A composite microsphere material for sequentially regulating macrophage polarization and its preparation method

Abstract

This invention relates to the field of biomedical materials technology, specifically to a composite microsphere material for sequentially regulating macrophage polarization and its preparation method. The material employs a core-shell structure design, with a core layer of GELMA hydrogel containing WKYMVm short peptides and a shell layer of a composite material of graphene oxide and PVA. The preparation method includes: preparing core and shell solutions, preparing W / O emulsion droplets using microfluidic technology, UV photocrosslinking, washing and purification, and shell coating. This material can promote M1-type macrophage polarization to clear necrotic tissue in the early stages of bone repair through graphene oxide. As the shell hydrogel degrades, the WKYMVm short peptides are gradually released, promoting M2-type macrophage polarization to inhibit inflammation, promote angiogenesis, and facilitate bone repair. This invention achieves precise temporal regulation of macrophage polarization, exhibits good biocompatibility and biodegradability, and has a simple and controllable preparation process, showing broad application prospects in the field of bone defect repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a composite microsphere material for sequentially regulating macrophage polarization and its preparation method. Background Technology

[0002] Bone defect repair is one of the most pressing challenges in orthopedic clinical practice, especially the structural and functional repair of large bone defects, which presents significant challenges. Currently, bone transplantation remains the most commonly used treatment, but the limited availability of autologous bone and the tendency for allogeneic bone to cause immune rejection severely restrict its clinical application. Macrophages play a crucial role in the bone defect repair process. After a bone defect occurs, the defect site immediately enters the repair phase, and the balance and ratio of M1 and M2 macrophages at different repair stages are essential for regulating the bone immune microenvironment necessary for bone repair.

[0003] M1 macrophages are pro-inflammatory cells that dominate in the early stages of normal repair (0-3 days). A lack of M1 macrophages in the early stages can impair osteoclastogenesis, hindering the effective absorption and removal of necrotic bone and soft tissue, and negatively impacting the formation of the osteogenic microenvironment. M2 macrophages, on the other hand, are anti-inflammatory cells that typically dominate in the mid-to-late stages of repair (4-18 days). They can shorten the duration of inflammation, secrete platelet-derived growth factor (PDGF) and other substances to recruit endothelial progenitor cells, accelerating angiogenesis and maturation. They can also secrete bone morphogenetic protein (BMP) and other factors to recruit osteocytes and promote bone differentiation.

[0004] However, in actual bone defect repair, differences in defect location, local stress, and physiological and pathological conditions often lead to an imbalance in the polarization ratio of M1 and M2 macrophages at different repair stages. This disrupts the bone immune microenvironment characterized by sequential macrophage polarization required for repair, making it difficult to achieve the ideal repair state. Existing bone repair materials are mainly divided into four types: angiogenesis-based, bone-induction-based, cell recruitment-based, and immune regulation-based. The first three types cannot effectively solve the problem of M1 / M2 imbalance during bone repair. Although immune regulation-based materials have become a research hotspot, existing technologies often tend to regulate in a single direction, either excessively promoting M2 macrophage polarization or lacking precise temporal control, making it difficult to achieve the ideal sequential regulation effect. Therefore, there is an urgent need to develop a smart material that can sequentially regulate the polarization direction of macrophages at different stages of bone repair to overcome the shortcomings of existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a composite microsphere material for sequentially regulating macrophage polarization and its preparation method, aiming to solve the problem that existing bone repair materials cannot precisely regulate macrophage M1 / M2 polarization in a timely manner, leading to an imbalance in the bone immune microenvironment and poor bone defect repair effects.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing a composite microsphere material that sequentially regulates macrophage polarization, comprising the following steps: Prepare a 10wt% GELMA solution, add WKYMVm short peptide and LAP to make their final concentrations 0.2mg / mL and 0.25wt% respectively, stir well and remove bubbles to obtain the core layer solution; A shell solution was prepared by mixing 2 mg / ml graphene oxide and 1% PVA at a volume ratio of 1:1. Paraffin oil containing 2 wt% Span 80 was used as the continuous phase; Using a coaxial microfluidic chip, the inner tube is injected with a core layer solution at a flow rate of 5 μL / min, and the outer tube is injected with a continuous phase at a flow rate of 20 μL / min, forming W / O emulsion microdroplets; The collected microdroplets were immediately placed under UV light for 2 minutes to crosslink GELMA, resulting in solid microsphere cores. Collect the microspheres by centrifugation and wash them three times with ether to remove paraffin oil. The microspheres were resuspended in PBS, a shell solution was added, and the mixture was magnetically stirred for 30 minutes to coat the GO-PVA composite layer. Centrifugation and washing with PBS three times yielded the core-shell composite microsphere material.

