BIOLEGEND, USA), CD86 (159203, BIOLEGEND, USA), and CD206 (141719, BIOLEGEND, USA), and washed with PBS. CD11b+ / CD86+ macrophages or CD11b+ / CD206+ macrophages were analyzed by flow cytometry (BD Biosciences, USA).

[0043] 4. RNA Sequencing (RNA-seq) and Data Analysis. As mentioned earlier, two groups of RAW 264.7 macrophages (LPS-stimulated cells treated with the PBC system: LPS+PBC group; LPS-stimulated cells treated with blank microgel microspheres: LPS+Gel group) were cultured for 3 days. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA sequencing was performed by Shanghai Jikai Gene Chemical Technology Co., Ltd. (China). The screening threshold for differentially expressed transcripts was P<0.05 and fold change ≥1. Functional and signaling pathway enrichment analyses were performed on differentially expressed transcripts using the Gene Oncology (GO) database. Differences in predefined gene sets were analyzed using GSEA (v4.1.0) software.

[0044] 5. Macrophage Mitochondrial Transfer Detection. Mitochondria were stained using MitoTracker Red CMXRos (C1049B, Beyotime, China) and MitoTracker Green FM (C1048, Beyotime, China). The staining solution was diluted with culture medium and added to the cells to be tested. After incubation at 37°C for 30 min, the cells were washed with PBS to remove excess stain. The staining results were observed using a fluorescence microscope, flow cytometry, and a microplate reader (Synergy H1, BioTek).

[0045] 6. Metabolic Studies. Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) of cells were measured using a Seahorse XFe96 analyzer (Seahorse Bioscience, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. For OCR measurement, treated macrophages were seeded in Seahorse XF DMEM medium (supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine). The following compounds were injected into the corresponding ports: 1 µM oligomycin (Port A), 1.5 µM FCCP (Port B), 0.5 µM rotenone, and antimycin A (Port C). For ECAR measurement, 10 mM glucose, 1.5 µM oligomycin, and 50 mM 2-deoxyglucose (2-DG) were added.

[0046] 7. In vitro osteogenesis.

[0047] 7.1 Inhibition of macrophage mitochondrial respiration. To investigate whether transferred mitochondria play a role in regulating energy metabolism, mitochondrial respiration in macrophages was inhibited: RAW 264.7 macrophages were treated for 3 h with 2 µM rotenone (Sigma-Aldrich, Cat#R8875) and 2 µM antimycin A (Sigma-Aldrich, Cat#A8674). These two inhibitors act on respiratory chain complex I and complex III, respectively.

[0048] 7.2 Cell Scratch Assay. RAW264.7 macrophages were cultured for 3 days according to the aforementioned groups (Gel group and PBC group). The culture supernatant was extracted and collected. Macrophage Conditioned Medium (MCM) and MC3T3-E1 cell-specific medium (CMM6-0501, OriCell, China) were mixed at a 1:1 ratio and used to culture MC3T3-E1 osteoblasts in six-well plates. After cell confluence, scratches were created on the MC3T3-E1 cells using a 200µL pipette tip, and cell growth was observed after 6 h and 12 h.

[0049] 7.3 Osteogenic Differentiation. Osteogenic differentiation was induced using the appropriate culture medium collected in Section 7.2, supplemented with 0.25 mM ascorbic acid, 10 nM sodium β-glycerophosphate, and 20 nM dexamethasone as osteogenic inducing factors. The conditioned medium was changed every 2 days during the first week, and every 3 days thereafter. MC3T3-E1 cells cultured for 7 and 14 days were stained using an alkaline phosphatase staining kit (C3206; Beyotime, China). Alizarin Red staining (ALIR-10001, OriCell, China) was performed on day 21.

[0050] 8. In vivo bone repair.

