Polysaccharide-tea polyphenol-metal compound as well as preparation method and application thereof

Abstract

The invention discloses a polysaccharide-tea polyphenol-metal compound, which is formed by dispersing a metal-tea polyphenol nano ligand in polysaccharide gel formed by covalent crosslinking of atractylodes macrocephala polysaccharide and sodium alginate, and the metal-polyphenol nano ligand is a nano complex formed by coordination of tea polyphenol and metal ions. The invention also discloses a preparation method and application thereof. The rhizoma atractylodis macrocephalae polysaccharide-sodium alginate-tea polyphenol derivative-metal composite system disclosed by the invention not only can improve the stability and release behavior of tea polyphenol in vivo, but also can regulate and control an immunometabolism microenvironment in a bone repair process, induce inflammation resolution and promote angiogenesis and bone matrix deposition, so that the bone repair effect is improved. Multiple synergistic effects of inflammation resistance, oxidation resistance, vascularization promotion and osteogenesis induction are realized, so that repair and regeneration of skull defects are remarkably accelerated, and a new intelligent immunometabolism regulation strategy is provided for repair of complex bone defects.

Description

Technical Field

[0001] This invention relates to a polysaccharide-tea polyphenol-metal complex, its preparation method and application, belonging to the field of biomedical materials and nanobiotechnology. Background Technology

[0002] Bone defects are a common and challenging clinical problem in orthopedics, often caused by factors such as trauma, infection, tumor resection, or congenital developmental abnormalities. Large or complex bone defects exceed the body's own regenerative limits, and without effective intervention, they often lead to delayed bone healing or permanent bone loss, severely impacting patients' functional recovery and quality of life. Bone tissue regeneration is essentially a dynamic biological process involving the synergistic effects of immune regulation and metabolic remodeling, encompassing complex interactions between osteoblasts, osteoclasts, and immune cells. Local immune metabolic disorders are considered a significant factor contributing to bone repair failure; therefore, promoting bone regeneration through material-mediated immune metabolic reprogramming has become a key direction in bone tissue engineering.

[0003] Tea polyphenols are derived from green tea leaf extracts and include epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epicatechin (EC), and epigallocatechin (EGC), with EGCG being the most abundant. These compounds generally possess antioxidant, anti-inflammatory, immunomodulatory, and osteogenic biological activities. Tea polyphenols can alleviate oxidative stress and inflammatory responses by scavenging reactive oxygen species (ROS), upregulating the activity of antioxidant enzymes (such as SOD, CAT, and GSH-Px), and inhibiting the release of inflammatory factors (IL-1β, IL-6, and TNF-α). Furthermore, tea polyphenols can promote osteogenic differentiation and matrix mineralization by regulating signaling pathways such as PI3K-AKT, Nrf2-ARE, and Wnt / β-catenin. However, tea polyphenols have poor stability in vivo, are easily oxidized and inactivated, have low permeability, and a short half-life, resulting in limited bioavailability. Summary of the Invention

[0004] The purpose of this invention is to provide a polysaccharide-tea polyphenol-metal complex. By constructing a polysaccharide-polyphenol-metal synergistic network structure with strongly negatively charged natural polysaccharides (such as tragacanth gum and sodium alginate) as the matrix, the stability and local retention of tea polyphenols under physiological conditions are improved, achieving the synergistic release of metal ions and polyphenol components. This composite system can regulate the inflammatory microenvironment, oxidative stress state, and local immune cell phenotype at the bone defect site, promoting the activation of osteogenic signaling pathways and matrix deposition, thereby enhancing bone repair and regeneration capabilities. This invention aims to provide a programmable structure and multiple activities for bone defect repair by constructing a polysaccharide-tea polyphenol-metal composite system with anti-inflammatory, antioxidant, and osteogenic promoting properties. The technical solution adopted by this invention is as follows: A polysaccharide-tea polyphenol-metal complex is formed by dispersing metal-tea polyphenol nanoligands in a polysaccharide gel covalently crosslinked with Atractylodes macrocephala polysaccharide and sodium alginate. The metal-tea polyphenol nanoligands are nanocomposites formed by coordination between tea polyphenols and metal ions.