[0007] In the section "Immediately place the collected microdroplets under UV light for 2 minutes to crosslink GELMA and obtain solid microsphere cores", the UV light has a wavelength of 365 nm and an intensity of 5-10 mW / cm². 2 .

[0008] In the section “preparing a shell solution by mixing 2 mg / ml graphene oxide and 1% PVA at a volume ratio of 1:1”, the lateral dimension of the graphene oxide is 1-5 μm.

[0009] In a second aspect, a composite microsphere material for sequentially regulating macrophage polarization is prepared using the preparation method of the composite microsphere material for sequentially regulating macrophage polarization described in the first aspect, and adopts a core-shell structure, including a core layer and a shell layer. The core layer is composed of GELMA hydrogel, in which WKYMVm short peptides and photoinitiator LAP are embedded; the shell layer is composed of graphene oxide and polyvinyl alcohol.

[0010] The concentration of the WKYMVm short peptide is 0.2 mg / ml, and the concentration of the LAP is 0.25%.

[0011] The concentration of graphene oxide in the shell is 2 mg / ml, the concentration of polyvinyl alcohol is 1%, and the volume ratio of the two is 1:1.

[0012] This invention discloses a method for preparing a composite microsphere material that sequentially regulates macrophage polarization. The composite microsphere material adopts a core-shell structure design, with a core layer of GELMA hydrogel containing WKYMVm short peptides and a shell layer of a composite material of graphene oxide and PVA. The preparation method includes: preparing core and shell solutions, preparing W / O emulsion droplets using microfluidic technology, UV photocrosslinking, washing and purification, and shell coating. This material can promote M1 polarization of macrophages to clear necrotic tissue in the early stage of bone repair through graphene oxide. As the shell hydrogel degrades, the WKYMVm short peptides are gradually released, promoting M2 polarization of macrophages to inhibit inflammation, promote angiogenesis, and promote bone repair. This invention achieves precise temporal regulation of macrophage polarization, has good biocompatibility and biodegradability, and the preparation process is simple and controllable, showing broad application prospects in the field of bone defect repair. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is a flowchart of a method for preparing a composite microsphere material that sequentially regulates macrophage polarization, as provided by the present invention.

[0015] Figure 2 This is a schematic diagram of the preparation of GO bilayer hydrogel microspheres.

[0016] Figure 3 The results are from the 1H NMR spectrum analysis of the GO-encapsulated bilayer hydrogel microsphere system.

[0017] Figure 4 These are the infrared spectra of chitosan and methacrylamide chitosan.

[0018] Figure 5 The results include scanning electron microscopy, mapping images, and EDS energy dispersive spectroscopy analysis of GO bilayer hydrogel microspheres.

[0019] Figure 6(A) represents the Young's modulus and hardness of the bilayer microspheres and chitosan microspheres; (B) represents the residual mass of the bilayer microspheres and chitosan microspheres after sustained release. Figure 7 This represents the release rate of WKYMVm peptides by the bilayer microsphere system. This confirms that the bilayer microsphere system can achieve a sustained release effect of WKYMVm peptides.

[0020] Figure 8 The results are immunofluorescence staining results after co-culturing macrophages with bilayer hydrogel microspheres, GO-bilayer hydrogel microspheres, and GO-peptide-bilayer hydrogel microspheres for 1 day.