[0051] 8.1 Establishment of a rat model of borderline mandibular bone defect. The experimental rats were male Sprague-Dawley (SD) rats, weighing 250–300 g. The rats were purchased from Spfbiotech biotechnology Ltd. The rats were anesthetized with isoflurane. The submandibular region was disinfected and prepared, and a parallel incision of approximately 3 cm was made along the lower border of the mandible. The subcutaneous skin was sequentially incised, and the muscles and periosteum were bluntly dissected to the bone surface, exposing the lower border of the mandible and the buccal and lingual sides of the bone surface. After exposing the middle part of the mandible, a full-thickness bone defect with a diameter of 4 mm was prepared on the buccal and lingual side of the mandible (lower part of the mandible) using a fissure drill. During the operation, physiological saline was used for cooling. According to the aforementioned experimental groups (Gel group and PBC group), biomaterials were implanted into the defect site, and the wound was tightly sutured.

[0052] 8.2 Micro-CT Analysis. Three rats from each group underwent micro-CT scans at 2, 4, and 8 weeks post-surgery. In short, mandibular bone samples were fixed in 4% paraformaldehyde (pH 7.5) and subjected to micro-CT scanning (SKYSCAN 1276; Bruker, Germany). Scanning parameters included: 8µm resolution, 180° rotation, 100kV voltage, and 500µA current. Three-dimensional reconstruction of the mandible and analysis of internal bone volume were performed using CT Analyzer and VG Studio 2.1 software (Heidelberg, Germany).

[0053] 8.3 Histological staining. All samples were decalcified after micro-CT scanning. After decalcification, samples were trimmed, graded dehydrated, and then embedded in paraffin. The defect area was sectioned to approximately 5 µm thick and stained with hematoxylin and eosin (H&E) and Masson's stain. Immunofluorescence staining was then performed: samples harvested 2 weeks post-surgery were stained with anti-CD68 primary antibody (1:100, MCA341GA, Bio-Rad, USA), anti-CD86 primary antibody (1:600, 32223-1, Proteintech, China), and anti-CD206 primary antibody (1:500, 18704-1-AP, Proteintech, China) to assess macrophage polarization.

[0054] 9. Metabolomics Analysis. Regenerated tissue from the defect area was collected and washed several times with cold PBS. The samples were then frozen in liquid nitrogen for at least 15 min and stored at -80°C. The tissue was homogenized for 5 min at 40 Hz using a high-throughput tissue homogenizer. The following solvents were added sequentially: 400 µL of a 1:1 mixture of ethyl acetate and ethanol, 200 µL of anhydrous methanol, 200 µL of a 3:1 mixture of anhydrous methanol and water, and 200 µL of a 3:1 mixture of dichloromethane and methanol. After each addition, the homogenizer was shaken for 2 min, centrifuged for 5 min, and the supernatant was collected. All steps were performed on ice. The supernatants from the four extractions for each sample were combined, and 1 mL of the combined supernatant was transferred to an OA-SYS nitrogen blower and dried at room temperature. The residue was resuspended in 100 µL of a 4:1 mixture of methanol and ultrapure water, vortexed for 2 min, sonicated for 10 min, and centrifuged at 12,000 rpm for 10 min. Finally, 60 µL of the solution was used for LC-MS / MS analysis. 20 µL of the supernatant from the remaining extract of each sample was thoroughly mixed and stored as part of the quality control (QC) group.

[0055] 10. Statistical Analysis. Variables are expressed as mean ± standard deviation. Statistical analysis and graphing were performed using Origin 9.1 or GraphPadPrism 7.0 software. Unpaired or paired two-tailed Student's t-tests were used for comparisons between two groups. All experiments were repeated at least three times. Five randomly selected fields of view were observed under a microscope.

[0056] II. Results.

[0057] 1. Synthesis and characterization of pomegranate biomimetic microgel / periosteum composite system.

[0058] First, polylactic acid (PLLA) films were prepared using spin-coating technology. For example... Figure 1 As shown in Figure A, scanning electron microscopy (SEM) reveals a relatively smooth surface morphology. After polydopamine (PDA) coating treatment, a distinct brown layer is visible deposited on the PLLA surface, confirming successful surface modification. Figure 1 B).

