Bone repair material based on metal-flavonoid compound coating and application thereof

Abstract

The invention relates to a bone repair material based on a metal-flavonoid compound coating and application of the bone repair material, and belongs to the technical field of biological medicine. According to the invention, metal ions and flavonoid compounds are organically combined, and a traditional bone repair material is successfully modified by virtue of excellent chelating ability and strong surface affinity of the flavonoid compounds. The metal-flavonoid compound coating is used for carrying out universal biological function modification on a traditional bone repair material, so that the diabetic bone microenvironment is accurately adjusted, and the regeneration of bone tissues is powerfully promoted. The bone repair material disclosed by the invention has good osteogenic potential under a high-glucose condition, and can greatly promote angiogenesis, so that the bone repair material has good application in the field of bone repair, especially in diabetic secondary bone injury.

Description

A bone repair material based on a metal-flavonoid coating and its application Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a bone repair material based on a metal-flavonoid compound coating and its application. Background Technology

[0002] Diabetes is one of the most pressing global health challenges of the 21st century. According to the latest statistics from the International Diabetes Federation, approximately 589 million adults aged 20 to 79 worldwide will have the disease in 2024, representing 11.1% of the population in this age group. As a chronic metabolic disease, diabetes can lead to multiple organ dysfunction, significantly increasing the risk of fractures, particularly in the skeletal system. Patients with type 2 diabetes, in particular, have a 1.2 to 1.7 times higher incidence of hip and spinal fractures than non-diabetic patients. In addition to increased fracture risk, diabetes often leads to delayed or nonunion of bone defects, severely impairing patients' quality of life and clinical prognosis. Currently, there are no bone regeneration materials specifically designed for diabetic patients. Given the urgent clinical need and the complexity of developing novel implantable biomaterials, surface functionalization and coating of existing implants with bioactive coatings have become a practical and efficient strategy. Such modifications can endow materials with new biological functions while maintaining their original mechanical properties, thereby improving bone regeneration under diabetic pathological conditions.

[0003] Currently, the design of bone repair materials for diabetic patients largely focuses on loading functional substances. This necessitates the design of bone repair materials with excellent drug, growth factor, and ion loading and sustained-release capabilities. However, these traditional materials are broadly diverse, including metals such as titanium (Ti) and 316L stainless steel; polymers such as polyetheretherketone (PEEK) and polycaprolactone (PCL); ceramics such as β-tricalcium phosphate (β-TCP) and calcium silicate (CS); and composite materials such as PCL / β-TCP composites. Existing functional substances may only modify a specific type of bone repair material, making it difficult to universally modify traditional bone repair materials that are widely used in clinical practice. Therefore, there is an urgent need for a strategy that can universally modify traditional bone repair materials while resisting oxidative stress, osteogenic dysfunction, and angiogenesis disorders caused by hyperglycemia. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a universally applicable method for modifying bone repair materials in the prior art.

[0005] To address the aforementioned technical problems, this invention proposes a bone repair material based on a metal-flavonoid coating and its applications. This invention organically combines metal ions with flavonoids, leveraging the excellent chelating ability and strong surface affinity of flavonoids to successfully modify traditional bone repair materials. The metal-flavonoid coating provides universal biofunctional modification of traditional bone repair materials, thereby precisely regulating the bone microenvironment in diabetes and effectively promoting bone tissue regeneration. The bone repair material of this invention exhibits good osteogenic potential under high glucose conditions and can significantly promote angiogenesis, thus showing promising applications in the field of bone repair, particularly in diabetic secondary bone injury.

[0006] The first objective of this invention is to provide a bone repair material based on a metal-flavonoid compound coating, the bone repair material comprising a substrate and a coating, the coating comprising metal ions adsorbed on the substrate and flavonoid compounds coordinated with the metal ions through phenolic hydroxyl groups.

[0007] Furthermore, the metal ion is selected from Mg. 2+ Co 2+ Cu 2+ Zn 2+ and Sr 2+ One or more of them.

[0008] Furthermore, the flavonoid compound is selected from one or more of luteolin, curcumin, and quercetin.

[0009] Furthermore, the substrate is selected from one or more of titanium, 316L stainless steel, polyetheretherketone, polycaprolactone, tricalcium β-phosphate, and calcium silicate.

[0010] A second objective of this invention is to provide an application of the above-mentioned bone repair material in the preparation of diabetic secondary bone injury.

[0011] A third objective of this invention is to provide a treatment product for diabetic secondary bone injury, the treatment product comprising the aforementioned bone repair material.

[0012] A fourth objective of this invention is to provide an application of the above-mentioned bone repair material in the preparation of bone injury treatment products.

[0013] A fifth objective of this invention is to provide a bone injury treatment product comprising the aforementioned bone repair material.

[0014] The sixth objective of this invention is to provide a method for preparing the above-mentioned bone repair material, wherein the preparation method involves adding a substrate and reacting it in the presence of flavonoids and metal ions.

[0015] Furthermore, the pH of the reaction is 9-11.

[0016] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0017] The bone repair material containing a metal-flavonoid coating of this invention exhibits strong osteogenic potential in high-glucose environments and provides strong support for promoting angiogenesis, thus showing promising applications in bone repair, particularly in diabetic secondary bone injury. Furthermore, the coating of this invention is universally applicable, capable of forming uniform and stable coatings on a wide variety of biomaterials, including metallic materials such as titanium (Ti) and 316L stainless steel, polymeric materials such as polyetheretherketone (PEEK) and polycaprolactone (PCL), ceramic materials such as β-tricalcium phosphate (β-TCP) and calcium silicate (CS), and composite materials such as PCL / β-TCP. Moreover, this method can also form uniform coatings on complex structural materials, such as three-dimensional porous scaffolds and clinical implants, fully demonstrating the broad applicability of this strategy. Attached Figure Description

