Preparation method and application of metal-organic framework composite coating loaded with active molecules on zinc alloy surface

Abstract

The application discloses a preparation method and application of a metal organic framework composite coating of active molecules loaded on a zinc alloy surface, and belongs to the technical field of biomedically degradable metal materials. The preparation method of the organic metal framework composite coating comprises the following steps: S1, preparing a ZnO nanostructure transition layer; S2, in-situ generating a metal organic framework coating induced by 2,5-dihydroxyterephthalic acid; and S3, preparing a metal organic framework composite coating loaded with deferoxamine. The application can effectively slow down the degradation rate of a zinc alloy substrate containing Mg, Ca and Sr, and makes the zinc alloy substrate containing Mg, Ca and Sr free of local corrosion, so that no toxicity is caused to cells and tissues around an implantation site. Slow release of the loaded deferoxamine can significantly accelerate blood supply reconstruction at a fracture / bone defect site, and can balance osteogenesis and osteoclastogenesis. The application solves the problems of uncontrollable degradation and lack of regulation on local immune inflammation and osteogenesis-osteoclastogenesis balance.

Description

Technical Field

[0001] This invention relates to the field of biomedical biodegradable metal materials technology, specifically to the preparation method and application of a metal-organic framework composite coating on a zinc alloy surface loaded with active molecules. Background Technology

[0002] Osteoporotic fracture repair suffers from problems such as an imbalance between osteogenic and osteoclastic processes, insufficient osteogenic formation, excessive osteoclastic processes, poor blood supply reconstruction, and persistent chronic inflammation. In clinical practice, titanium alloy internal fixation devices are commonly used but have drawbacks such as stress shielding, low bioactivity, and inability to regulate the pathological microenvironment.

[0003] Biodegradable zinc alloys are biodegradable, have a moderate degradation rate compared to magnesium and iron, are highly biosafe, and their degradation products, zinc ions, can promote bone growth and inhibit osteoclast resorption at certain concentrations, making them an ideal implant material for osteoporotic fracture repair.

[0004] However, in actual clinical applications, zinc alloys still have the following key issues to be addressed: During implantation, zinc alloys suffer from uncontrollable degradation, namely, the rapid release of zinc ions and the degradation pattern mainly consisting of pitting and local corrosion that significantly damage mechanical properties, as well as a lack of regulation of local immune inflammation and the dynamic balance between osteogenic and osteoclastogenic processes. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying a metal-organic framework composite coating loaded with active molecules on the surface of zinc alloys. This invention constructs a metal-organic framework-loaded deferoxamine composite functional coating on the surface of zinc alloys containing Mg, Ca, and Sr. This coating can effectively regulate the degradation of zinc alloys containing Mg, Ca, and Sr, the release of zinc ions, and the regulation of corrosion modes, and achieves the controllable release of DFO. This enables the realization of biofunctional effects such as promoting osteolysis, inhibiting osteoclast formation, immune regulation, and angiogenesis, thus solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface includes the following steps:

[0008] S1. Preparation of ZnO nanostructure transition layer:

[0009] Zinc alloy samples containing Mg, Ca, and Sr were cleaned, polished, and dried with sandpaper before use. The cleaned and dried zinc alloy samples containing Mg, Ca, and Sr were placed in a polytetrafluoroethylene-lined reactor. A hydrothermal reaction solution to induce the growth of a ZnO nanostructure transition layer was added to completely immerse the zinc alloy samples containing Mg, Ca, and Sr. The reactor was then sealed and tightened. The reactor was placed in a constant temperature oven for treatment to construct a ZnO nanostructure transition layer on the surface of the zinc alloy containing Mg, Ca, and Sr. The samples were then removed, gently rinsed with deionized water, and dried before use.

[0010] S2, 2,5-Dihydroxyterephthalic acid induces in-situ formation of metal-organic framework coatings:

[0011] A zinc alloy containing Mg, Ca, and Sr with a ZnO nanostructure transition layer on its surface is immersed in a reaction solution containing organic ligands to carry out an in-situ coordination reaction to generate a metal-organic framework crystal coating. After immersion, it is gently rinsed with deionized water, dried, and placed in a vacuum drying oven for later use.

[0012] S3. Preparation of metal-organic framework composite coating loaded with deferroamine;

[0013] The zinc alloy sample containing Mg, Ca, and Sr with a metal-organic framework crystal coating was immersed in a deferoxamine solution, allowing the deferoxamine molecules in the solution to fully diffuse into the tiny pores of the metal-organic framework to complete drug loading. After loading, the sample was gently rinsed with deionized water, dried, and stored in a vacuum drying oven for later use.

[0014] Preferably, in step S1, the hydrothermal reaction solution is a solution of 0.02M Zn(NO3)2·6H2O and 0.5M NaOH after thorough stirring, and the reaction vessel is placed in a constant temperature oven for hydrothermal treatment for 4 hours.

[0015] Preferably, in S3, the deferoxamine solution is a solution in which 9.43 mg / mL of Mg(NO3)2·6H2O, 0.19 mg / mL of Zn(NO3)2·6H2O, 4.51 mg / mL of 2,5-dihydroxyterephthalic acid, and 1.00 mg / mL of deferoxamine are fully dissolved.

[0016] Preferably, in step S1, the zinc alloy sample containing Mg, Ca, and Sr is mechanically polished using sandpaper with a grit size of 400-800 mesh to remove the oxide layer and impurities on the surface of the zinc alloy sample containing Mg, Ca, and Sr. The polished zinc alloy sample containing Mg, Ca, and Sr is then placed in an ultrasonic cleaner and cleaned sequentially with anhydrous ethanol and deionized water for 10 minutes each time. Anhydrous ethanol is used to remove oil stains from the surface of the zinc alloy sample containing Mg, Ca, and Sr, while deionized water is used to remove residual ethanol and other water-soluble impurities. The cleaned zinc alloy sample containing Mg, Ca, and Sr is then dried with nitrogen to remove any residual liquid from the surface.