[0059] Fourier transform infrared (FTIR) spectroscopy further validated the surface functionalization. The unmodified PLLA film exhibited characteristic peaks at 1188 cm⁻¹ and 1757 cm⁻¹, corresponding to the stretching vibrations of the COC and C=O bonds, respectively. After PDA coating, an additional absorption peak appeared at 1615 cm⁻¹, which is attributed to the bending vibrations of NH and / or NH₂ groups present in the PDA matrix. Figure 1 C). This spectral change confirms that the PDA was successfully integrated onto the PLLA film.

[0060] In terms of mechanical properties, PLLA / PDA films exhibit higher strength and ductility compared to uncoated PLLA films. Figure 1 D). The tensile strength increased from 1.07±0.05 kPa for PLLA to 1.19±0.02 kPa for PDA modification. Figure 1 E). Furthermore, the water contact angle decreased significantly after PDA treatment, indicating improved surface hydrophilicity. Figure 1 F).

[0061] To assess its adhesion ability under physiological conditions, an overlap shear test was performed using wet bone tissue. The results showed that PDA significantly enhanced adhesion: the PLLA / PDA membrane achieved an adhesion strength of 29.19 ± 1.98 kPa, while the unmodified PLLA membrane only achieved 5.70 ± 1.89 kPa. Figure 1 G). Atomic force microscopy (AFM) images ( Figure 1The H and I results show that the PLLA / PDA surface exhibits a smooth undulating morphology and displays electrical response behavior, which may be caused by the polarization characteristics of PLLA under the influence of the PDA interface.

[0062] In summary, the above physicochemical analysis confirms that the PDA-coated PLLA membrane possesses the key characteristics required for guided bone regeneration (GBR) applications: improved mechanical strength, enhanced surface wettability, and significantly improved adhesion to biological tissues.

[0063] To ensure that the hydrogel microspheres possess both biocompatibility and conductivity, we first optimized the doping concentration of PEDOT:PSS using the CCK-8 method. Figure 2 A). Compared with the control group, the addition of 0.1% PEDOT:PSS did not significantly change cell morphology or density, indicating good cell compatibility at this concentration. Therefore, 0.1% PEDOT:PSS was selected as the optimal formulation for subsequent experiments.

[0064] Microsphere size is a key parameter affecting cell attachment and biological performance, and it can be controlled by adjusting the flow rate in microfluidic systems. For example... Figure 2 As shown in Figure B, increasing the continuous phase flow rate proportionally increases microsphere yield while keeping the dispersed phase flow rate constant. At the optimal setting of a continuous phase flow rate of 3 µL / min and a dispersed phase flow rate of 1 µL / min, the microspheres exhibit a uniform diameter of approximately 170 µm. This size range has been shown to support effective cell adhesion, growth, and spatial distribution, which are crucial for therapeutic efficacy. The degradation curve of the GelMA / PEDOT:PSS hydrogel reaches equilibrium around day 24, while its maximum swelling ratio is reached within the first 12 hours after immersion in phosphate-buffered saline (PBS). Figure 2 (C and 2D). These results demonstrate its good mechanical integrity and water retention capacity, further confirming its suitability for defect filling. The morphology of cured GelMA and GelMA / PEDOT:PSS microspheres is as follows: Figure 2 As shown in Figure E. SEM analysis confirmed that both microspheres exhibit an interconnected porous network structure, and PEDOT:PSS is clearly visible and uniformly distributed on the surface. Size distribution analysis ( Figure 2 F) shows that the GelMA / PEDOT:PSS microspheres have a size ranging from 160 to 170 µm, maintaining compatibility for minimally invasive injection via syringe. Zeta potential measurements show that after adding PEDOT:PSS, the potential decreased from -2.96 ± 0.63 mV (GelMA) to -5.54 ± 0.29 mV (F). Figure 2 G), indicating an increase in electrostatic repulsion between microspheres and improved dispersion stability. Cyclic voltammetry (CV) analysis ( Figure 2H) further verified the improved conductivity: the conductivity of the GelMA / PEDOT:PSS hydrogel was 0.106±0.010 mS / cm, significantly higher than that of GelMA alone (0.046±0.008 mS / cm). Figure 2 I). Mechanical test results ( Figure 2 J) indicates that the addition of PEDOT:PSS improved the compressive stress and maximum strain. The compressive strength of the GelMA / PEDOT:PSS hydrogel significantly increased to 141.60 ± 2.50 kPa (J). Figure 2 (K), further confirming that the addition of conductive polymers improves the mechanical robustness of the material.