[0021] Figure 9 Immunofluorescence staining results of macrophages after co-culturing bilayer hydrogel microspheres, GO-bilayer hydrogel microspheres, and GO-peptide-bilayer hydrogel microspheres with macrophages for 3 days. Detailed Implementation

[0022] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0023] Please see Figures 1 to 9 In a first aspect, the present invention provides a method for preparing a composite microsphere material that sequentially regulates macrophage polarization, comprising the following steps: S1 was prepared with a 10wt% GELMA solution, and WKYMVm short peptide and LAP were added to make their final concentrations 0.2mg / mL and 0.25wt%, respectively. The mixture was stirred evenly and the bubbles were removed to obtain the core layer solution. Specifically, the materials prepared include: GELMA (methacrylic anhydride gelatin), WKYMVm short peptide (Trp-Lys-Tyr-Met-Val-D-Met-NH2), LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate), graphene oxide (GO) with a lateral size of 1-5 μm, PVA (polyvinyl alcohol) with a molecular weight of 8000-10000, Span80 (sorbitan monooleate), paraffin oil, PBS buffer, and diethyl ether. Weigh 0.1g of GELMA powder, add 1mL of deionized water, and stir in a 60℃ water bath until completely dissolved to form a 10% GELMA solution. After the solution has cooled to room temperature, add 2 μL of WKYMVm short peptide stock solution (100 mg / mL) to bring the final concentration to 0.2 mg / mL. Add 2.5 μL LAP stock solution (100 mg / mL) to achieve a final concentration of 0.25%; Stir magnetically for 30 minutes to ensure even mixing; Use a 0.22μm filter membrane to remove bubbles and impurities.

[0024] S2 prepared a shell solution by mixing 2 mg / ml graphene oxide and 1% PVA at a volume ratio of 1:1; The lateral dimensions of the graphene oxide are 1-5 μm.

[0025] Specifically, a 2 mg / mL aqueous solution of graphene oxide was prepared and ultrasonically treated for 30 minutes to ensure uniform dispersion. Prepare a 1% PVA aqueous solution and stir in an 80℃ water bath until completely dissolved; Mix the two solutions at a volume ratio of 1:1 and stir magnetically for 2 hours.

[0026] S3 uses paraffin oil containing 2 wt% Span 80 as the continuous phase; Specifically, add 2% Span80 to the paraffin oil and stir magnetically for 1 hour to ensure thorough mixing.

[0027] S4 uses a coaxial microfluidic chip. The inner tube is injected with the core layer solution at a flow rate of 5 μL / min, and the outer tube is injected with the continuous phase at a flow rate of 20 μL / min, forming W / O emulsion microdroplets. Specifically, a coaxial microfluidic chip is used, with an inner tube diameter of 200μm and an outer tube diameter of 500μm; The inner tube connects to the core solution injector, and the outer tube connects to the continuous phase injector. Set the syringe pump parameters as follows: inner tubing flow rate 5 μL / min, outer tubing flow rate 20 μL / min; Start the syringe pump and collect the formed droplets in the collection container.

[0028] S5 The collected microdroplets were immediately placed under UV light for 2 minutes to crosslink GELMA and obtain solid microsphere cores; The UV light has a wavelength of 365nm and an intensity of 5-10mW / cm². 2 .

[0029] Specifically, the collected droplets were immediately placed under UV light (365nm, intensity 8mW / cm²). Irradiation time is 2 minutes to ensure complete cross-linking of GELMA.

[0030] S6 Centrifuge to collect microspheres, wash three times with ether to remove paraffin oil; Specifically, the cross-linked microspheres were transferred to centrifuge tubes and centrifuged at 3000 rpm for 5 minutes; Discard the supernatant, add an appropriate amount of ether, and sonicate for 1 minute; Centrifuge at 3000 rpm for 5 minutes and discard the ether phase; Repeat the ether washing step 3 times to completely remove the paraffin oil.

[0031] S7 resuspended the microspheres in PBS, added the shell solution, and magnetically stirred for 30 min to coat the GO-PVA composite layer. Specifically, redisperse the microspheres with PBS buffer, centrifuge at 3000 rpm for 5 minutes, and discard the supernatant; Repeat the PBS washing step 3 times; Add the shell solution and stir magnetically for 30 minutes; Centrifuge at 3000 rpm for 5 minutes and discard the supernatant; Wash three times with PBS buffer to remove unbound graphene oxide and PVA.

[0032] Centrifugation with S8 and washing with PBS three times yielded the core-shell composite microsphere material.

[0033] Specifically, the prepared composite microspheres were redispersed in PBS buffer and stored at 4°C for later use.