[0018] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0019] Figure 1 illustrates the construction, screening, and formation mechanism of metal-flavonoid coatings. A is a schematic diagram of the metal-flavonoid coating screening process; B is a macroscopic image of β-TCP modified with self-assembled coatings (composed of different metal ions and flavonoid compounds), scale bar: 1 mm; C is the alkaline phosphatase (ALP) activity of BMSCs cultured on β-TCP scaffolds with different metal-flavonoid coatings under high glucose (HG) conditions, n=4; D is the total tubular structure length formed by HUVECs cultured on β-TCP scaffolds with different metal-flavonoid coatings under high glucose (HG) conditions, n=4; E is the molecular dynamics simulation of copper-quercetin (CQ) formation on the β-TCP surface; F is the Cu in the system. 2+ The relationship between quercetin density and its distance relative to the β-TCP surface; G is Cu 2+ The interaction energy between quercetin and β-TCP changes over time; H is a schematic diagram of the CQ coating formation process; I and J are verifications of the universality of the CQ coating on different types and structures of biomaterials, scale bar: 1 mm;

[0020] Figure 2 shows the characterization of the screened CQ-coated β-TCP (β-TCP@CQ) scaffolds; where A is a representative macroscopic photograph of β-TCP and β-TCP@CQ, scale bar: 1 mm; B is a representative scanning electron microscope image of β-TCP and β-TCP@CQ, scale bar: top view 0.5 mm and 200 nm, respectively; cross-sectional view: 0.5 mm; C is a representative micro-CT image of β-TCP and β-TCP@CQ, scale bar: 0.5 mm; D is the elemental distribution in β-TCP@CQ determined by energy dispersive spectroscopy (EDS), confirming the presence of Ca, P, O, C and Cu, scale bar: 0.2 mm. mm; E is the full X-ray photoelectron spectroscopy (XPS) scan of β-TCP@CQ; F is the high-resolution XPS spectrum of Cu2p in β-TCP@CQ; G is the bonding strength of the β-TCP@CQ coating evaluated by nano-scratch test; H is the evaluation of the degradation performance of β-TCP and β-TCP@CQ, n=4; I is the release of quercetin and Cu from β-TCP@CQ over time. 2+ Ion release curves, n=4; J and K are the determination of the scavenging activities of β-TCP and β-TCP@CQ on DPPH and ABTS•+ free radicals, n=5; L is the scavenging ability of β-TCP and β-TCP@CQ on reactive oxygen species (ROS) such as H2O2, •O2- and •OH, n=5;

[0021] Figure 3 shows the potential of CQ coating to enhance angiogenesis promotion by β-TCP under high glucose (HG) conditions; A represents the cell viability of HG-damaged HUVECs after 1, 3, and 7 days of treatment with β-TCP or β-TCP@CQ scaffolds (n=5); B and C represent the intracellular reactive oxygen species (ROS) levels in HG-damaged HUVECs treated with β-TCP or β-TCP@CQ scaffolds, detected by DCFH-DA staining and corresponding quantitative analysis (n=5, scale bar: 100 μm); D and E represent the migration ability of HG-damaged HUVECs treated with β-TCP or β-TCP@CQ scaffolds (scale bar: 100 μm); FH represents representative tubular structure formation images and corresponding quantitative analysis of HG-damaged HUVECs treated with β-TCP or β-TCP@CQ scaffolds (n=4, scale bar: 100 μm). μm; IK is a qRT-PCR analysis of the expression of typical pro-angiogenic factors (VEGFA, eNOS, HIF-1α) in HG-damaged HUVECs treated with β-TCP or β-TCP@CQ stents, n=5;

[0022] Figure 4 shows that the CQ coating enhanced the osteogenic potential of β-TCP under high glucose (HG) conditions. A represents the survival rate of HG-damaged BMSCs after 1, 3, and 7 days of treatment with β-TCP or β-TCP@CQ scaffolds (n=5); B and C represent the intracellular ROS levels in HG-damaged BMSCs treated with β-TCP or β-TCP@CQ scaffolds, detected by DCFH-DA staining and corresponding quantitative analysis (n=5, scale bar: 100 μm); D and E represent ALP staining and corresponding semi-quantitative analysis in HG-damaged BMSCs treated with β-TCP or β-TCP@CQ scaffolds (n=5, scale bar: 100 μm). μm; FH is the qRT-PCR analysis of typical osteogenic markers (OPN, RUNX2, OCN) expression in HG-damaged BMSCs treated with β-TCP or β-TCP@CQ scaffolds, n=5; IK is the WB analysis of OPN and RUNX2 expression in HG-damaged BMSCs treated with β-TCP or β-TCP@CQ scaffolds, n=3; L and M are representative immunofluorescence staining and corresponding semi-quantitative analysis of OCN and RUNX2 in HG-damaged BMSCs treated with β-TCP or β-TCP@CQ scaffolds, scale bar: 100 μm;

[0023] Figure 5 shows the establishment of a type 2 diabetic rat model as assessed by the intraperitoneal glucose tolerance test (IPGTT) and the intraperitoneal insulin tolerance test (IPITT).

[0024] Figure 6 shows the osteogenic effect of β-TCP@CQ in diabetic rats. A is a representative micro-computed tomography (CT) image showing the three-dimensional reconstruction of the diabetic skull defect area after β-TCP or β-TCP@CQ implantation. The newly formed bone tissue is green and the implanted scaffold is purple. Scale bar: 1 mm. B–D are quantitative analyses of osteogenic regeneration parameters in the defect area based on micro-CT data, including bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular bone thickness (Tb.Th), n=5. E and F are representative histological images of the diabetic bone defect area after β-TCP or β-TCP@CQ treatment, showing the results of hematoxylin-eosin (H&E) staining and Masson's trichrome staining, respectively. Scale bar: 200 μm.