[0065] To investigate the response of macrophages to the pomegranate biomimetic composite system (PBC), we placed PBC components in a low-adhesion culture plate and seeded macrophages onto its surface. Cell behavior was monitored by immunofluorescence staining at different time points. Figure 3 To assess the biocompatibility of the microspheres, we performed a Calcein-AM / PI activity assay, supplemented by DAPI and phalloidin staining. Figure 3 The results showed that pre-osteoblasts adhered effectively to the surface of the microspheres and maintained a healthy morphology, with no deformation such as curling, shrinkage, or fragmentation observed. Notably, no dead cells were detected in the field of view, indicating excellent cellular compatibility.

[0066] By day 5, we observed a particularly interesting phenomenon: some microspheres appeared to interconnect, aggregating into plaque-like structures that expanded as cells grew. This behavior may be attributed to the addition of PEDOT:PSS. PEDOT:PSS is a conductive polymer whose role in promoting nerve regeneration and repair has been extensively studied due to its high conductivity. In our study, this similar "self-assembly" behavior between microspheres and macrophages may help enhance intercellular communication and structural integration, potentially supporting more organized tissue regeneration.

[0067] 2. Early inflammatory response regulation and ROS clearance.

[0068] To simulate the early inflammatory microenvironment of bone repair, macrophages were pretreated with lipopolysaccharide (LPS) and then co-cultured with either non-conductive GelMA microspheres (LPS+Gel group) or a conductive PBC system (LPS+PBC group). Macrophages untreated with LPS or exposed to hydrogel served as the control group (Ctrl). To assess the immunomodulatory effect of the PBC system on macrophage polarization, we examined the expression of M1 and M2 phenotypic markers. Quantitative PCR (qPCR) analysis showed that, compared to the LPS+Gel group, the expression of M1-related markers, including inducible nitric oxide synthase (iNOS), was significantly reduced in the LPS+PBC group. Figure 4 A) and tumor necrosis factor-α (TNF-α) Figure 4 B). Conversely, the expression of M2-related markers, such as interleukin-10 (IL-10), was significantly elevated. Figure 4 C) and arginase-1 (Arg-1) Figure 4 D).

[0069] These transcriptional results were further supported by flow cytometry analysis. Flow cytometry results showed that in the LPS+PBC group, the proportion of CD86+ (M1 type) macrophages decreased from 29.2% to 23.6%, while the proportion of CD206+ (M2 type) macrophages increased from 41.5% to 53.4%. Figure 4 E). Immunofluorescence staining results also confirmed this: in the LPS+PBC group, the green fluorescence of CD86 was weakened, while the red fluorescence of CD206 was enhanced (E). Figure 4 (F) further confirmed the polarization of macrophages towards the anti-inflammatory M2 phenotype. These results collectively indicate that under inflammatory conditions, the PBC system can significantly promote macrophage polarization towards the M2 phenotype, thereby supporting its potential to regulate the immune microenvironment and promote bone repair.