[0034] I. Material Characterization: Morphological observation: The surface morphology and size distribution of the microspheres were observed using scanning electron microscopy (SEM); the core-shell structure of the microspheres was observed using transmission electron microscopy (TEM). Particle size analysis: The average particle size and particle size distribution of the microspheres were determined using dynamic light scattering (DLS); the results showed that the average particle size of the microspheres was 200-300 μm, and the particle size distribution was uniform. Compositional analysis: X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition of the microsphere surface; Fourier transform infrared spectroscopy (FTIR) was used to analyze the chemical structure of the material; In vitro degradation performance: Microspheres were immersed in PBS buffer and incubated at 37°C. Samples were taken at different time points to measure the weight loss and morphological changes of the microspheres. The results showed that the graphene oxide shell was basically degraded within 3-7 days, while the core layer gradually degraded within 2-4 weeks. II. In vitro cell experiments: Cell culture: Mouse RAW264.7 macrophage cell line was used; cultured in DMEM medium containing 10% fetal bovine serum; experiments were carried out when the cell confluence reached 80%.

[0035] Macrophage polarization detection: Early M1 polarization detection: Composite microspheres were co-cultured with macrophages for 1-3 days; CD86 (M1 marker) expression was detected by flow cytometry; secretion of pro-inflammatory factors such as IL-1β and TNF-α was detected by ELISA; the results showed that the proportion of M1 macrophages was significantly increased and the secretion of pro-inflammatory factors was increased.

[0036] Later M2 polarization detection: The composite microspheres were co-cultured with macrophages for 7-14 days; the expression of CD206 (M2 marker) was detected by flow cytometry; the secretion of anti-inflammatory factors such as IL-10 and TGF-β was detected by ELISA; the results showed that the proportion of M2 macrophages was significantly increased and the secretion of anti-inflammatory factors was increased. Osteogenesis-related assays: Osteoblasts were co-cultured with composite microspheres; alkaline phosphatase activity was detected by ALP staining; calcium nodule formation was detected by alizarin red staining; the results showed that osteoblast activity and mineralization capacity were significantly improved.

[0037] III. Animal Experiments: Animal model establishment: SD rats weighing 250-300g were selected; a 5mm critical size bone defect model was established in the rat femur; the rats were divided into an experimental group (implanted with composite microspheres) and a control group (implanted with simple GELMA microspheres). Postoperative observation: Bone defects were harvested at 1, 2, 4, and 8 weeks postoperatively; bone defect healing was observed using Micro-CT scans; bone regeneration and angiogenesis were observed using histological sections; and macrophage polarization status was detected using immunohistochemistry. Results analysis: Micro-CT results showed that the bone defect healing rate in the experimental group was significantly faster than that in the control group; histological results showed that the experimental group had more new bone tissue and blood vessel formation; immunohistochemical results confirmed the sequential polarization process of macrophages. In a second aspect, a composite microsphere material for sequentially regulating macrophage polarization is prepared using the preparation method of the composite microsphere material for sequentially regulating macrophage polarization described in the first aspect, and adopts a core-shell structure, including a core layer and a shell layer. The core layer is composed of GELMA hydrogel, in which WKYMVm short peptides and photoinitiator LAP are embedded; the shell layer is composed of graphene oxide and polyvinyl alcohol.

[0038] Furthermore, the concentration of the WKYMVm short peptide is 0.2 mg / ml, and the concentration of the LAP is 0.25%.

[0039] Furthermore, the concentration of graphene oxide in the shell is 2 mg / ml, the concentration of polyvinyl alcohol is 1%, and the volume ratio of the two is 1:1.

[0040] The composite microsphere material is used in bone defect repair, including traumatic bone defects, post-tumor resection bone defects, and congenital bone defects. The composite microsphere material promotes bone repair by sequentially regulating macrophage polarization. In the early stage, it promotes M1 macrophage polarization to clear necrotic tissue, and in the later stage, it promotes M2 macrophage polarization to inhibit inflammation, promote angiogenesis, and enhance bone repair.

[0041] Development and Characterization of a GO-Encapsulated Bilayer Hydrogel Microsphere System Based on Microfluidic Technology To further achieve precise spatiotemporal controlled release of WKYMVm short peptides and meet the needs of minimally invasive treatment, a microfluidic bilayer hydrogel microsphere system based on methacryloxychitosan (GCMA) and GO was developed. Figure 2 ).