[0017] Preferably, in step S1, the drying conditions of the drying oven are set to a temperature of 80°C, a vacuum degree of less than 0.1 MPa, and a duration of 2 hours, so that the moisture on the surface and inside of the zinc alloy sample containing Mg, Ca, and Sr is completely evaporated.

[0018] Preferably, in step S1, after the hydrothermal treatment is completed, the zinc alloy sample containing Mg, Ca, and Sr is taken out of the reaction vessel and gently rinsed with deionized water to remove the residual reaction solution and unreacted solutes on the surface. The rinsed zinc alloy sample containing Mg, Ca, and Sr is placed in a vacuum drying oven and dried at 80°C for 1 hour to remove the adsorbed moisture on the surface.

[0019] Preferably, in step S2, the reaction solution containing the organic ligand is a 2,5-dihydroxyterephthalic acid ligand reaction solution with a concentration of 4.51 mg / mL. When the ZnO nanostructure transition layer undergoes a coordination reaction with the 2,5-dihydroxyterephthalic acid ligand and gradually transforms in situ to form a metal-organic framework crystal coating, a magnetic stirrer is used to continuously stir at a speed of 200 rpm. This ensures that the 2,5-dihydroxyterephthalic acid molecules uniformly contact the surface of the zinc alloy sample containing Mg, Ca, and Sr. After the coordination reaction, the coordination bonds between the 2,5-dihydroxyterephthalic acid ligand on the ZnO surface and the Zn²⁺ ions are further extended, forming a three-dimensional network structure metal-organic framework coating.

[0020] Preferably, in step S2, after the in-situ conversion is completed, the sample is gently rinsed with deionized water to remove the residual 2,5-dihydroxyterephthalic acid solution and unreacted solutes on the surface. The rinsed zinc alloy sample containing Mg, Ca, and Sr is placed in a vacuum drying oven and dried at 80°C for 1 hour to remove the adsorbed moisture on the surface.

[0021] According to another aspect of the present invention, an application is provided for a metal-organic framework composite coating prepared by the method described above for preparing a metal-organic framework composite coating loaded with active molecules on a zinc alloy surface, wherein the metal-organic framework composite coating is applied to angiogenesis, bone repair and fracture healing.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention constructs a ZnO nanostructure transition layer and a metal-organic framework coating on the surface of a zinc alloy containing Mg, Ca, and Sr. This effectively slows down the degradation rate of the zinc alloy matrix containing Mg, Ca, and Sr, allowing for the stable and slow release of zinc ions. The zinc alloy substrate containing Mg, Ca, and Sr exhibits no localized corrosion and does not cause toxicity to cells and tissues surrounding the implantation site. Due to the micro-nano structure of the metal-organic framework coating, the loaded deferoxamine is released slowly, which can significantly accelerate the regeneration of blood supply to the fracture / bone defect site, promote angiogenesis and regeneration, and regulate the release of active molecules and metal ions, thus maintaining a dynamic balance between osteogenic and osteoclastogenic processes. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the construction and function of the DFO@MOF functional coating zinc-based intramedullary nail of the present invention;

[0025] Figure 2 These are characterization diagrams of the material surface morphology and physicochemical properties of the present invention;

[0026] Figure 3 The images show the surface morphology, phase composition, and release behavior of the material of the present invention after immersion for 21 days.

[0027] Figure 4 This figure shows the effect of the DFO@MOF-ZMCS extract of the present invention on the cell viability and adhesion behavior of BMSCs.

[0028] Figure 5 This figure shows the effect of the DFO@MOF-ZMCS extract of the present invention on the activity and adhesion behavior of HUVECs cells.

[0029] Figure 6 This is an in vitro evaluation diagram of the ability of the DFO@MOF-ZMCS composite coating of the present invention to promote endothelial cell migration.

[0030] Figure 7 This image shows the in vitro and in vivo evaluation of the angiogenesis-promoting ability of the DFO@MOF-ZMCS composite coating of the present invention.

[0031] Figure 8 This is an immunofluorescence analysis diagram showing that the DFO@MOF-ZMCS composite coating of the present invention promotes the expression of angiogenesis-related proteins.

[0032] Figure 9 This is a diagram showing the effect of the DFO@MOF-ZMCS composite coating of the present invention on the expression of angiogenesis-related genes and proteins;

[0033] Figure 10This is an in vitro evaluation diagram of the ability of DFO@MOF-ZMCS to promote the migration of BMSCs according to the present invention;

[0034] Figure 11 This diagram illustrates how DFO@MOF-ZMCS promotes early ALP expression and late mineralization formation in BMSCs during osteogenic differentiation.

[0035] Figure 12 Immunofluorescence image of DFO@MOF-ZMCS upregulating osteogenic-related proteins RUNX2 and BMP-2 in BMSCs according to the present invention;

[0036] Figure 13 This is a diagram showing the expression of genes and proteins related to the promotion of osteogenic differentiation of BMSCs by DFO@MOF-ZMCS in this invention;

[0037] Figure 14 This is a diagram illustrating the ability of DFO@MOF-ZMCS of the present invention to inhibit osteoclast differentiation and bone resorption.

[0038] Figure 15 This is a diagram illustrating the inhibition of osteoclast-related gene and protein expression by DFO@MOF-ZMCS of the present invention.