[0005] Preferably, in the metal-tea polyphenol nanoligand, the metal ion is Zn. 2+ Fe 3+ or Cu 2+ The tea polyphenols are epigallocatechin gallate (EGCG), and the molar ratio of metal ions to tea polyphenols is 1:(1-6), preferably 1:(3-4); the mass ratio of Atractylodes macrocephala polysaccharide to sodium alginate in the gel is 1:1-3:1; the mass ratio of metal-tea polyphenol nanoligands to gel (based on dry matter) is 1:(10-2), preferably 1:5.

[0006] Preferably, Atractylodes macrocephala polysaccharide and sodium alginate form a polysaccharide gel network through an EDC / NHS reaction; the carboxyl, hydroxyl or vicinal diol structures in the polysaccharide gel can undergo secondary coordination with the metal ions in the metal-tea polyphenol nanoligands, so that the nanoligands are anchored in the polysaccharide gel network through metal-polysaccharide coordination bonds, thereby constructing a stable polysaccharide-tea polyphenol-metal composite system.

[0007] This invention also discloses a method for preparing the above-mentioned polysaccharide-tea polyphenol-metal complex, the steps of which include: (1) Dissolve tea polyphenols in anhydrous ethanol and zinc acetate in water to prepare solutions of equal concentration. Mix the two solutions in equal volumes, stir and react, then centrifuge to collect the precipitate, which is the metal-tea polyphenol nano ligand. (2) Atractylodes macrocephala polysaccharide and sodium alginate were mixed in a mass ratio of 1:1–3:1, dissolved in MES buffer, and cross-linking agent EDC / NHS was added. The mixture was stirred to obtain polysaccharide gel. The polysaccharide gel was dialyzed and then freeze-dried. (3) The metal-tea polyphenol nanoligands were centrifuged or lyophilized and then resuspended in deionized water to obtain a metal-tea polyphenol nanoligand dispersion. A pre-prepared polysaccharide gel (constructed from Atractylodes macrocephala polysaccharide and sodium alginate) was added to the dispersion and gently incubated under light-protected conditions to allow the nanoligands to adsorb onto the polysaccharide gel and coordinate with the metal-polysaccharide. Unbound nanoligands were then removed by centrifugation or washing. Subsequently, the resulting composite system was transferred to MES buffer and an EDC / NHS system was added to allow the carboxyl groups in the polysaccharide network to undergo a condensation reaction with the hydroxyl or amino groups on the adjacent polysaccharide chains, thereby further forming a covalently cross-linked polysaccharide gel network and obtaining a stable polysaccharide-tea polyphenol-metal complex. There is no restriction on the order of steps (1) and (2).

[0008] Preferably, the concentration of the anhydrous ethanol solution of tea polyphenols in step (1) is 1-6 mg·mL. -1 The concentration of the aqueous solution of zinc acetate is 1 mg·mL. -1 Stir at room temperature for 8–12 h.

[0009] Preferably, in step (2), the molar ratio of EDC, NHS and polysaccharide is EDC:NHS:–COOH=1.5:1:1, the pH of the MES buffer is 5.8–6.0, after adding EDC / NHS, activate for 20–30 min, then adjust the pH of the reaction system to 7.2–7.4, and continue the reaction for 4–6 h.

[0010] Preferably, dialysis is performed for 24–48 h using a dialysis bag with a molecular weight cutoff of 3 kDa.

[0011] Preferably, in step (3), the mass ratio of polysaccharide gel to metal-tea polyphenol nanoligand is 1:(10-2), and the mixture is incubated in the dark at 22-25 °C for 4-12 h; the pH of the MES buffer is 5.8-6.0, and after adding EDC / NHS, it is activated for 20-30 min, and then the pH of the reaction system is adjusted to 7.2-7.4, and the reaction is continued for 2-4 h.

[0012] The present invention also discloses the application of the above-mentioned polysaccharide-tea polyphenol-metal complex in the preparation of drugs for bone defect repair.

[0013] This study used C57BL / 6J mice as experimental animals to establish a skull defect model in order to evaluate the role of polysaccharide-tea polyphenol-metal complex in immune metabolism regulation and bone tissue regeneration.