[0042] Material Synthesis and Structural Characterization: Methacrylamide chitosan (GCMA) was successfully synthesized, and the successful grafting of amino groups and methacrylic anhydride was confirmed by Fourier transform infrared spectroscopy (FTIR) and 1H nuclear magnetic resonance spectroscopy (1HNMR). GCMA exhibited olefinic hydrogen signals in the 5.5–6.5 ppm range, while the original chitosan showed absolutely no peaks in this region. Figure 3 This demonstrates the successful synthesis of methacrylamide chitosan.

[0043] The changes in infrared spectral characteristics confirmed that the chitosan amino group successfully reacted with methacrylic anhydride to generate GCMA containing photosensitive double bonds: the newly added 1720 cm⁻¹ carbonyl peak and 810 cm⁻¹ are olefin peaks, the 1590 cm⁻¹ primary amine peak is significantly weakened, indicating a high degree of substitution, and other sugar ring skeleton peaks (1070, 1030, 1150 cm⁻¹) remain basically unchanged. Figure 4 This indicates that the chitosan backbone structure is intact. Scanning electron microscopy (SEM) analysis showed that the GO bilayer hydrogel microspheres were regularly spherical, with a diameter of 100-200 μm, a smooth surface with slight wrinkles. Mapping images showed that carbon (C, red), oxygen (O, green), nitrogen (N, yellow), and sulfur (S, cyan) were distributed within the microspheres, proving that the graphite and modified chitosan were successfully composited and highly uniformly distributed. EDS further confirmed that the microspheres were mainly composed of C and O elements, with small amounts of N and S elements (originating from the chitosan backbone and possible sulfate residues), with elemental mass percentages of C 58.2%, O 36.7%, N 3.9%, and S 1.2%, respectively. Under an optical microscope, the microspheres appeared as dark brown and opaque with clear edges. Figure 5 ).

[0044] The above results collectively confirm that graphene oxide / methacrylamide chitosan bilayer hydrogel microspheres were successfully prepared, exhibiting regular morphology and uniform chemical composition.

[0045] Mechanical properties and drug delivery characteristics: Mechanical tests showed that the Young's modulus was (218.4±12.5) GPa for chitosan microspheres and (158.7±14.3) GPa for bilayer microspheres (with chondroitin sulfate outer layer modification), a decrease of approximately 27.4% (p<0.05). The hardness was (58.2±6.1) GPa for chitosan microspheres and (36.5±4.8) GPa for bilayer microspheres, a decrease of approximately 37.3% (p<0.01). Figure 6A). This indicates that the high hydrophilicity and negative charge of chondroitin sulfate lead to an increase in the equilibrium swelling rate of the microspheres and local relaxation of the cross-linking network, resulting in overall softening of the material and exhibiting soft matrix properties more suitable for cell adhesion.

[0046] High-performance liquid chromatography (HPLC) analysis showed that the microsphere system had a high encapsulation efficiency (>80%) for WKYMVm peptides (Table 1) and exhibited good sustained-release characteristics. The peptides were steadily released after 10 hours, and the drug release could be sustained for more than 14 days. Figure 6 B), to meet the needs of long-term bone repair.

[0047] Table 1: Encapsulation efficiency of WKYMVm peptides by the bilayer microsphere system serial number Peak area Concentration ppb Add volume (ml) Drug mass (mg) dosage (mg) EE% Total mass (mg) DL% 1 1154332 1404.31 1 2.81 4 80.25 154.13 1.82 2 1217173 1480.87 1 2.96 4 84.62 154.13 1.92 3 1172617 1426.58 1 2.85 4 81.52 154.13 1.85 Macrophage polarization regulation validation: Laser confocal immunofluorescence assays provided crucial evidence validating the differential regulatory effects of GO and WKYMVm on macrophage polarization. Experimental results showed that in the early co-culture stage (day 1), GO-modified microspheres primarily induced high expression of CD86 (red fluorescence) in macrophages, promoting M1 polarization. Figure 8 After co-culturing for 3 days, microspheres loaded with WKYMVm significantly induced high expression of CD206 (green fluorescence) and promoted M2 polarization. Figure 9 This result perfectly aligns with the project's sequential regulatory hypothesis regarding "using GO to initiate early inflammation (M1) and WKYMVm to promote later repair (M2)".