[0039] Figure 16 This image shows the imaging and micro-CT evaluation of the DFO@MOF-ZMCS composite coating of the present invention in promoting in vivo repair of osteoporotic fractures. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] To address the existing problems of uncontrollable degradation, rapid zinc ion release, and degradation primarily characterized by pitting and localized corrosion that significantly impair mechanical properties, coupled with a lack of regulation of local immune inflammation and osteogenic-osteoclast dynamic balance, please refer to [the relevant documentation / reference needed]. Figures 1-16 This embodiment provides the following technical solution:

[0042] A method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface includes the following steps:

[0043] S1. Preparation of ZnO nanostructure transition layer:

[0044] Zinc alloy samples containing Mg, Ca, and Sr were sanded, cleaned, and dried before being placed in a drying oven for later use. A hydrothermal reaction solution (0.02 M Zn(NO3)2·6H2O and 0.5 M NaOH) was prepared to induce the growth of a ZnO nanostructure transition layer and stirred thoroughly until dissolved. The cleaned and dried zinc alloy samples containing Mg, Ca, and Sr were placed in a polytetrafluoroethylene-lined reactor, and the hydrothermal reaction solution was added to completely immerse the zinc alloy samples containing Mg, Ca, and Sr. The reactor was then sealed and tightened. The reactor was placed in a constant temperature oven (90℃) for hydrothermal treatment for 4 hours to construct a ZnO nanostructure transition layer on the surface of the zinc alloy containing Mg, Ca, and Sr. The samples were then removed, gently rinsed with deionized water, and dried for later use.

[0045] S2, 2,5-dihydroxyterephthalic acid (DHTA) induces in-situ generation of metal-organic framework (MOF) coatings;

[0046] A DHTA ligand reaction solution with a concentration of 4.51 mg / mL was prepared. A ZnO-modified zinc alloy containing Mg, Ca, and Sr was immersed in the DHTA solution and soaked at room temperature for 30 minutes to allow the ZnO nanostructure transition layer to undergo a coordination reaction with the DHTA ligand and gradually transform in situ to form a metal-organic framework coating. After soaking, the coating was gently rinsed with deionized water, dried, and placed in a vacuum drying oven for later use.

[0047] S3. Preparation of metal-organic framework (MOF) composite coating loaded with deferroamine (DFO);

[0048] Prepare the DFO solution: 9.43 mg / mL Mg(NO3)2·6H2O, 0.19 mg / mL Zn(NO3)2·6H2O, 4.51 mg / mL DHTA, and 1.00 mg / mL deferoxamine. After thorough dissolution, immerse the sample in the prepared DFO solution and let it stand at room temperature for 24 hours to allow the DFO molecules in the solution to fully diffuse into the micropores of the MOF to complete drug loading. After loading, gently rinse with deionized water, dry, and store in a vacuum drying oven for later use.

[0049] Specifically, a ZnO nanostructure transition layer is first introduced onto the material surface to improve interfacial stability and coating adhesion; subsequently, a deferoxamine metal-organic framework composite coating is loaded to construct a composite material system that combines mechanical support, controllable degradation, and angiogenesis promotion capabilities (see...). Figure 1 Through in vitro cell experiments and animal models of osteoporotic fractures, this study will systematically evaluate its promoting effects on angiogenesis, bone repair, and fracture healing, and explore its potential mechanisms through imaging, histological, and molecular biological analyses, providing theoretical and experimental basis for the surface functionalization strategy of biodegradable zinc-based internal fixation materials.

[0050] like Figure 2 As shown, to verify the successful construction of the ferroamine metal-organic framework composite coating on the ZMCS alloy surface, the surface morphology, microstructure, and chemical composition of the sample were systematically characterized. SEM results (e.g., ...) are presented below. Figure 2 As shown in A, Figure 2 (A shows the scanning electron microscope (SEM) morphology of the surfaces of three groups of samples: ZMCS, MOF-ZMCS, and DFO@MOF-ZMCS.) The original ZMCS surface is relatively smooth with obvious parallel processing textures. After modification with a metal-organic framework (MOOr Framework), the MOF-ZMCS surface is continuously covered by a uniform granular micro / nano structure, indicating that the MOOr Framework coating has been successfully deposited on the alloy surface. After further loading with a MOOr Framework, the DFO@MOF-ZMCS surface still maintains a complete and continuous coating layer, while the particle structure becomes denser, and the surface roughening characteristics are further enhanced. The corresponding surface energy dispersive spectroscopy (EDS) results show that the C, N, O, Mg, and Zn elements on the coating surface are all relatively uniformly distributed, suggesting that the organic / inorganic components are well integrated in the surface functional layer. TEM, HRTEM, and SAED results further reveal the microstructural characteristics of the coating (e.g., ...). Figure 2 As shown in BC, Figure 2 B is the surface energy dispersive spectroscopy (EDS) elemental distribution map of the sample, showing the spatial distribution of elements such as Zn, Mg, C, N, and O; Figure 2 C represents the EDS energy spectrum of the sample surface, used for elemental composition analysis. Both MOF-ZMCS and DFO@MOF-ZMCS exhibit irregular granular structures. Clear lattice fringes can be observed under HRTEM, and SAED shows typical ring diffraction characteristics, indicating that the constructed coating has a certain degree of crystallinity. Notably, the sample after DFO loading still retains similar lattice and diffraction characteristics to MOF-ZMCS, indicating that the introduction of DFO did not destroy the basic crystal structure of the metal-organic framework coating. TEM-EDS elemental distribution results further confirm that C, N, O, Mg, and Zn elements are uniformly distributed within the particles, indicating that the composite of DFO and the metal-organic framework coating is relatively uniform. EDS energy spectrum quantitative analysis results (e.g.) Figure 2 As shown in DE, Figure 2 D represents the X-ray diffraction (XRD) patterns of each group of samples, used to characterize the crystal phase composition of the material surface. The symbols in the figure correspond to the diffraction peaks of different crystal phases. Figure 2E represents the Fourier transform infrared (FTIR) spectra of DFO, ZMCS, MOF-ZMCS, and DFO@MOF-ZMCS (used to analyze characteristic absorption peaks of functional groups on the material surface). This further confirmed the changes in surface elemental composition. Compared with MOF-ZMCS, the N and O content on the surface of DFO@MOF-ZMCS was significantly increased, with N increasing from 0.56±0.11 at.% to 1.20±0.01 at.% and O from 20.40±0.89 at.% to 24.86±1.02 at.%; meanwhile, the Zn content decreased from 5.70±0.24 at.% to 2.63±0.15 at.%. This result indicates that after DFO loading, the nitrogen-rich, oxygen-containing functional group-rich organic components further covered the surface of the metal-organic framework coating, thereby weakening the matrix element signal. FTIR spectra (e.g.) Figure 2 As shown in Figure F, MOF-ZMCS exhibits characteristic absorption peaks related to organic ligands compared to ZMCS. Furthermore, DFO@MOF-ZMCS shows or enhances characteristic absorption signals related to carboxyl groups, amide groups, and C–O bonds, indicating that DFO has been successfully introduced into the metal-organic framework coating. XRD results (as shown in Figure F) further demonstrate this. Figure 2 As shown in G), each group of samples retained the characteristic diffraction peaks of the Zn matrix and the alloy's second phase. Meanwhile, the samples modified with the metal-organic framework exhibited new diffraction signals, while the overall diffraction peak shape remained largely intact after DFO loading. This indicates that DFO primarily binds to the metal-organic framework surface in a loaded form, without altering its basic crystal framework. XPS full spectrum (as shown in G) Figure 2 Characteristic peaks such as Mg 1s, Zn 2p, O 1s, C 1s and N 1s can be clearly observed in the (shown in H). Among them, the O 1s and N 1s signals of DFO@MOF-ZMCS are further enhanced, which further proves that DFO has been successfully fixed on the MOF-ZMCS surface.