[0014] To improve the stability of tea polyphenols and their local retention capacity at bone defect sites, this system introduces Atractylodes macrocephala polysaccharide and sodium alginate as natural polysaccharide carriers. Atractylodes macrocephala polysaccharide exhibits significant immunomodulatory activity, promoting macrophage polarization towards the anti-inflammatory M2 phenotype, increasing the expression of anti-inflammatory factors such as IL-10 and Arg-1, and reducing TNF-α levels, thereby improving the bone repair microenvironment. Sodium alginate possesses excellent gelling properties and osteoblast compatibility; its carboxyl group can bind with multivalent metal ions (such as Zn). 2+ Fe 3+ Cu 2+ Cross-linking (e.g., zinc ... 2+ It can promote osteoblast differentiation and inhibit osteoclast formation; Fe 3+ Participates in cellular redox metabolism and maintenance of mitochondrial function; Cu 2+This can activate angiogenesis-related enzyme systems (such as LOX and VEGF pathways), promote angiogenesis, and provide nutritional support for bone regeneration. Experimental results show that covalent polysaccharide gels loaded with metal-tea polyphenol nanocomposites (MPN) can significantly promote macrophage polarization from M1 to M2 type in the early postoperative period, reduce the expression levels of pro-inflammatory factors such as TNF-α and IL-1β, and increase the expression of anti-inflammatory factors such as Arg-1 and IL-10, thereby effectively improving the local inflammatory microenvironment.

[0015] Regarding the repair effect, μCT scan results showed that the defect area of ​​the composite gel treatment group exhibited abundant new bone tissue formation, with a significantly higher bone volume fraction (BV / TV) than the control group. Histological HE and Masson staining further confirmed the significant thickening of the new bone plate and collagen deposition. Immunohistochemical results indicated that the complex significantly upregulated the expression levels of osteogenic-related proteins Runx2, ALP, and OPN, while promoting CD31 and VEGF-positive angiogenesis, demonstrating a good synergistic effect of osteogenic and angiogenesis.

[0016] The material described in this invention can self-assemble into a negatively charged polysaccharide-based composite network structure with multi-level dynamic stability and functional responsiveness through metal-phenol coordination between metal ions and the hydroxyl groups of tea polyphenols, as well as cross-linking with the carboxyl or hydroxyl groups of polysaccharides. Its surface is rich in functional groups such as phenolic hydroxyl, carboxyl, and hydroxyl groups, facilitating further functionalization. Due to its negatively charged properties, it can selectively target positively charged inflamed tissues and neovascularization areas, achieving precise retention and sustained release.

[0017] Furthermore, the polyphenol-metal coordination structure in this system possesses redox adaptive properties, enabling the release of Zn under local oxidative stress conditions. 2+ Fe 3+ or Cu 2+ The invention also provides the application of the above-mentioned composite snow swallow polysaccharide microspheres in the preparation of a preparation for treating ulcerative colitis. These microspheres contain bioactive ions that activate osteogenic signaling pathways (BMP / Smad, PI3K-AKT) and maintain cellular energy homeostasis.

[0018] In summary, this invention constructs a complex system of Atractylodes macrocephala polysaccharide-sodium alginate-tea polyphenol derivative-metal, which not only improves the stability and release behavior of tea polyphenols in vivo, but also induces inflammation differentiation, promotes angiogenesis and bone matrix deposition by regulating the immune metabolic microenvironment during bone repair. This achieves multiple synergistic effects of anti-inflammatory, antioxidant, pro-angiogenic and osteogenic induction, thereby significantly accelerating the repair and regeneration of skull defects and providing a new intelligent immune metabolic regulation strategy for the repair of complex bone defects.

[0019] Compared with the prior art, the polysaccharide-tea polyphenol-metal complex proposed in this invention has the following significant advantages: (1) Constructing a multi-level functional network: A stable polysaccharide framework is formed by covalent cross-linking of Atractylodes macrocephala polysaccharide with sodium alginate, and a metal-tea polyphenol nanocoordination structure (MPN) is introduced inside it to achieve the coupling of structural stability and bioactivity.

[0020] (2) Precise regulation of immune metabolism: The complex can promote the transformation of macrophages into repair-type (M2), regulate glucose metabolism and oxidative phosphorylation levels, restore the homeostasis of the post-inflammatory microenvironment, and provide an immune prerequisite for bone formation.

[0021] (3) Synergistic promotion of osteogenic and vascularization: Fe 3+ Zn 2+ Cu 2+ Plasma is gradually released during material degradation, activating osteogenic-related genes such as Runx2 and ALP, while promoting angiogenesis mediated by the HIF-1α / VEGF pathway.

[0022] (4) Significant antioxidant and tissue protection effects: Tea polyphenol components (EGCG, EGC, ECG, EC) can scavenge ROS, inhibit NF-κB pathway activation, reduce oxidative stress damage, and promote cell migration and ECM remodeling.