[0025] Figure 7 illustrates the immunohistochemical analysis used to validate the promoting effect of β-TCP@CQ on bone regeneration in diabetes and its potential mechanism. A shows the osteogenic potential of β-TCP@CQ assessed by immunohistochemical staining of OCN and RUNX2 (scale bar: 25 μm); B shows the angiogenic potential of β-TCP@CQ assessed by immunohistochemical staining of CD31 and FLT1 (scale bar: 25 μm); C shows the quantitative statistical analysis of OCN and RUNX2 expression levels (n=6); D shows the quantitative statistical analysis of CD31 and FLT1 expression levels (n=6); E shows the effect of immunohistochemical staining on the activation of ATP7A and SOD3 by β-TCP@CQ (scale bar: 25 μm); F shows the immunohistochemical staining of p-PI3K and p-Akt to explore the activation effect of β-TCP@CQ on the PI3K-Akt signaling pathway (scale bar: 25 μm); G and H show the quantitative statistical analysis of the expression levels of ATP7A, SOD3, p-PI3K, and p-Akt (n=6). Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0027] Example 1: Construction, optimization and formation mechanism analysis of metal-flavonoid coatings

[0028] (1) The preparation of a copper-quercetin (CQ) coating was carried out as an example. 50 mL of quercetin aqueous solution (0.06 mol / L, solution A) was prepared, and the pH was adjusted to 10 with NaOH. Another 50 mL of copper chloride solution (0.03 mol / L, solution B) was prepared. The substrate material was placed in a 5 mL round-bottom centrifuge tube, followed by the addition of 1.5 mL of solution A and 1.5 mL of solution B. The mixture was shaken at 100 rpm for 3 hours. The resulting coating was rinsed with deionized water and then freeze-dried to obtain a scaffold with the desired coating. Other metal-flavonoid coatings were prepared using the same method, only requiring the replacement of the corresponding flavonoid and metal chloride precursors.

[0029] (2) Selection of metal-flavonoid coatings: Under high glucose (HG) conditions, the osteogenic and angiogenic activities of bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) were evaluated to screen for different metal-flavonoid coatings. Specifically, BMSCs isolated from the bone marrow of newborn rats were seeded onto various metal-flavonoid coated scaffolds in 24-well plates, with 2 × 10⁶ cells per well. 4Cells were cultured at 5 × 10⁶ cells per well after cell adhesion. The culture medium was then replaced with one containing 50 mM high glucose (HG) and osteogenic inducing factor, and cultured for another 7 days. Alkaline phosphatase (ALP) activity was quantitatively determined using a commercial kit (Beyotime, Beijing, China) with p-nitrophenyl phosphate (pNPP) as the chromogenic substrate. ALP activity values ​​were corrected for total cellular protein content, which was determined using the bisquinoline carboxylic acid (BCA) method. In the tubular structure formation assay, human umbilical vein endothelial cells (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) were cultured at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 4 × 10⁶ cells per well on metal-flavonoid coated scaffolds. After adhesion, the medium was replaced with high-glucose medium and cultured for 3 days. Cells were then enzymatically digested to obtain cells suitable for subsequent tubular structure formation experiments. Matrigel (ABW, China) cells were thawed on ice at 4°C and added to pre-chilled 48-well plates, then incubated at 37°C for 30 minutes to gel. The suspension containing HUVECs was then seeded at 4 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells in Matrigel-coated wells and incubated at 37°C for 6 hours. The total tubular length was then observed under a microscope and quantitatively analyzed using ImageJ software (National Institutes of Health).

[0030] (3) Molecular dynamics (MD) simulation analysis of the coating mechanism: MD simulations were performed using the Forcite module in Materials Studio. Quercetin and Cu 2+ The molecular structure was constructed and its geometry optimized. The simulation chamber was generated using an amorphous cell module, containing 148 quercetin molecules and 20 Cu molecules. 2+ Ions. The COMPASS III force field was used throughout the simulation. The system density was obtained by running for 500 ps in an isothermal-isobaric (NPT) ensemble at 298 K, followed by placing the system on the (001) surface. Then, equilibration was performed for 2 ns in a canonical (NVT) ensemble at 298 K. The last 2 ns of the NVT trajectory were used for radial distribution function (RDF) and mass density analysis.

[0031] To construct a metal-flavonoid coating on the surface of conventional bone repair materials for the treatment of diabetic bone defects, β-tricalcium phosphate (β-TCP) was selected as a representative material. Mg was chosen. 2+ Co 2+ Cu 2+ Zn 2+ and Sr 2+As representative osteogenic / angiogenic metal ions, luteolin, curcumin, and quercetin are representative flavonoids. Under high glucose (HG) conditions, the osteogenic activity of different coating-modified β-TCP was evaluated using bone marrow mesenchymal stem cells (BMSCs), and its angiogenic activity was evaluated using human umbilical vein endothelial cells (HUVECs) to determine the optimal coating formulation (Figure 1A).

[0032] Multiple metal-flavonoid combinations were applied to β-TCP scaffolds to screen for their osteogenic and angiogenic potential. Despite variations in color among the different metal-flavonoid coatings, all scaffolds exhibited uniform surface coverage (Fig. 1B). Subsequently, bone marrow mesenchymal stem cells (BMSCs) were co-cultured with the various coated scaffolds under high glucose conditions. All scaffolds significantly increased alkaline phosphatase (ALP) activity in BMSCs, with strontium-luteolin and CQ showing the strongest effects, increasing ALP activity by 57.8% and 60.6%, respectively (Fig. 1C). Regarding in vitro angiogenic potential, copper-curcumin and CQ showed the best performance, increasing the total tubular length by 59.3% and 61.3%, respectively, compared to the high glucose group (Fig. 1D). In conclusion, under high glucose conditions, the CQ coating exhibited the most significant osteogenic and angiogenic potential.

[0033] To further investigate the bonding mechanism between the CQ coating and β-TCP, molecular dynamics (MD) simulations were performed by constructing a system of β-TCP crystals containing Cu, quercetin, and exposed crystal faces. Conformational analysis showed that Cu... 2+ First, the molecules adsorb onto the β-TCP surface, then coordinate with the phenolic hydroxyl groups of quercetin, thereby achieving molecular growth along the crystal plane (Figure 1E). Furthermore, the conjugation between the quercetin benzene ring and the β-TCP surface promotes continuous stacking, further driving the growth of CQ on the crystal surface and forming a coating approximately 4 nm thick (Figure 1F). To elucidate the intermolecular interactions in the system, the binding energies between Cu, quercetin, and the β-TCP crystal surface were calculated (Figure 1G). All binding energies are negative, indicating that Cu... 2+ Or the adsorption of quercetin on the β-TCP surface and Cu 2+ The self-assembly process between Cu and quercetin can both occur spontaneously. Furthermore, Cu... 2+ The binding energy between quercetin and the β-TCP surface may be much greater than that between Cu and Cu. 2+ The binding energy between Cu and quercetin indicates that Cu 2+ Quercetin and quercetin tend to preferentially adsorb on the β-TCP surface and coordinate with each other, subsequently self-assembling to form a CQ nanoscale coating (Figure 1H, where Figure 1H shows the coating structure formed after self-assembly, and does not indicate the order in which metal ions and flavonoids bind to β-TCP).