[0051] Overall, the morphological, structural, and chemical composition analysis results consistently indicate that the DFO-loaded metal-organic framework composite coating has been successfully constructed on the surface of ZMCS alloy. Furthermore, the coating retains a relatively complete microstructure and crystal characteristics after DFO loading, which lays a material science foundation for subsequent research on degradation, drug release, and biological functions.

[0052] To further evaluate the stability and degradation behavior of the material in the in vitro environment, a 21-day immersion experiment was conducted on the sample. SEM results (e.g.) were obtained. Figure 3 As shown in A, Figure 3A shows the surface morphology and local magnification of ZMCS, MOF-ZMCS, and DFO@MOF-ZMCS samples after 21 days of in vitro immersion. The results indicate that varying degrees of corrosion product deposition occurred on the surfaces of all groups after immersion. The functional coating group showed a more continuous deposition layer with locally denser covering structures. XRD results after immersion (e.g., ...) Figure 3 As shown in B, Figure 3 B shows the X-ray diffraction (XRD) patterns of each group of samples after 21 days of immersion, used to analyze the crystal phase composition of surface deposits or corrosion products. Characteristic peaks related to surface deposits were detected, indicating that a certain phosphate deposit layer formed on the material surface during the immersion process; ion release analysis showed (e.g.) Figure 3 As shown in C, Figure 3 C represents Zn during the soaking process of different samples. 2+ (Release concentration versus time curves for each Zn group) 2+ The cumulative release concentration gradually increases with prolonged soaking time; simultaneously, the DFO release curve (e.g.) Figure 3 As shown in D, Figure 3 D is the curve showing the cumulative release concentration of DFO over time during the immersion of DFO@MOF-ZMCS samples. This indicates that the DFO@MOF-ZMCS group exhibits sustained-release characteristics, with relatively rapid release initially followed by a stable release. The pH changes in the immersion solution (e.g.) Figure 3 As shown in E, Figure 3 E (the pH value of the soaking solution changes over time) shows that the pH of each group remained in the neutral to slightly alkaline range, without significant and drastic fluctuations.

[0053] Overall, DFO-loaded metal-organic framework composite coatings can form stable micro / nano-structured functional layers on the surface of ZMCS alloys and exhibit continuous Zn absorption under in vitro immersion conditions. 2+ The release of DFO and the relatively stable environmental response characteristics indicate that the composite coating has good structural stability and controllable release capability, providing a material basis for subsequent research on angiogenesis and osteogenic repair.

[0054] To evaluate the biocompatibility of DFO-loaded metal-organic framework composite coated zinc alloys and its impact on bone repair-related cell behaviors, BMSCs and HUVECs were cultured in vitro. The effects of the material extract on cell activity, proliferation, and adhesion / spreading ability were assessed using live / dead staining, cell viability assays, and cytoskeleton fluorescence staining systems. Figure 4-5 (as shown)

[0055] Live / dead cell staining results (e.g.) Figure 4 A, Figure 5 As shown in A, Figure 4A shows the results of BMSCs live / dead staining, with green representing live cells and red representing dead cells; Figure 5 (A shows the HUVECs live / dead staining results) indicating that BMSCs and HUVECs were in good overall survival after treatment with gold extracts in each combination. Cells were predominantly green fluorescent, with only a small number of red dead cells observed, suggesting that the material extracts did not produce significant cytotoxicity. Quantitative analysis further confirmed (e.g.) Figure 4 B Figure 5 As shown in B, Figure 4 B represents the statistical analysis of the proportion of cell death. Figure 5 (B represents the cell death rate statistics). The cell death rate in the DFO@MOF-ZMCS group remained at a low level, with no significant increase compared to the control group, indicating good cell compatibility.