[0023] (5) Good biosafety and biodegradability: The covalent polysaccharide matrix is ​​derived from natural sources and possesses excellent biocompatibility and controllable degradation; the metal ion content is within a safe trace range and does not trigger systemic toxic reactions. Attached Figure Description

[0024] Figure 1 The images show scanning electron microscope (SEM) images and bright-field images of the Atractylodes macrocephala polysaccharide-sodium alginate covalent gel prepared in Example 1 of this invention; the left image shows a scanning electron microscope (SEM) image of the polysaccharide-tea polyphenol-metal composite gel prepared in this invention. The upper right image is a schematic diagram of the process of manipulating the polysaccharide-tea polyphenol-metal sol material using a syringe. The lower right image shows the macroscopic appearance of the gel after it has been formed in a mold.

[0025] Figure 2 Transmission electron microscopy (TEM) image and particle size distribution (DLS) image of the zinc-based tea polyphenol nanoligand (EGCG–Zn MPN) prepared in Example 2 of this invention; Figure 3The image shows a scanning electron microscope (SEM) image and elemental mapping of the polysaccharide-tea polyphenol-metal complex prepared in Example 2 of this invention. Figure 4 The rheological property curves of the polysaccharide-tea polyphenol-metal complex of the present invention are shown below. Figure 5 The tensile stress-strain curves of the composite gels prepared in Examples 1 and 2 of the present invention are shown in Figure 5. The upper figure in Figure 5 is a photograph of the material mechanics testing device, and the lower figure in Figure 5 is the force-displacement curve of the polysaccharide-tea polyphenol-metal complex and epigallocatechin gallate-zinc material in mechanical testing.

[0029] Figure 6 The release curves of metal ions and polyphenol components of the composite materials prepared in Examples 1 and 2 of this invention are shown.

[0030] Figure 7 The promoting effect of the complex in Example 3 of this invention on osteogenic differentiation of osteoblasts (MC3T3-E1) is shown by ALP staining and Alizarin Red S staining.

[0031] Figure 8 This is a μCT three-dimensional reconstruction image of the complex in a mouse skull defect model in Example 4 of the present invention.

[0032] Figure 9 These are HE and Masson staining images of the complex-treated group and the control group in Example 4 of the present invention.

[0033] Figure 10 The images show the immunohistochemical images of the complex treatment group and the control group in Example 4 of this invention (IL-10, TNF, IL-1, IL-6 (positive expression)). Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments, but the description of the embodiments does not limit the scope of protection of the present invention in any way.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Furthermore, while this document may provide examples of parameters containing specific values, it should be understood that the parameters need not be exactly equal to the corresponding values, but may approximate the corresponding values ​​within acceptable error tolerances or design constraints.

[0036] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.

[0037] Example 1: Preparation of covalent Atractylodes macrocephala-sodium alginate polysaccharide gel Weigh out Atractylodes macrocephala polysaccharide (APS) and sodium alginate (SA), mix them at a mass ratio of 1:1 to 3:1, add 50 mM MES buffer (pH 5.8–6.0) to dissolve, and adjust the solid content to 5–10 mg / mL to obtain a polysaccharide mixed solution. Under magnetic stirring, add carbodiimide crosslinking agent EDC·HCl and N-hydroxysuccinimide (NHS) to make the EDC:NHS:–COOH ratio (based on the total molar number of carboxyl groups in APS and SA) 1.5:1:1. React at pH 5.8–6.0 for 30 min to activate the carboxyl groups; then slowly adjust the pH of the solution to 7.2–7.4 with 0.1–0.5 mol / L NaOH (MES buffer system), and continue the reaction at room temperature for 4–6 h to form a homogeneous and transparent covalently crosslinked hydrogel. The resulting hydrogel was placed in a MWCO 3.5kDa dialysis bag and dialyzed in deionized water for 24–48 h (with water changed periodically during the period) to remove residual small molecules. It was then freeze-dried to obtain Atractylodes macrocephala polysaccharide-sodium alginate covalently cross-linked polysaccharide gel (RAMP).

[0038] like Figure 1 As shown, the prepared covalent Atractylodes macrocephala-sodium alginate polysaccharide gel exhibits a typical three-dimensional porous honeycomb structure under scanning electron microscopy (SEM). The pore size is uniformly distributed in the range of 50–150 μm, with smooth pore walls and good interconnectivity, forming an open network structure that facilitates the diffusion of nutrient molecules and cell adhesion. Bright-field images show that the gel has high transparency and soft texture in the hydrated state, and can be smoothly extruded in a syringe, exhibiting excellent injectability and flowability. Furthermore, the visualization images further demonstrate that it has complete formation, uniform structure, and no bubble defects.