[0034] Finally, to demonstrate the universality of this formation mechanism, researchers validated the coating capability of CQ on bone repair materials with different chemical compositions and structures (Figures 1I and 1J). The results showed that CQ can form uniform coatings on the surfaces of a variety of biomaterials, including metals such as titanium (Ti) and 316L stainless steel; polymers such as polyetheretherketone (PEEK) and polycaprolactone (PCL); ceramics such as β-tricalcium phosphate (β-TCP) and calcium silicate (CS); and composite materials such as PCL / β-TCP. Furthermore, this method can also form uniform CQ coatings on complex structural materials, such as three-dimensional porous scaffolds and clinical implants, fully demonstrating the broad applicability of this strategy.

[0035] Example 2: Preparation and Antioxidant Performance Evaluation of Copper-Quercetin (CQ) Coated β-TCP (β-TCP@CQ) Scaffold

[0036] (1) Preparation of copper-quercetin (CQ) coated β-TCP (β-TCP@CQ) scaffold: Prepare 50 mL of quercetin aqueous solution (0.06 mol / L, solution A), and adjust the pH to 10 with NaOH. Take another 50 mL of copper chloride solution (0.03 mol / L, solution B). Place the β-TCP scaffold in a 5 mL round-bottom centrifuge tube, and then add 1.5 mL of solution A and 1.5 mL of solution B. Shake the mixture at 100 rpm for 3 hours. Rinse the resulting coating with deionized water and freeze-dry to obtain a scaffold with the desired coating.

[0037] (2) Characterization of CQ-coated β-TCP (β-TCP@CQ): The overall scaffold morphology was observed using an optical microscope (Olympus, Japan). The microstructure of the scaffold was analyzed using a scanning electron microscope (SEM; JSM-7800F, JEOL, Japan) equipped with an energy dispersive spectroscopy (EDS) instrument for elemental distribution imaging. The three-dimensional structure and porosity were analyzed using high-resolution micro-computed tomography (micro-CT; Skyscan 1276, Bruker, Belgium). The elemental valence states were determined using X-ray photoelectron spectroscopy (XPS; PHI 5000 VersaProbe III, ULVAC-PHI, Japan). The UV-Vis absorption spectra of quercetin and CQ solutions were recorded using a UV-Vis spectrophotometer (Cary 5000, Agilent, USA). Fourier transform infrared (FTIR) spectra were further acquired using a Fourier transform infrared (FTIR) instrument (Bruker, Germany). Scratch tests were performed on the β-TCP@CQ disk using a calibrated NanoTest system (Micro Materials Ltd., Rexham, UK) and a diamond Rockwell conical indenter (tip radius 9 μm). During the progressively loaded scratch tests, the applied load increased linearly to a maximum of 500 mN. The mechanical properties of the scaffold were evaluated using uniaxial compression tests on a universal testing machine (Instron, USA) at a loading rate of 2 mm / min. The Young's modulus of compression was calculated from the linear region of the stress-strain curve. In in vitro degradation and ion release studies, the scaffold was immersed in neutral Tris-HCl buffer and removed on days 1, 3, 7, and 14, dried, and weighed. Degradation rate was calculated as a percentage of weight loss. The release of quercetin from the supernatant collected at each time point was determined by UV-Vis spectrophotometry, and the release of copper ions was determined by inductively coupled plasma mass spectrometry (ICP-MS; Model 7850, Agilent Technologies, Singapore).

[0038] (3) Antioxidant performance evaluation of β-TCP@CQ: A DPPH stock solution with a concentration of 1 mg / mL was prepared and diluted 50 times before use. The scaffold was immersed in 2 mL of the diluted DPPH solution and incubated at 37°C for 30 minutes. The absorbance of the solution at 517 nm before and after incubation was measured. A 7.4 mmol / L ABTS solution was prepared and mixed with an equal volume of 2.6 mmol / L potassium persulfate solution. The mixture was reacted at room temperature in the dark for 18 hours to generate ABTS•+ free radicals, and then diluted 50 times for later use. The scaffold material was incubated with 2 mL of the diluted ABTS•+ solution at 37°C for 30 minutes, and the absorbance at 734 nm before and after incubation was measured.

[0039] The physicochemical properties and antioxidant capacity of the selected β-TCP@CQ scaffolds were comprehensively evaluated. Macroscopic images showed that a uniform brownish-yellow CQ coating formed on the surface of the β-TCP@CQ scaffolds compared to the original β-TCP (Fig. 2A). Scanning electron microscopy (SEM) further revealed that the β-TCP surface was relatively smooth and flat, while the β-TCP@CQ scaffolds exhibited a rough and uniform CQ coating (Fig. 2B). Micro-computed tomography (micro-CT) analysis showed that the CQ coating did not alter the internal pore structure of the β-TCP, maintaining its porosity (Fig. 2C). Energy dispersive spectroscopy (EDS) detected the presence of calcium (Ca), phosphorus (P), oxygen (O), carbon (C), and copper (Cu) in the β-TCP@CQ scaffolds. The detection of carbon and copper further confirmed the successful introduction of the CQ coating (Fig. 2D). In addition, X-ray photoelectron spectroscopy (XPS) confirmed the presence of Ca, P, O, C, and Cu, with copper being the most abundant element. 2+ Its existence in form indicates that it is through Cu 2+ It coordinates with quercetin (Figures 2E and 2F).