[0056] CCK-8 test results (such as) Figure 4 C Figure 5 As shown in C, Figure 4 C represents the changes in relative cell viability detected by CCK-8 assay; Figure 5 C (Cells were detected by CCK-8 assay for relative cell viability) showed that, compared with the Ctrl and ZMCS groups, both the MOF-ZMCS and DFO@MOF-ZMCS groups improved relative cell viability to varying degrees. Among them, DFO@MOF-ZMCS showed the highest cell metabolic activity, suggesting that the DFO-loaded metal-organic framework coating can further promote cell proliferation activity while maintaining material safety.

[0057] Cytoskeleton fluorescence staining results (e.g.) Figure 4 D、 Figure 5 As shown in D, Figure 4 D represents the cytoskeleton fluorescence staining results: F-actin is green and DAPI in the cell nucleus is blue. Figure 5 D represents cytoskeleton fluorescence staining (F-actin green, DAPI blue). The results showed that cells in the Ctrl and ZMCS groups had relatively limited spreading, with some cells appearing round or short spindle-shaped. In the MOF-ZMCS group, cells gradually extended, and F-actin stress fibers were more clearly defined. In contrast, the DFO@MOF-ZMCS group showed the most complete cell spreading, with cells exhibiting typical spindle or polygonal shapes and a more regular cytoskeleton arrangement. Corresponding quantitative analysis results (e.g.) Figure 4 E, Figure 5 As shown in E, Figure 4 E represents the quantitative analysis of relative cell adhesion area, scale bar = 100 μm, data are expressed as mean ± standard deviation, n = 3; Figure 5E represents the quantitative analysis of cell adhesion area. Scale bar = 100 μm. Data are expressed as mean ± standard deviation, n = 3. The results showed that the cell adhesion area of ​​the DFO@MOF-ZMCS group was significantly higher than that of the other groups (P < 0.05), indicating that the composite coating can effectively enhance cell adhesion and spreading ability.

[0058] The above results indicate that the zinc alloy extract of the metal-organic framework composite coating loaded with DFO exhibits good cell compatibility with both BMSCs and HUVECs, and can significantly promote cell viability and adhesion spreading behavior. The DFO@MOF-ZMCS group showed the best overall performance, suggesting that it, through multi-ion release and synergistic action with DFO, is beneficial for maintaining and enhancing the function of osteogenic and angiogenesis-related cells during bone repair.

[0059] To systematically evaluate the regulatory role of DFO-loaded metal-organic framework (CAM) composite coated zinc alloys on angiogenesis, this study analyzed three aspects: endothelial cell migration, vascular network formation, and expression of angiogenesis-related molecules. The analysis was validated using an in vivo CAM model, constructing a complete chain of evidence from both functional phenotype and molecular mechanism dimensions (e.g., Figure 6 –9 (as shown in Figure 9).

[0060] In the evaluation of cell migration ability, the results of the scratch healing test (such as...) Figure 6 As shown in A, Figure 6 A shows the results of the scratch healing and Transwell migration assays, recording changes in the scratch area and the distribution of migrating cells at 0 h and 12 h, respectively. The results indicate that at 0 h, the scratch width was consistent across all groups; after 12 h of culture, cells from all groups migrated towards the scratch area, but the degree of closure varied significantly. The Ctrl group showed relatively slow migration, with a large blank area still visible in the center of the scratch; the ZMCS group showed significantly faster migration and a significantly smaller scratch width; the MOF-ZMCS group showed further enhanced migration, with cells tending to form a continuous monolayer; in contrast, the DFO@MOF-ZMCS group showed the most significant scratch closure at 12 h, with more complete cell coverage and a denser cell arrangement in the defect area. Quantitative analysis of the corresponding migration rate (e.g., ...) Figure 6 As shown in B, Figure 6 B represents the quantitative analysis results of scratch healing rate. The results showed that the DFO@MOF-ZMCS group was significantly higher than the Ctrl group and the other material groups (P < 0.001), and superior to the DFO-only group, suggesting that DFO, combined with the metal-organic framework structure, can synergistically enhance endothelial cell migration ability. Transwell migration experiments further validated this trend. Figure 6 A): The Ctrl group had fewer transmembrane cells; the number of migrating cells gradually increased in the ZMCS and MOF-ZMCS groups; the DFO@MOF-ZMCS group had a more dense and uniform distribution of migrating cells, and the statistical results of the number of migrating cells (e.g.) Figure 6 As shown in C, Figure 6 C represents the statistical analysis results of the number of migrating cells in Transwell. The results showed that the DFO@MOF-ZMCS group was significantly higher than that of the other groups (P < 0.001), indicating that the composite coating can effectively enhance the active migration behavior of endothelial cells.

[0061] In the evaluation of vascular network formation capacity, the results of Matrigel's in vitro tube-forming experiments (such as...) Figure 7 As shown in A, Figure 7 A shows the results of Matrigel's in vitro tubular formation experiment. The Ctrl group formed only a few short and discontinuous tubular structures; the ZMCS group formed preliminary vascular-like structures, but with limited branching; the MOF-ZMCS group formed a clearer network structure with significantly increased branching; the DFO@MOF-ZMCS group formed a dense and highly branched vascular network with more complete lumen connections and significantly increased network complexity. Quantitative results of total vessel length (e.g.) Figure 7 As stated in B, Figure 7 B (quantitative analysis of the total length of vascular-like structures) further confirmed that the DFO@MOF-ZMCS group was significantly higher than the Ctrl group and other treatment groups (P < 0.001), suggesting that it has a significant promoting effect on early structural reconstruction of angiogenesis.