[0039] The porous three-dimensional network and good rheological properties of the gel provide ideal physical support and microenvironment for the uniform embedding of the metal-tea polyphenol complex and osteoblast migration.

[0040] Example 2 Preparation of polysaccharide-tea polyphenol-metal complex ECG, EC, EGC, and EGCG were dissolved in anhydrous ethanol to prepare solutions with a concentration of 5 mg / mL. -1 The stock solution was prepared; simultaneously, zinc acetate (Zn(CH3COO)2) was dissolved in deionized water to prepare a solution of the same concentration (5 mg·mL⁻¹). -1A metal ion solution was prepared. Subsequently, the two precursor solutions were mixed at a volume ratio of 1:1 to maintain the overall ratio of ethanol to water in the reaction system at 1:1 (v / v). The mixture was gently stirred at room temperature for 8–12 h, allowing the phenolic hydroxyl groups in the tea polyphenol molecules to react with Zn. 2+ Ions coordinate with each other, driving the self-assembly of the system to form a zinc-based tea polyphenol coordination nanostructure (ECG / EC / EGC / EGCG–ZnMPN). After the reaction, the dispersion was centrifuged at 14,000 rpm for 10 min, the supernatant was discarded, and the mixture was washed three times with deionized water to remove unreacted components and residual solvent. The resulting precipitate was resuspended in deionized water to obtain a light brown, transparent nanoparticle dispersion. The Zeta potential of EGCG–Zn MPN was -18 to -30 mV, indicating that the particle surface was negatively charged and the system had good electrostatic stability.

[0041] like Figure 2 As shown, the obtained EGCG–Zn MPN nanoparticles are nearly spherical and uniformly dispersed, with particle sizes concentrated in the range of 120–160 nm and PDI < 0.2. TEM images show that their boundaries are clear, their surfaces are smooth, and there is no obvious agglomeration, indicating that the metal–phenol coordination structure is uniform.

[0042] Subsequently, the covalent Atractylodes macrocephala-sodium alginate polysaccharide gel prepared in Example 1 was placed in EGCG-Zn nanodispersion and mixed at a ratio of 1 g gel to 10–30 mL dispersion. The mixture was then gently shaken and incubated for 4–12 h under light-protected conditions to allow the nanoparticles to fully penetrate the gel pores and undergo weak coordination adsorption with the carboxyl and hydroxyl sites in the gel network. After washing with deionized water to remove unadsorbed particles, the composite system was transferred to MES buffer (25–50 mM, pH 5.8–6.0), and EDC / NHS (molar ratio 1.5:1:1) was added for activation for 20–30 min. The pH was then adjusted to 7.2–7.4, and the reaction continued for 2–4 h to allow the remaining carboxyl groups in the gel to further form amide bonds with the hydroxyl or amino groups of the polyphenol molecules, thereby achieving stable fixation of MPN.

[0043] After thorough washing and freeze-drying for 48 h, the resulting composite material yielded a light brown, structurally intact Atractylodes macrocephala polysaccharide-sodium alginate-tea polyphenol-metal composite gel, named EGCG-Zn@RAMP. Figure 3 As shown, scanning electron microscope (SEM) images reveal uniformly distributed nanoparticle structures on both the surface and interior of the composite material; in the corresponding elemental mapping, the Zn signal is continuously distributed throughout the gel network, indicating that the metal-phenol coordination structure is successfully embedded and stably exists within the gel pores.

[0044] like Figure 4As shown, rheological performance analysis indicates that the storage modulus (G′) of the composite gel is much higher than the loss modulus (G″), exhibiting typical elastic-dominated characteristics, which suggests that its three-dimensional cross-linked structure is stable and has good resistance to deformation. Figure 5 The tensile stress-strain curves of the gels prepared in Examples 1 and 2 are shown. The comparison results show that the fracture stress and maximum strain of the material are significantly increased after the introduction of metal-phenol coordination, indicating that the dynamic metal-coordination effect enhances the toughness and energy dissipation capacity of the gel.