[0040] The interfacial bonding strength between the CQ coating and β-TCP was evaluated using a nano-scratch test. The results showed that the bonding force between the CQ coating and the β-TCP substrate was approximately 2.2 N, indicating good adhesion (Figure 2G). More importantly, the β-TCP@CQ scaffold exhibited stable degradation performance, with a biodegradation rate of approximately 4.13 wt% within 14 days (Figure 2H). Furthermore, the β-TCP@CQ scaffold was able to continuously release Cu. 2+ Ions and quercetin. After 14 days, the cumulative release of quercetin reached 41.15 ± 4.22 μg / mL, while Cu... 2+ The cumulative release of ions reached 4.26 ± 0.79 μg / mL (Figure 2I).

[0041] Notably, the β-TCP scaffold, lacking intrinsic free radical scavenging ability, was compared with the β-TCP@CQ scaffold, which exhibited significant scavenging activity against 1,1-diphenyl-2-picrylhydrazine (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, with scavenging rates of approximately 38% and 61%, respectively (Figures 2J and 2K). Furthermore, the β-TCP@CQ scaffold also demonstrated significant scavenging ability against various reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂) and superoxide anion radicals (•O₂). 2- The scavenging rates of α and β free radicals (•OH) were approximately 58%, 86%, and 65%, respectively (Figure 2L).

[0042] Example 3: Pro-angiogenic capacity of β-TCP@CQ in a diabetic environment

[0043] β-TCP@CQ's ability to promote angiogenesis in a diabetic environment

[0044] (1) Cell viability assay: HUVECs were subjected to a concentration of 5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of cells / well on β-TCP and β-TCP@CQ scaffolds, placed in 96-well plates, and cultured in high-glucose medium. Cell viability was assessed on days 1, 3, and 7 using the Cell Counting Kit-8 (CCK-8, Yeasen, Shanghai, China). The procedure was brief: the culture medium was removed, and fresh medium containing 10 μL of CCK-8 reagent per well was added. After incubation at 37°C for 1.5 hours, absorbance at 450 nm was measured using a microplate reader (Epoch 2NS, BioTek, USA).

[0045] (2) Detection of intracellular reactive oxygen species (ROS): HUVECs were stored at 5 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per 24-well plate on β-TCP and β-TCP@CQ scaffolds and cultured in high-glucose medium for 3 days. Cells were then harvested and stained with 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Beyotime, Shanghai, China) to assess intracellular ROS levels. A brief procedure was performed as follows: cells were incubated with DCFH-DA at 37°C for 30 min in fresh medium, washed twice with DMEM, and counterstained with Hoechst (Beyotime, China) for 5 min. Fluorescence images were acquired using the Operetta CLS high-content imaging system (PerkinElmer, USA), and fluorescence intensity was quantified using ImageJ software (NIH, USA).

[0046] (3) Scratch assay: Human umbilical vein endothelial cells (HUVECs) were pretreated with different scaffolds under high glucose conditions, digested, and seeded in 6-well plates to form a monolayer of confluent cells. A straight scratch was made on the cell monolayer using a 200 μL pipette tip. Images were taken immediately and 24 hours later using an inverted microscope (CKX53, Olympus, Japan). Cell migration rate was quantitatively analyzed using ImageJ software combined with pseudo-color mapping.

[0047] (4) Tubular structure formation experiment: HUVECs pretreated with different scaffold materials under high sugar conditions were collected and inoculated into 24-well plates coated with Matrigel (ABW, China). After incubation at 37°C for 6 hours, images of the tubular structures were taken using a microscope, and the number of nodes and total tube length were quantitatively analyzed using ImageJ software.

[0048] (5) Quantitative Real-Time PCR (qRT-PCR): Total RNA was extracted from HUVECs cultured on scaffolds for 3 days using a commercial RNA extraction kit. Complementary DNA (cDNA) was synthesized using a SYBR premix kit. The mRNA expression levels of vascular endothelial growth factor A (VEGFA), endothelial nitric oxide synthase (eNOS), and hypoxia-inducible factor-1α (HIF-1α) were detected by qRT-PCR. Relative expression levels were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene.

[0049] In the treatment of diabetic bone defects, promoting endothelial cell activation under high glucose (HG) conditions is crucial for establishing a functional vascular network and ensuring adequate nutrient supply to damaged tissues. Therefore, the pro-angiogenic effect of β-TCP@CQ on human umbilical vein endothelial cells (HUVECs) under high glucose conditions was evaluated. Compared with the control group, the HG group showed significantly reduced HUVEC survival on days 3 and 7. However, β-TCP@CQ treatment significantly reversed this decline, demonstrating a strong protective effect on cell survival. In contrast, β-TCP alone did not produce a similar effect, and the HUVEC survival rate in the β-TCP group was similar to that in the HG group (Figure 3A). Given that a high glucose environment induces excessive reactive oxygen species (ROS) production in HUVECs, which is the main reason for the decreased cell survival, intracellular ROS levels were detected using DCFH-DA staining (Figure 3B). β-TCP alone was ineffective in clearing ROS, and there was no significant difference in ROS levels between the β-TCP group and the HG group. Conversely, β-TCP@CQ significantly reduced ROS accumulation in HUVECs under high glucose conditions. Quantitative analysis showed that the ROS level in HUVECs treated with β-TCP@CQ was approximately 1 / 9 of that in the HG group and the β-TCP group, and there was no significant difference compared with the control group (Figure 3C).

[0050] Furthermore, a high-glucose environment impaired the migration ability of HUVECs. However, β-TCP@CQ significantly enhanced the migration ability of HUVECs, with a migration rate significantly higher than that of the high-glucose group and the β-TCP group (Figs. 3D and 3E). In addition, although a high-glucose environment inhibited the formation of tubular structures in HUVECs, β-TCP@CQ still promoted significant angiogenesis under high-glucose conditions (Fig. 3F). Quantitative analysis showed that β-TCP@CQ significantly increased the number of vascular junctions and total vessel length, further confirming its pro-angiogenic effect (Figs. 3G and 3H). Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that β-TCP@CQ significantly restored the expression levels of key pro-angiogenic genes (including VEGFA, eNOS, and HIF-1α), which were originally suppressed under high-glucose conditions. In contrast, there was no significant difference in the expression levels of these genes between the high-glucose group and the β-TCP group (Figs. 3I–3K). In summary, these results indicate that the CQ coating significantly alleviates high glucose-induced angiogenesis inhibition and enhances the pro-angiogenic potential of β-TCP under hyperglycemic conditions.