[0062] In vivo CAM model results further confirm the angiogenesis potential of the material (e.g. Figure 7 As stated in C Figure 7 C is an image of chicken embryo chorioallantoic membrane (CAM) angiogenesis experiment. Compared with the Ctrl group, the vascular density of the ZMCS and MOF-ZMCS groups gradually increased, while the DFO@MOF-ZMCS group formed more neovascular branches around the implantation area, with a wider vascular extension range. Statistical analysis of total vascular length (e.g.) Figure 7 As stated in D, Figure 7 D (quantitative analysis of total blood vessel length in the CAM model) showed that the DFO@MOF-ZMCS group was significantly higher than other groups (P < 0.001), indicating that the material not only promotes the formation of blood vessel-like structures in vitro, but also has a good angiogenesis-promoting effect in vivo.

[0063] To further elucidate its molecular basis, immunofluorescence was used to detect the expression of key angiogenesis proteins CD31 and VEGF (e.g., Figure 8 As shown in the figure), the results indicate that with the enhancement of material function, the fluorescence signals of CD31 and VEGF gradually increase, with the DFO@MOF-ZMCS group showing the most significant expression (as shown in the figure). Figure 8 As shown in A, Figure 8A shows the immunofluorescence staining results of CD31 (green) and VEGF (yellow) in HUVECs after treatment with different material extracts, nuclear DAPI staining (blue), and F-actin labeling of the cytoskeleton (red); relative fluorescence intensity quantitative analysis (e.g.) Figure 8 As shown in B, Figure 8 B (the quantitative analysis results of the relative fluorescence intensity of CD31 and VEGF) further confirmed that the expression levels of CD31 and VEGF in this group were significantly higher than those in other groups (P < 0.01).

[0064] In transcriptional and protein-level validation, qRT-PCR results showed that the relative expression levels of CD31 and VEGF mRNA were significantly upregulated in the DFO-loaded MOF-ZMCS group (e.g., Figure 9 As shown in AB, Figure 9 AB (qRT-PCR detection of relative expression levels of CD31 and VEGF mRNA) showed a trend consistent with immunofluorescence results; Western blot results further showed that the CD31 and VEGF protein band in the DFO-loaded MOF-ZMCS group were the most intense (e.g., ...). Figure 9 As shown in C, Figure 9 C represents Western blot detection of VEGF and CD31 protein expression, with GAPDH used as an internal control), corresponding to grayscale quantitative analysis (e.g. Figure 9 As shown in DE, Figure 9 DE (Western blot gray-scale quantitative analysis results) indicated that its protein expression level was significantly higher than that of other groups (P < 0.01), suggesting that this material can synergistically enhance the expression of angiogenesis-related factors at the transcriptional and translational levels.

[0065] The combined results of the functional experiments and molecular assays show that DFO-loaded metal-organic framework composite coated zinc alloys can significantly enhance endothelial cell migration, promote vascular network formation, and upregulate the expression of key angiogenesis factors CD31 and VEGF at the transcriptional and protein levels. This effect may be related to the activation of HIF-1α-related pathways by the hypoxia-mimicking effect induced by DFO and the synergistic regulation of endothelial cell function by multiple Zn / Mg / Ca / Sr ions, providing an important foundation for the construction of a vascularized microenvironment during bone repair.

[0066] Specifically, using bone marrow mesenchymal stem cells (BMSCs) as the target, this study systematically evaluated the osteogenic effect of the DFO@MOF-ZMCS composite coating at four levels: cell recruitment / migration, early osteogenic differentiation, matrix mineralization maturation, and expression of key osteogenic molecules. This aimed to verify its potential ability to achieve angiogenesis-osteogenic coupling regulation during bone repair (e.g., Figure 10-13 (as shown)

[0067] First, the effects of different treatments on the migration ability of BMSCs were evaluated using scratch healing and Transwell migration assays. The scratch healing assay showed that the scratch width was consistent across groups at 0 h of culture. By 12 h, cells in the Ctrl group showed limited migration into the defect area, and the scratch area remained relatively clear. In contrast, the scratch closure in the ZMCS group was significantly accelerated, suggesting that the zinc alloy extraction environment can enhance the migration behavior of BMSCs to some extent. Furthermore, migration was further enhanced in the DFO-ZMCS group, while the DFO@MOF-ZMCS group showed the most complete cell coverage in the defect area and the least clear scratch boundary, exhibiting a more significant "bridging" closure trend. DFO alone also promoted migration, but overall it was weaker than the DFO@MOF-ZMCS group. Quantitative analysis results were consistent with morphological observations; the migration rate of the DFO@MOF-ZMCS group was significantly higher than that of the Ctrl and other treatment groups (e.g., Ctrl and Transwell). Figure 10 As shown in A–B Figure 10 A shows representative images from the scratch healing experiment (0 h and 12 h), comparing the coverage of the scratch area by Ctrl, ZMCS, DFO-ZMCS, DFO@MOF-ZMCS and DFO-treated BMSCs. Figure 10 B represents the quantitative analysis of scratch healing migration rate); the Transwell experiment further verified the above conclusions: the Ctrl group had the fewest transmembrane cells; the ZMCS group and the DFO-ZMCS group gradually increased; the DFO@MOF-ZMCS group had the most densely packed transmembrane cells, and quantitative statistics showed that its number of migrating cells was significantly higher than that of the other groups (e.g., Figure 10 As shown in C, Figure 10 C represents representative staining images and statistics of the number of transmembrane-migrating cells in the Transwell migration assay. The above results suggest that the DFO@MOF-ZMCS composite coating can effectively enhance the migration ability of BMSCs, providing favorable conditions for the recruitment and colonization of bone-derived stem cells in the bone injury area, which is an important prerequisite for subsequent osteogenic differentiation and tissue regeneration.