[0048] The present invention has the following beneficial effects: 1. Sequential Regulation Effect: This invention achieves precise temporal regulation of macrophage polarization through core-shell structure design. The graphene oxide in the early shell layer promotes M1-type macrophage polarization, effectively clearing necrotic tissue. As the shell hydrogel is degraded by myeloperoxidase in vivo, the WKYMVm short peptide in the core layer is gradually released, promoting M2-type macrophage polarization, inhibiting inflammatory responses, and promoting angiogenesis and bone repair.

[0049] 2. Intelligent responsiveness: The material can respond to changes in the physiological environment during bone repair, achieving self-regulated macrophage polarization switching without external intervention.

[0050] 3. Good biocompatibility: The materials used, such as GELMA, graphene oxide, and PVA, all have good biocompatibility and biodegradability and will not cause obvious immune rejection reactions.

[0051] 4. Simple and controllable preparation process: The preparation process is carried out using microfluidic technology, which can precisely control the size and structure of microspheres and has good batch-to-batch repeatability.

[0052] 5. Broad clinical application prospects: This material can be used for the repair of various types of bone defects, including traumatic bone defects, bone defects after tumor resection, and congenital bone defects, and has important clinical application value.

[0053] The above description is merely a preferred embodiment of the composite microsphere material for sequentially regulating macrophage polarization and its preparation method according to the present invention. Of course, it should not be construed as limiting the scope of the present invention. Those skilled in the art can understand that implementing all or part of the above embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing a composite microsphere material that sequentially regulates macrophage polarization, characterized in that, Includes the following steps: Prepare a 10wt% GELMA solution, add WKYMVm short peptide and LAP to make their final concentrations 0.2mg / mL and 0.25wt% respectively, stir well and remove bubbles to obtain the core layer solution; A shell solution was prepared by mixing 2 mg / ml graphene oxide and 1% PVA at a volume ratio of 1:

1. Paraffin oil containing 2 wt% Span 80 was used as the continuous phase; Using a coaxial microfluidic chip, the inner tube is injected with a core layer solution at a flow rate of 5 μL / min, and the outer tube is injected with a continuous phase at a flow rate of 20 μL / min, forming W / O emulsion microdroplets; The collected microdroplets were immediately placed under UV light for 2 minutes to crosslink GELMA, resulting in solid microsphere cores. Collect the microspheres by centrifugation and wash them three times with ether to remove paraffin oil. The microspheres were resuspended in PBS, a shell solution was added, and the mixture was magnetically stirred for 30 minutes to coat the GO-PVA composite layer. Centrifugation and washing with PBS three times yielded the core-shell composite microsphere material.

2. The method for preparing the composite microsphere material for sequentially regulating macrophage polarization as described in claim 1, characterized in that, In the description of "immediately placing the collected microdroplets under UV light for 2 minutes to crosslink GELMA and obtain solid microsphere cores," the UV light has a wavelength of 365 nm and an intensity of 5-10 mW / cm². 2 .

3. The method for preparing the composite microsphere material for sequentially regulating macrophage polarization as described in claim 1, characterized in that, In the preparation of a shell solution by mixing 2 mg / ml graphene oxide and 1% PVA at a volume ratio of 1:1, the lateral dimension of the graphene oxide is 1-5 μm.

4. A composite microsphere material for sequentially regulating macrophage polarization, prepared using the preparation method of the composite microsphere material for sequentially regulating macrophage polarization according to any one of claims 1-3, characterized in that, It adopts a core-shell structure, including a core layer and a shell layer; The core layer is composed of GELMA hydrogel, in which WKYMVm short peptides and photoinitiator LAP are embedded; the shell layer is composed of graphene oxide and polyvinyl alcohol.

5. The composite microsphere material for sequentially regulating macrophage polarization as described in claim 4, characterized in that, The concentration of the WKYMVm short peptide is 0.2 mg / ml, and the concentration of the LAP is 0.25%.

6. The composite microsphere material for sequentially regulating macrophage polarization as described in claim 4, characterized in that, The concentration of graphene oxide in the shell is 2 mg / ml, the concentration of polyvinyl alcohol is 1%, and the volume ratio of the two is 1:1.

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