[0051] Example 4: β-TCP@CQ bone-promoting capacity in diabetic environments

[0052] (1) Cell viability assay: BMSCs were sputtered at 5 × 10⁻⁶ ppm. 3 Cells were seeded at a density of 100 cells / well on β-TCP and β-TCP@CQ scaffolds in 96-well plates and cultured in high-glucose medium. Cell viability was assessed on days 1, 3, and 7 using a CCK-8 assay kit.

[0053] (2) Intracellular reactive oxygen species detection: Bone marrow mesenchymal stem cells (BMSCs) were cultured at 5 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of 1,000 cells on β-TCP and β-TCP@CQ scaffolds in 24-well plates and cultured in high-glucose medium for 3 days. Cells were then harvested and stained with DCFH-DA to assess intracellular reactive oxygen species levels.

[0054] (3) ALP staining: BMSCs were stained at a density of 2 × 10⁶ cells / well. 4 Cells were seeded at a density of 1,000 cells per well in 24-well plates, with β-TCP or β-TCP@CQ scaffolds placed in the upper chamber of the Transwell insert. Cells were cultured for 7 days in high-glucose medium containing osteogenic inducing factors. They were then fixed with 4% paraformaldehyde for 15 minutes and stained for 30 minutes using a commercial ALP staining kit (Beyotime, Shanghai, China). The formation of the purple precipitate was observed under an optical microscope (CKX53; Olympus, Japan).

[0055] (4) qRT-PCR: BMSCs were PCR-prepared at 5 × 10⁻⁶ mmol / L. 4Cells were seeded at a density of 100 cells / well on β-TCP and β-TCP@CQ scaffolds in 12-well plates and cultured for 7 or 14 days in high-glucose medium containing osteogenic inducing factors. Total RNA was extracted and reverse transcribed into cDNA, which was used as a template for PCR amplification. The mRNA expression levels of osteopontin (OPN), Runt-associated transcription factor 2 (RUNX2), and osteocalcin (OCN) were quantitatively detected using specific primers. Relative gene expression levels were normalized using GAPDH as an internal control.

[0056] (5) Western blot (WB): High glucose (HG) stimulated and scaffold-treated BMSCs were lysed on ice for 30 min with RIPA lysis buffer containing protease inhibitors. Total protein concentration was determined using a bisquinoline carboxylic acid (BCA) protein quantification kit. Before electrophoresis, protein samples were mixed with 4×Laemmli buffer and boiled at 95°C for 10 min. After separation by SDS-PAGE, proteins were transferred to 0.22 μm PVDF membranes (Millipore, USA) using wet transfer. The membranes were blocked at room temperature with TBST solution containing 5% bovine serum albumin (BSA) for 2 h, followed by overnight incubation at 4°C with primary antibodies against RUNX2 and OPN. After washing, the membranes were incubated at room temperature with horseradish peroxidase (HRP)-labeled secondary antibody for 1 h. Protein bands were detected using a chemiluminescence imaging system, and the gray values ​​of the bands were quantified using ImageJ software, with β-actin as an internal control for normalization.

[0057] (6) Immunofluorescence staining: BMSCs were stained at a density of 1×10⁻⁶ cells / well. 4 Cells were seeded at a density of 1,000 cells per well in 24-well plates, with β-TCP or β-TCP@CQ scaffolds placed in the upper chamber of the Transwell insert. Cells were cultured in high-glucose medium containing osteogenic inducing factors for 7 days (for RUNX2 staining) or 14 days (for OCN staining). Cells were fixed with 4% paraformaldehyde for 20 minutes and washed three times with PBS. Cells were permeabilized with PBS containing 0.1% Triton X-100 for 20 minutes, followed by blocking with 5% BSA for 1 hour at room temperature. Cells were incubated overnight at 4°C with primary antibodies against RUNX2 or OCN, and then incubated for 2 hours at room temperature with fluorescently labeled secondary antibodies. Samples were mounted with anti-quenching mounting medium containing DAPI. Fluorescence images were acquired using a laser scanning confocal microscope (CSU-W1 SoRa; Nikon, Germany), and fluorescence intensity was quantitatively analyzed using ImageJ software.

[0058] (7) Quantitative Real-Time PCR (qRT-PCR): Total RNA was extracted from BMSCs cultured on scaffolds for 3 days using a commercial RNA extraction kit. Complementary DNA (cDNA) was synthesized using a SYBR premix kit. The mRNA expression levels of osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2), and osteopontin (OPN) were detected by qRT-PCR. Relative expression levels were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene.

[0059] Under hyperglycemic conditions, the osteogenic differentiation capacity of bone marrow mesenchymal stem cells (BMSCs) is significantly impaired, which is considered a key factor contributing to the difficulty in effectively repairing diabetic bone defects. Against this backdrop, the role of β-TCP@CQ in activating BMSCs under high glucose (HG) conditions was further investigated. Notably, β-TCP@CQ significantly reversed the decline in BMSC viability induced by the HG environment. In the HG group, cell viability decreased to approximately 70% of that in the control group on both days 3 and 7. However, treatment with the β-TCP@CQ scaffold significantly reversed this decline and significantly promoted cell proliferation, while β-TCP scaffold alone did not have a similar effect (Figure 4A). Regarding oxidative stress, persistent high glucose conditions led to excessive accumulation of reactive oxygen species (ROS) in BMSCs, as shown by DCFH-DA staining. However, β-TCP@CQ significantly cleared these elevated ROS levels, while β-TCP alone did not show a significant ROS scavenging effect (Figures 4B and 4C).