[0068] Based on the migration advantage, ALP staining and activity assays were used to evaluate the early osteogenic differentiation level of BMSCs. ALP staining results showed that the Ctrl group had lighter staining and limited positive areas; the ZMCS and MOF-ZMCS groups showed significantly increased blue-purple deposition, suggesting that the material-related microenvironment can initiate and enhance early osteogenic differentiation. Notably, the DFO@MOF-ZMCS group showed the strongest ALP positive signal, with deeper staining and more uniform distribution, indicating a more significant promoting effect on early osteogenic differentiation; while the DFO group alone also enhanced ALP-related performance, it was generally weaker than the composite coating group; quantitative ALP activity results further confirmed that the DFO@MOF-ZMCS group was significantly higher than the control groups such as Ctrl, ZMCS, and MOF-ZMCS (e.g., ...). Figure 11 As shown in A–B Figure 11 A shows representative ALP staining images (overall plate view and micrograph) of BMSCs from different treatment groups and the formation of mineralized nodules as shown by Alizarin Red S staining; Figure 11 B represents the quantitative results of ALP activity, suggesting that the composite coating can significantly improve the osteogenic function of BMSCs in the early stage of osteogenic differentiation. Subsequently, the formation of mineralized nodules was observed by ARS staining to assess the matrix mineralization capacity in the late stage of osteogenic differentiation. ARS staining showed that only a small amount of scattered red mineralization was observed in the Ctrl group; the number and area of ​​mineralized nodules increased in the ZMCS group and the MOF-ZMCS group, and the mineralization distribution became more continuous; the DFO@MOF-ZMCS group showed the most significant mineralization, with a wider range of red deposition and denser nodules, demonstrating a stronger mineralization maturation capacity; although the DFO group alone showed some improvement compared to the Ctrl group, the degree of mineralization was still lower than that of the DFO@MOF-ZMCS group; semi-quantitative results showed that the mineralization level of the DFO@MOF-ZMCS group was significantly higher than that of the other treatment groups (e.g., Figure 11 As shown in C, Figure 11 C represents the semi-quantitative results of Alizarin Red S staining, indicating that the composite coating not only promotes early osteogenic differentiation but also further drives the formation and maturation of mineralized nodules, exhibiting a continuous enhancing effect throughout the entire process of osteogenic differentiation.

[0069] To further validate its osteogenic role at the cellular phenotype and key protein expression levels, immunofluorescence staining was used to detect the expression of osteogenic-related core factors RUNX2 and BMP-2. Immunofluorescence results showed that the fluorescence signals of RUNX2 and BMP-2 were weak in the Ctrl group; the signals were enhanced in the ZMCS and MOF-ZMCS groups; and the DFO@MOF-ZMCS group showed the strongest fluorescence for both, along with more extended cell morphology and clearer F-actin stress fiber structure, suggesting that it not only upregulates the expression of osteogenic-related proteins but also promotes cell adhesion, spreading, and phenotypic stability. Quantitative fluorescence intensity analysis further confirmed that the expression of RUNX2 and BMP-2 in the DFO@MOF-ZMCS group was significantly higher than in other groups (e.g., ...). Figure 12 As shown in A–B Figure 12 A shows representative images of RUNX2 and BMP-2 immunofluorescence staining; Figure 12 B represents the quantitative analysis of the relative fluorescence intensity of RUNX2 and BMP-2, which supports their role in promoting osteodifferentiation from the perspective of protein localization and expression.

[0070] Finally, a systematic validation of osteogenic differentiation-related molecules was performed at the transcriptional and protein levels. qRT-PCR results showed that, compared with Ctrl, both ZMCS and MOF-ZMCS upregulated the expression of osteogenic-related genes such as RUNX2, BMP-2, and OSX. The DFO@MOF-ZMCS group showed the greatest upregulation, with a statistically significant difference (e.g., ...). Figure 13 As shown in A–C. Figure 13 A–C represent the relative mRNA expression levels of RUNX2, BMP-2, and OSX detected by qRT-PCR, suggesting that the composite coating can significantly enhance transcriptional activation for osteogenic differentiation. Western blot results further showed that osteogenic-related proteins OSX, OPN, and OCN were most strongly expressed in the DFO@MOF-ZMCS group, and the gray-scale quantification results were consistent with the trend of qRT-PCR (e.g., ...). Figure 13 As shown in DG, Figure 13 D represents the protein bands of OSX, OPN, OCN, and internal control GAPDH detected by Western blot. Figure 13 E–G represents the corresponding protein grayscale quantitative analysis, indicating that the composite coating can simultaneously enhance the osteogenic differentiation process at the protein expression level, promoting the advancement of cells from early differentiation to the matrix secretion and mineralization maturation stage.

[0071] In summary, the DFO@MOF-ZMCS composite coating exhibits optimal effects in promoting BMSC migration and recruitment, enhancing early osteogenic ALP activity, accelerating late-stage mineralization nodule formation, and upregulating the expression of key osteogenic molecules such as RUNX2 / BMP-2 / OSX-OPN-OCN, demonstrating a stable and continuous promoting effect throughout the entire osteogenic differentiation process. Combined with the aforementioned evidence chain for pro-angiogenesis, the DFO@MOF-ZMCS composite coating is expected to improve the bone repair microenvironment and strengthen angiogenesis-osteogenic coupling through a synergistic mechanism of "pro-angiogenesis and pro-osteoogenesis," providing sufficient cellular and molecular evidence for subsequent in vivo bone repair efficacy verification.

[0072] After verifying that the DFO@MOF-ZMCS composite coating has significant pro-angiogenic and osteogenic effects, its regulatory role on osteoclast differentiation and bone resorption function was further evaluated to comprehensively analyze its impact on bone homeostasis during bone remodeling (e.g., Figure 14 –15 (as shown in Figure 15).