[0045] like Figure 6 As shown, the release curves of metal ions and polyphenol components in the composite material exhibit a clear continuous release characteristic, Zn 2+ The gel and EGCG were stably released within 24–72 h, and the release rate was positively correlated with the degree of gel swelling, consistent with the characteristics of the Fick diffusion model. This sustained-release behavior helps maintain long-term antioxidant and immunomodulatory effects in vivo, providing continuous stimulation for subsequent osteogenic induction and tissue repair.

[0046] Example 3: Establishment of an osteoblast model and the osteogenic effect of tea polyphenol-metal complex. To verify the osteogenic activity of the polysaccharide-tea polyphenol-metal complex described in this invention at the osteoblast level in vitro, primary cranial osteoblasts were isolated from C57BL / 6 newborn mice and cultured and functionally analyzed in vitro. C57BL / 6 newborn mice aged 1–3 days were used, and the skull was isolated under aseptic conditions with all attached soft tissue removed. The skull was then cut into approximately 1 mm pieces. 3 Small fragments of cells were thoroughly washed with sterile PBS, then a mixed enzyme solution containing 0.25% trypsin and 0.1% type I collagenase was added. The cells were continuously digested at a constant temperature of 37 °C, with repeated gentle pipetting to promote cell release. After digestion, α-MEM culture medium containing 10% fetal bovine serum (FBS) was added to terminate the reaction. The cells were filtered through a 70 μm sterile filter to remove tissue debris, and after centrifugation to remove the supernatant, the resulting cells were resuspended in α-MEM complete medium (containing 10% FBS and 1% penicillin and streptomycin), seeded in culture dishes, and treated with different materials at a concentration of 10 μg / ml. The cells were cultured in a 37 °C, 5% CO2 incubator, and passaged when the cell confluence reached approximately 80%. The osteogenic phenotype of the cells was identified by alkaline phosphatase (ALP) activity staining and Alizarin Red mineralized nodule staining to confirm the purity and activity of the primary osteoblasts.

[0047] like Figure 7As shown, ALP staining results indicated that cells in the EGCG–Zn MPN group exhibited a deeper purple color, suggesting significantly enhanced ALP activity; Alizarin Red staining results showed that this group formed more and denser red mineralized nodules. Compared with the control group and the single-group treatment, the complex treatment significantly promoted osteoblast mineralization differentiation and matrix deposition, further verifying that the tea polyphenol-metal complex described in this invention has excellent osteogenic induction effects.

[0048] Example 4: Establishment of a skull defect model and the therapeutic effect of the polysaccharide-tea polyphenol-metal complex. To verify the biological effects of the polysaccharide-tea polyphenol-metal complex described in this invention in promoting bone defect repair, a mouse cranial defect model was established and in vivo repair experiments were conducted. Twenty-four male wild-type C57BL / 6 mice, aged 8–10 weeks and weighing 18–22 g, were used in the experiment. All mice were acclimatized for one week in a specific pathogen-free (SPF) grade animal facility, with an ambient temperature maintained at (21 ± 2)℃, relative humidity at (45 ± 10)%, and a light / dark cycle of 12 h light / 12 h dark. During the experiment, mice had free access to standard feed and sterile drinking water. After the acclimatization period, the mice were randomly divided into four groups (n = 6): a model control group, an EGCG-Zn group, a RAMP group (comparative material group), and an EGCG-Zn@RAMP group (complex group of this invention). Under anesthesia, a standard full-thickness cranial defect with a diameter of 3 mm was prepared in the center of the mouse parietal bone using a trephine. The model control group only had the defect established without treatment; the other treatment groups had the corresponding material implanted at the defect site. Post-surgery, the mice were returned to their original cages and housed in groups with free access to food and water. The animals were sacrificed 12 weeks post-surgery, and skull samples were collected for further analysis.

[0049] Figure 8 These are μCT three-dimensional reconstruction images of skull defects in different treatment groups in Example 4 of the present invention. It can be seen that the complex group formed continuous and dense new bone tissue in the defect area. Figure 9 The results of HE and Masson staining were shown. The newly formed bone plate in the complex group had an intact structure, dense collagen fibers, and significantly reduced inflammatory cell infiltration. Figure 10The images show immunohistochemical staining of the complex group (IL-10, TNF-α, IL-1β, IL-6) in Example 4 of this invention. The results indicate that the expression of the anti-inflammatory factor IL-10 was significantly upregulated in the complex group, while the positive expression of the pro-inflammatory factors TNF-α, IL-1β, and IL-6 was significantly reduced. These results demonstrate that the polysaccharide-tea polyphenol-metal complex prepared in this invention can effectively regulate local immune metabolic balance, significantly inhibit inflammatory responses, and promote new bone formation, exhibiting excellent osteogenic induction and immunomodulatory properties. All animal experiments were strictly conducted in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 2011) and approved by the Nanjing University Laboratory Animal Ethics Committee.