[0060] The osteogenic differentiation capacity of BMSCs damaged by high glucose was further evaluated. ALP staining showed that β-TCP@CQ reversed the inhibitory effect of high glucose on osteogenic differentiation of BMSCs (Figure 4D). Quantitative analysis showed that ALP expression levels in BMSCs treated with β-TCP@CQ recovered to levels comparable to those in the control group, while there was no significant difference between the β-TCP group and the high glucose group (Figure 4E). In addition, qRT-PCR results confirmed that β-TCP@CQ effectively reversed the downregulation of key osteogenic differentiation genes OPN, RUNX2, and OCN induced by high glucose, with their expression levels increasing by 1.0, 1.7, and 1.1-fold, respectively (Figures 4F-H). Western blot analysis showed that the protein levels of RUNX2 and OPN further validated the osteogenic inhibition caused by high glucose, and confirmed that β-TCP@CQ significantly increased protein expression levels compared to the high glucose group, while β-TCP alone did not have this effect (Figures 4I-K). Furthermore, immunofluorescence staining showed that β-TCP@CQ could activate the expression of OCN and RUNX2. In high-glucose-damaged BMSCs treated with β-TCP@CQ, OCN showed strong green fluorescence, and RUNX2 showed strong red fluorescence, with intensities comparable to the control group; while the high-glucose group and β-TCP group showed only weak fluorescence signals (Figure 4L and M).

[0061] Example 5: β-TCP@CQ promotes bone defect repair in diabetic rats

[0062] (1) In vivo therapeutic effect of β-TCP@CQ on bone regeneration in diabetes: skull defect model and scaffold implantation in type 2 diabetic rats. A type 2 diabetes mellitus (T2DM) model was established using 6-week-old male Wistar rats by intraperitoneal injection of streptozotocin (STZ) after a high-fat diet. The successful establishment of the diabetic phenotype was confirmed by intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT). In the IPGTT, rats were fasted for 12 hours and then intraperitoneally injected with 50% (w / v) glucose solution (2.0 g / kg), with blood glucose levels measured every 30 minutes for 2 hours. In the IPITT, rats were fasted for 4 hours and then intraperitoneally injected with insulin (0.75 U / kg, 0.2 mL), with blood glucose levels monitored every 30 minutes for 2 hours. After confirming the diabetic phenotype, standard skull defect surgery was performed. Rats were anesthetized with isoflurane and fixed in a supine position. The skin was incised along the midsagittal line, and the periosteum was bluntly dissected to expose the parietal bone. A circular, full-thickness critical-size defect with a diameter of 5 mm was created on the parietal bone using a drill. Animals were randomly divided into three groups: a blank control group (modeling only), a β-TCP scaffold implantation group, and a β-TCP@CQ scaffold implantation group.

[0063] (2) MicroCT Analysis: Eight weeks after implantation, parietal bone samples were removed and fixed with 4% paraformaldehyde. A microCT scanner (Skyscan 1276; Bruker, Belgium) was used with a scanning voltage of 70 kV, a current of 112 μA, a matrix size of 1024, and a slice thickness of 0.048 mm. Three-dimensional reconstruction was performed using CTAn software, with the scaffold and newly formed bone displayed in different colors. The region of interest (ROI) was defined as the original defect area (5 mm in diameter), and bone mineral density (BMD), bone volume fraction (BV / TV), bone surface density (BS / TV), and trabecular bone thickness (Tb.Th) were quantified.

[0064] Histological analysis: Samples were fixed in 4% paraformaldehyde for 48 hours, followed by decalcification in 10% EDTA (pH 7.4) solution, with the solution changed every 72 hours for 4 to 6 weeks until complete decalcification. Decalcified tissues were dehydrated with graded ethanol, cleared in xylene, and embedded in paraffin. Sections with a thickness of 5 μm were cut along the sagittal or coronal planes. For hematoxylin-eosin (H&E) staining, after dewaxing, sections were stained with hematoxylin for 5 minutes, rinsed and blued, and counterstained with eosin for 2 minutes. After dehydration and clearing, sections were mounted with neutral resin. Stained sections were observed under a light microscope to assess inflammatory cell infiltration, new bone formation, and tissue integrity. For Masson trichrome staining, sections were first stained with Weigert iron hematoxylin to visualize cell nuclei, then differentiated in acidic ethanol, followed by Biebrich scarlet-acid fuchsin staining for 3 minutes, and finally methylene blue staining for 5 minutes to visualize collagen fibers. Subsequently, the samples were briefly differentiated in 1% acetic acid, followed by dehydration and clearing, and then mounted with neutral resin. The proportion of collagen-positive areas was quantitatively analyzed using ImageJ software to assess collagen deposition in the bone matrix.

[0065] After confirming that β-TCP@CQ can promote osteogenic differentiation of BMSCs and angiogenic differentiation of HUVECs under hyperglycemic conditions, its potential to promote bone defect repair in diabetes mellitus in vivo was further investigated. A skull defect model was established in type 2 diabetes mellitus (T2DM) rats. The successful establishment of the T2DM rat model was verified by intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT). As shown in Figure 5, the persistent hyperglycemia in the T2DM group confirmed the successful establishment of the model. In addition, IPITT analysis showed that the T2DM group exhibited an insulin resistance phenotype, with significantly lower insulin sensitivity than the control group. Bone repair was assessed at week 8 after scaffold implantation at the skull defect site. Bone regeneration was assessed by microCT. In the control group, only a small amount of new bone formation was observed at the defect edge. In contrast, both β-TCP and β-TCP@CQ promoted new bone formation in the defect area, and the osteogenic effect of β-TCP@CQ was significantly better than that of β-TCP (Figure 6A). Quantitative micro-CT analysis confirmed that β-TCP@CQ exhibited superior osteogenic properties. Compared with the β-TCP group and the control group, the β-TCP@CQ group showed significantly higher bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular thickness (Tb.Th). Specifically, the BMD, BV / TV, and Tb.Th of the β-TCP@CQ group were increased by 1.07 times, 1.68 times, and 1.69 times, respectively, compared with the β-TCP group. Notably, although the BV / TV value of the β-TCP group was significantly higher than that of the control group, there was no significant difference in BMD and Tb.Th, indicating that the quality of newly formed bone in the β-TCP group was poor (Figures 6B-D).