[0070] Given that macrophages are key mediators of post-injury inflammatory responses, and that reactive oxygen species (ROS) play a crucial role in influencing macrophage polarization, we also assessed intracellular ROS levels. Figure 4 As shown in Figure G, compared with the LPS+Gel group, the ROS level in the LPS+PBC group was significantly reduced, indicating that the PBC system can effectively alleviate oxidative stress in an inflammatory environment. This antioxidant effect can be attributed to the catechol and amino groups present in PDA, which are known for their strong free radical scavenging activity. Furthermore, the addition of conductive hydrogel material promotes the bioelectrical recovery of oxidized catechol groups, enabling them to maintain sustained redox activity over time. These properties collectively enable the PBC system to maintain a favorable redox balance, thereby creating an environment conducive to osteogenic formation.

[0071] 3. The PBC system promotes mitochondrial translocation in macrophages.

[0072] To investigate the potential mechanism of action of the PBC system, we performed RNA sequencing and compared the gene expression profiles between the LPS+Gel group and the LPS+PBC group. (Heatmap visualization follows.) Figure 5 A) and volcano map analysis ( Figure 5 B) shows that, compared with the LPS+Gel group, the LPS+PBC group had 208 upregulated genes and 42 downregulated genes (differentially expressed genes, DEGs).

[0073] Gene Ontology (GO) enrichment analysis identified the top 30 significantly enriched items, particularly microtubule-associated complexes (...). Figure 5 C). This suggests that mitochondrial translocation may be activated because microtubules are crucial for intracellular transport and serve as tracks for mitochondrial movement. Their polarity guides mitochondrial transport and plays a vital role in intercellular organelle communication. This emerging mechanism—intercellular mitochondrial translocation—is gaining increasing attention due to its role in cellular metabolism, immune regulation, wound healing, and other physiological processes. Further supporting this mechanism is gene set enrichment analysis (GSEA), which showed that differentially expressed genes (DEGs) in the LPS+PBC group were significantly enriched in metabolic pathways, including oxidative phosphorylation and the respiratory electron transport chain (…). Figure 5 DE).

[0074] To validate these transcriptomic findings, we used the Seahorse Cell Energy Metabolism Analyzer to analyze RAW264.7 macrophages. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) maps were obtained. Figure 5 FG indicates that the LPS+PBC group promoted ATP production ( Figure 5 H), Basic breathing ( Figure 5 I) Maximum breathing ( Figure 5 J) and reserve respiratory capacity ( Figure 5 Enhanced K) – all hallmarks of oxidative phosphorylation – simultaneously inhibited glycolytic activity. These data suggest that co-culturing with PBCs enhances the metabolic flexibility and respiratory efficiency of macrophages.

[0075] To explore whether mitochondrial translocation contributes to the observed metabolic enhancement, we used a dual staining method to visualize mitochondria in the co-culture system. Mitotracker Deep Red was used to stain the mitochondria of RAW264.7 macrophages. Simultaneously, Mitotracker Green was used to stain MC3T3-E1 cells (a mature, undifferentiated pre-osteoblast cell line widely used as a standard in vitro model for bone tissue engineering) to assess mitochondrial distribution during osteogenic differentiation. Mitochondrial translocation from macrophages (upper chamber) to osteoblasts (lower chamber) was then evaluated.

[0076] We first used a microplate reader to quantify the mitochondrial signal in the macrophage conditioned medium, and the results showed that the fluorescence signal in the LPS+PBC group was significantly higher. Figure 5 LM). Flow cytometry analysis of MC3T3-E1 cells co-cultured with these macrophages confirmed increased mitochondrial signaling. Figure 5 N). Furthermore, fluorescence microscopy detected a significant increase in red fluorescently labeled mitochondria in MC3T3-E1 cells of the LPS+PBC group (N). Figure 5 O), confirming the enhancement of mitochondrial transfer.

[0077] 4. Osteogenic effect of the PBC system through mitochondrial transfer.

[0078] To assess the functional impact of mitochondrial translocation on osteogenic activity, researchers cultured MC3T3-E1 cells using macrophage culture supernatant with different treatments. Figure 6 A). Scratch assays showed that, compared with the control group, the LPS+PBC group significantly accelerated the migration of MC3T3-E1 cells at both 6h and 12h. Figure 6 B–C).