[0050] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A polysaccharide-tea polyphenol-metal complex, characterized in that, The polysaccharide gel is formed by dispersing metal-polyphenol nanocomplexes in a polysaccharide gel covalently crosslinked by atractylodes rhizome polysaccharide and sodium alginate, wherein the metal-polyphenol nanocomplexes are nanocomposites formed by polyphenol and metal ions through coordination.

2. The polysaccharide - tea polyphenol - metal complex according to claim 1, characterized in that, The metal-ion is Zn 2+ , Fe 3+ or Cu 2+ , the tea polyphenol is epigallocatechin gallate, the molar ratio of the metal-ion and the tea polyphenol is 1:1-6; the mass ratio of atractylodes rhizome polysaccharide and sodium alginate in the polysaccharide gel is 1:1-3:1; the mass ratio of the metal-tea polyphenol nano-ligand and the polysaccharide gel is 1:2-10.

3. The polysaccharide - tea polyphenol - metal complex according to claim 2, characterized in that, The atractylodes rhizome polysaccharide and sodium alginate form a polysaccharide gel network through EDC / NHS reaction; the carboxyl, hydroxyl or vicinal diol structure in the polysaccharide gel and the metal ions in the metal-polyphenol nanocomplexes undergo secondary coordination, so that the nanocomplexes are anchored in the polysaccharide gel network through metal-polysaccharide coordination bonds to form a polysaccharide-polyphenol-metal complex.

4. Process for the preparation of a polysaccharide - tea polyphenol - metal complex according to any one of claims 1 to 3, characterized in that, The steps include: (1) Dissolve polyphenol in anhydrous ethanol and zinc acetate in water to prepare solutions with the same concentration, mix equal volumes of the two solutions, stir and react, centrifuge to obtain the precipitate, which is the metal-polyphenol nanocomplex; (2) Mix atractylodes rhizome polysaccharide and sodium alginate according to a mass ratio of 1:1-3:1, dissolve in MES buffer, add crosslinking agent EDC / NHS, stir and react to obtain a polysaccharide gel, and then freeze-dry the polysaccharide gel after dialysis; (3) Resuspend the metal-polyphenol nanocomplexes in deionized water after centrifugation or freeze-drying to obtain a metal-polyphenol nanocomplex dispersion, add the previously prepared polysaccharide gel to the dispersion, and incubate under light shielding conditions to allow the nanocomplexes to adsorb to the polysaccharide gel and undergo metal-polysaccharide coordination; then dissolve the obtained complex system in MES buffer and react under the condition of EDC / NHS to obtain a stable polysaccharide-polyphenol-metal complex. Steps (1) and (2) have no order restriction.

5. The preparation method according to claim 4, characterized in that, The concentration of the tea polyphenol solution in step (1) is 1-6 mg·mL -1 The concentration of the zinc acetate aqueous solution is 1 mg·mL -1 and stirred at room temperature for 8-12 h.

6. The preparation method according to claim 4, characterized in that, In step (2), the molar ratio of EDC, NHS and polysaccharide is EDC:NHS:–COOH=1.5:1:1, the pH of the MES buffer is 5.8-6.0, and after adding EDC / NHS, the pH of the reaction system is adjusted to 7.2-7.4, and the reaction is continued for 4-6 h.

7. The preparation method according to claim 6, characterized in that, Dialysis is performed using a dialysis bag with a molecular weight cut-off of 3 kDa for 24-48 h.

8. The preparation method according to claim 4, characterized in that, In step (3), the mass ratio of polysaccharide gel to metal-polyphenol nanocomplexes is 1:2-10, and the incubation is carried out at 22-25℃ under light shielding for 4-12 h; the pH of the MES buffer is 5.8-6.0, and after adding EDC / NHS, the pH of the reaction system is adjusted to 7.2-7.4, and the reaction is continued for 2-4 h.

9. Use of the polysaccharide-polyphenol-metal complex of any one of claims 1-3 in the preparation of a drug for repairing bone defects.

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