[0066] Histological analysis was then performed to assess the quality of new bone formation. Hematoxylin and eosin (H&E) staining revealed severely impaired bone healing in the diabetic state, characterized by limited new bone formation and extensive fibrous tissue infiltration. In contrast, the β-TCP@CQ group showed significantly reduced inflammatory infiltration and abundant new bone formation (Figure 6E). Masson's trichrome staining further revealed enhanced collagen deposition in the β-TCP@CQ group, and its quantitative results were significantly higher than those of the other groups (Figure 6F).

[0067] Example 6: β-TCP@CQ promotes osteogenic differentiation in diabetic rats

[0068] (1) In vivo therapeutic effect of β-TCP@CQ on bone regeneration in diabetes: skull defect model and scaffold implantation in type 2 diabetic rats. A type 2 diabetes mellitus (T2DM) model was established using 6-week-old male Wistar rats by intraperitoneal injection of streptozotocin (STZ) after a high-fat diet. The successful establishment of the diabetic phenotype was confirmed by intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT). In the IPGTT, rats were fasted for 12 hours and then intraperitoneally injected with 50% (w / v) glucose solution (2.0 g / kg), with blood glucose levels measured every 30 minutes for 2 hours. In the IPITT, rats were fasted for 4 hours and then intraperitoneally injected with insulin (0.75 U / kg, 0.2 mL), with blood glucose levels monitored every 30 minutes for 2 hours. After confirming the diabetic phenotype, standard skull defect surgery was performed. Rats were anesthetized with isoflurane and fixed in a supine position. The skin was incised along the midsagittal line, and the periosteum was bluntly dissected to expose the parietal bone. A circular, full-thickness critical-size defect with a diameter of 5 mm was created on the parietal bone using a drill. Animals were randomly divided into three groups: a blank control group (modeling only), a β-TCP scaffold implantation group, and a β-TCP@CQ scaffold implantation group.

[0069] (2) Histological analysis: Samples were fixed in 4% paraformaldehyde for 48 hours, followed by decalcification in 10% EDTA (pH 7.4) solution, with the solution changed every 72 hours for 4 to 6 weeks until complete decalcification. Decalcified tissues were dehydrated by gradient ethanol, cleared in xylene, and embedded in paraffin. Sections with a thickness of 5 μm were cut along the sagittal or coronal plane.

[0070] (3) For immunohistochemical staining, antigen retrieval was performed using citrate buffer (pH 6.0) under microwave heating for 15 minutes, followed by cooling to room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes, while nonspecific binding was blocked with 5% goat serum at room temperature for 1 hour. Sections were incubated overnight at 4°C with primary antibodies against OCN, RUNX2, CD31, FLT1, ATP7A, SOD3, p-PI3K, and p-Akt. After washing, sections were incubated at 37°C with HRP-labeled secondary antibody for 1 hour. DAB substrate was used for staining for 3-5 minutes, followed by hematoxylin counterstaining, dehydration, and mounting. Positive signals appeared brown and were photographed under a light microscope. Quantitative analysis of the target region was performed using ImageJ software.

[0071] Immunohistochemical staining was used to detect osteogenic and angiogenesis markers (including OCN, RUNX2, CD31, and FLT1). The results showed that β-TCP@CQ significantly upregulated the expression of these four markers in diabetic bone defects (Figures 7A and 7B). Quantitative analysis indicated that β-TCP only caused a moderate increase, while β-TCP@CQ significantly upregulated their expression levels, consistent with the material's superior osteogenic and angiogenesis functions (Figures 7C and 7D). Furthermore, immunohistochemical results showed that β-TCP@CQ activated the ATP7A, SOD3, and PI3K-Akt signaling pathways in vivo (Figures 7E and 7F). Quantitative analysis further confirmed that the expression levels in the β-TCP@CQ group were significantly higher than those in the β-TCP group or the blank control group (Figures 7G and 7H). In summary, these results indicate that under diabetic conditions, β-TCP@CQ promotes bone defect repair by regulating copper homeostasis. Specifically, the coating can activate ATP7A, promote copper efflux, stimulate SOD3, and further activate the FLT1 / PI3K-Akt signaling pathway, thereby driving osteogenic and angiogenesis processes.

[0072] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A bone repair material based on a metal-flavonoid compound coating, characterized in that, The bone repair material includes a substrate and a coating, wherein the coating includes metal ions adsorbed on the substrate and flavonoids that coordinate with the metal ions through phenolic hydroxyl groups.

2. The bone repair material according to claim 1, characterized in that, The metal ion is selected from Mg. 2+ Co 2+ Cu 2+ Zn 2+ and Sr 2+ One or more of them.

3. The bone repair material according to claim 1, characterized in that, The flavonoids are selected from one or more of luteolin, curcumin and quercetin.

4. The bone repair material according to claim 1, characterized in that, The substrate is selected from one or more of titanium, 316L stainless steel, polyetheretherketone, polycaprolactone, tricalcium β-phosphate, and calcium silicate.

5. The method for preparing the bone repair material according to any one of claims 1-4, characterized in that, The preparation method involves adding a substrate and reacting it in the presence of flavonoids and metal ions.

6. The preparation method according to claim 5, characterized in that, The pH of the reaction is 9-11.

7. The application of the bone repair material according to any one of claims 1-4 or the bone repair material prepared by the preparation method according to claim 5 or 6 in the treatment of diabetic secondary bone injury.

8. A product for treating secondary bone injury in diabetes, characterized in that, The treatment product for diabetic secondary bone injury includes the bone repair material according to any one of claims 1-4 or the bone repair material prepared by the preparation method according to claim 5 or 6.

9. The use of the bone repair material according to any one of claims 1-4 or the bone repair material prepared by the preparation method according to claim 5 or 6 in the preparation of bone injury treatment products.

10. A bone injury treatment product, characterized in that, The bone injury treatment product includes the bone repair material according to any one of claims 1-4 or the bone repair material prepared by the preparation method according to claim 5 or 6.

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