[0079] Osteogenic differentiation was assessed by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining. Figure 6 D). The LPS+PBC group showed higher ALP activity and calcium deposition, indicating enhanced osteogenic potential. However, these effects were significantly attenuated when macrophages were pretreated with mitochondrial respiration inhibitors (rotenone / antimycin A, ROT / AA) at days 7, 14, and 21 of culture, suggesting that mitochondrial function plays a crucial role in mediating osteogenic promotion.

[0080] To further confirm this, researchers measured the oxygen consumption rate (OCR) of MC3T3-E1 cells after co-culturing with macrophages from different treatment groups (LPS+Gel group, LPS+PBC group, and ROT / AA pretreated macrophage group) for 24 hours. MC3T3-E1 cells co-cultured with macrophages from the LPS+PBC group exhibited significantly enhanced aerobic respiration capacity, an effect that was eliminated after ROT / AA pretreatment of the macrophages. Figure 6 E). OCR analysis showed similar trends in ATP production, basal respiration, and maximal respiratory capacity. Figure 6 F–H).

[0081] In summary, these findings indicate that the PBC system enhances mitochondrial translocation from macrophages to osteoblasts, thereby regulating the metabolic activity and osteogenic capacity of MC3T3-E1 cells. This reveals a previously underexplored mechanism for immunomodulatory osteogenic action through energy coupling via intercellular mitochondrial exchange.

[0082] 5. The PBC system promotes in vivo repair of bone defects. To evaluate the regulatory effect of the PBC system on bone regeneration, we used a rat mandibular critical-size bone defect model. The experimental groups included a Gel group (blank GelMA microspheres) and a PBC group. We hypothesized that the PBC system, after implantation, could act as a functional barrier, modulating the local immune microenvironment and promoting robust bone regeneration.

[0083] To assess macrophage polarization in vivo, we performed dual immunofluorescence staining against the universal macrophage marker (CD68) and subtype markers: CD86 (M1 type) and CD206 (M2 type). In the PBC group, CD86 expression was decreased compared to the gel group. Figure 7 A) indicates that there are fewer pro-inflammatory M1 macrophages in the defect area. In contrast, CD206 expression is increased in the PBC group ( Figure 7 B) indicates that more M2 macrophages, which promote regeneration, infiltrate.

[0084] Micro-CT imaging at weeks 2, 4, and 8 post-implantation showed progressive bone regeneration in the PBC group, characterized by significantly improved mineralization and marked reduction in the defect area. By week 8, the initial defect area in the PBC group was almost completely repaired, while the Gel group showed only limited bone regeneration, likely due to its lack of functional bioactivity. Figure 7 C).

[0085] Histological analysis using hematoxylin and eosin (H&E) staining and Masson's trichrome staining confirmed the findings of micro-CT. Figure 7 D). At week 4, the PBC group showed significant new bone formation along the defect boundary, and ossification was evident by week 8. In contrast, the Gel group showed dense fibrous connective tissue and extensive inflammatory cell infiltration, indicating poor regenerative capacity.

[0086] To explore the metabolic basis of tissue regeneration, we performed a large-scale, non-targeted metabolomics analysis of regenerated bone tissue. Heatmap comparisons showed that 34 metabolites were upregulated and 15 metabolites were downregulated in the PBC group. Figure 7 E). Subsequent KEGG pathway enrichment analysis ( Figure 7F) Pathways significantly enriched in glutathione metabolism and carbon metabolism were identified. Both pathways may be involved in redox regulation and energy homeostasis, warranting further investigation.

[0087] In summary, this invention discloses a pomegranate-inspired microgel / periosteum composite (PBC) system with sustained antioxidant function. This biomimetic structure mimics the structural and functional antioxidant hierarchy of the pomegranate by combining a protective and osteoconductive polydopamine / poly-L-lactic acid (PDA / PLLA) membrane with electroactive microgel "seeds." The PBC system exhibits excellent biocompatibility, mechanical strength, electrochemical stability, sustained redox cycling capacity, and strong immunomodulatory potential. Importantly, this study discovered a previously undescribed mechanism—the PBC system promotes the transfer of mitochondria from M2 polarized macrophages to osteoblast progenitor cells via a microtubule-dependent pathway. This intercellular mitochondrial exchange significantly enhances the metabolic activity and osteogenic differentiation capacity of osteoblast progenitor cells, ultimately accelerating bone regeneration. By integrating structural biomimicry, sustained antioxidant activity, immunomodulation, and mitochondrial bioengineering, the PBC system provides a novel and comprehensive strategy for immune-mediated bone tissue repair.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pomegranate-inspired bionic microgel / periosteum composite material, characterized in that, include: Bionic periosteum layer: composed of poly-L-lactic acid (PLLA) modified with polydopamine (PDA); Conductive microgel layer: Dispersed on the surface of the periosteal layer, it is formed by a conductive hydrogel containing poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) (PEDOT:PSS) and methacrylamide gelatin (GelMA), with a microgel particle size of 160–170 μm.

2. The material according to claim 1, characterized in that, The PDA coating thickness of the biomimetic periosteum layer is 0.5–5 μm, and the adhesion strength is ≥25 kPa.

3. The material according to claim 1, characterized in that, The conductive microgel contains 0.05–0.2% PEDOT:PSS by mass; the conductive hydrogel has a conductivity of 0.08–0.12 mS / cm; and the conductive microgel has a compressive strength of 130–150 kPa.

4. A method for preparing the pomegranate biomimetic microgel / periosteum composite material according to any one of claims 1 to 3, characterized in that, include: (1) Preparation of PLLA / PDA membrane: PLLA particles were dissolved in a mixed solvent of dichloromethane (DCM) and N,N-dimethylformamide (DMF) to form a 10wt% suspension. After spin coating, the membrane was immersed in a mixed solution containing 95% PDA solution and 5% ethanol and soaked at 4°C for 24h. After washing, the membrane was obtained. (2) Preparation of GelMA prepolymer: Dissolve gelatin in PBS, stir at 50°C to dissolve, add methacrylic anhydride (MA) dropwise, react at 50°C for 2 h, dialyze and freeze dry to obtain the product; (3) GelMA / PEDOT:PSS conductive hydrogel microspheres were prepared by microfluidic technology: the dispersed phase was a GelMA / PEDOT:PSS prepolymer solution, the continuous phase was a 2:1 mixture of mineral oil and Span80, the flow rate of the continuous phase was 1 μL / min, the flow rate of the dispersed phase was 2 μL / min, and microspheres with a size of 160-170 μm were obtained after UV curing. (4) The PLLA / PDA membrane from step (1) and the microspheres from step (3) are bonded together by adhesion to obtain a composite material.

5. The method for preparing the composite material according to claim 4, characterized in that, The volume ratio of DCM to DMF in the mixed solvent in (1) is 9:

1.

6. The method for preparing the composite material according to claim 4, characterized in that, The final volume concentration of PEDOT:PSS in the conductive hydrogel microspheres in (3) is 0.1%.

7. The method for preparing the composite material according to claim 4, characterized in that, In step (3), the GelMA / PEDOT:PSS prepolymer solution contains 1 wt% photoinitiator Irgacure 2959.

8. The use of the pomegranate biomimetic microgel / periosteum composite material according to any one of claims 1 to 3 in the preparation of bone defect repair drugs.

9. The use of the pomegranate biomimetic microgel / periosteum composite material according to any one of claims 1 to 3 in the preparation of medical devices for bone defect repair.

10. The application according to claim 9, characterized in that, The material enhances bone regeneration efficiency by promoting M2 polarization of macrophages and the transfer of mitochondria to osteoblasts.

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