[0073] First, osteoclast formation in each group was observed using TRAP staining. The results showed that the Ctrl group produced a large number of TRAP-positive multinucleated cells, which stained a deep purplish-red color, and were large and densely distributed. In contrast, the ZMCS and MOF-ZMCS groups showed a decrease in the number of TRAP-positive cells, but multinucleated cell formation was still evident. Notably, the DFO@MOF-ZMCS group showed a significant decrease in the number of TRAP-positive cells, with multinucleated cell formation being significantly inhibited and staining intensity weakened. The DFO group alone also showed a certain inhibitory trend, but overall it was weaker than the DFO@MOF-ZMCS group (e.g., ...). Figure 14 As shown in A, Figure 14 A shows representative TRAP-stained images, comparing osteoclast formation in the Ctrl, ZMCS, MOF-ZMCS, DFO@MOF-ZMCS, and DFO groups.

[0074] TRAP absorbance analysis results showed that the absorbance of the DFO@MOF-ZMCS group was significantly lower than that of Ctrl and other treatment groups (e.g., Figure 14 As shown in B, Figure 14 B represents the quantitative analysis of TRAP absorbance, indicating that osteoclast differentiation activity was significantly inhibited; further quantitative analysis of osteoclast number and osteoclast area showed that the number of osteoclasts and the area of ​​single cells in the DFO@MOF-ZMCS group were significantly decreased (e.g., Figure 14 As shown in C–D, Figure 14 C represents the number of osteoclasts per unit area; Figure 14 D represents the quantitative analysis of osteoclast area, indicating that the composite coating not only reduces the number of osteoclasts formed, but also inhibits their maturation and functionalization process.

[0075] To further verify its inhibitory effect at the molecular level, qRT-PCR was used to detect the expression of osteoclast-related genes. The results showed that the expression levels of TRAP, RANK, and CTSK were higher in the Ctrl group; slightly decreased in the ZMCS and MOF-ZMCS groups; and significantly downregulated in the DFO@MOF-ZMCS group, with statistically significant differences (e.g., ...). Figure 15 As shown in A–C. Figure 15 A–C represent the relative mRNA expression levels of TRAP, RANK, and CTSK detected by qRT-PCR, suggesting that the composite coating can inhibit the activation of osteoclast differentiation signaling pathways at the transcriptional level. Western blot results further confirmed that the expression of osteoclast-related proteins RANK and TRAP was significantly reduced in the DFO@MOF-ZMCS group, and the grayscale analysis results were consistent with the mRNA expression trend (e.g., ...). Figure 15 As shown in D–F, Figure 15 D represents the Western blot detection of RANK and TRAP protein expression and the internal control GAPDH band; Figure 15 E–F represent the corresponding protein grayscale quantitative analysis, indicating that the composite coating can continuously inhibit osteoclast differentiation and function at the protein expression level.

[0076] In summary, the DFO@MOF-ZMCS composite coating not only promotes angiogenesis and osteogenic differentiation, but also significantly inhibits the expression of osteoclast formation and bone resorption-related molecules, exhibiting a bidirectional regulatory characteristic of "promoting osteoogenesis and inhibiting osteoclast formation." This is beneficial for restoring the dynamic balance between osteogenic and osteoclast formation during bone remodeling, thereby providing a more stable microenvironment for osteoporotic bone repair.

[0077] After completing the in vitro evidence chain of "promoting angiogenesis, promoting osteoporosis, and inhibiting osteoclastosis," to further verify the real repair efficacy of the DFO@MOF-ZMCS composite coating in the osteoporotic microenvironment, an internal fixation model of osteoporotic fracture was established. X-ray follow-up, in vitro specimen observation, and micro-CT three-dimensional reconstruction and quantitative analysis of bone microstructure were performed at 6 and 12 weeks postoperatively (e.g., Figure 16 (As shown).

[0078] X-ray results showed that at 6 weeks postoperatively, the fracture line in the Ti6Al4V group was still relatively clear, with relatively insufficient callus formation; the ZMCS and MOF-ZMCS groups showed increased callus formation compared to the control, but the continuity of the fracture ends was still not ideal; the DFO@MOF-ZMCS group showed blurred fracture lines, larger callus volume, and more obvious bone bridge formation, suggesting that it could promote stable connection of the fracture ends earlier; by 12 weeks postoperatively, fracture repair had further progressed in all groups, but the DFO@MOF-ZMCS group showed more complete callus remodeling and better bone continuity, with the fracture end structure becoming more complete (e.g., Figure 16 As shown in A, Figure 16 A shows a comparison of X-ray images at 6 and 12 weeks postoperatively. Consistent with imaging observations, the fracture healing score in the DFO@MOF-ZMCS group was significantly higher than that in the other groups at both 6 and 12 weeks postoperatively, suggesting that the composite coating can continuously accelerate the fracture healing process (e.g., Figure 16 As shown in B, Figure 16 B represents the Lane-Sandhu fracture healing score. Ex vivo specimen observation further supports the above results: the DFO@MOF-ZMCS group showed more continuous callus formation and greater coverage in the fracture area, with a more stable bridging structure at the fracture ends. In contrast, other groups still showed varying degrees of discontinuity in the fracture end contours or insufficient callus connection (e.g., ...). Figure 16 As shown in C, Figure 16 C shows a gross observation of the ex vivo specimen of the fracture area after postoperative sampling. Micro-CT three-dimensional reconstruction showed that at 6 weeks postoperatively, the DFO@MOF-ZMCS group had richer new bone tissue and denser trabecular structure in the fracture area; at 12 weeks postoperatively, the trabecular continuity and overall morphology of this group were stronger, suggesting that it not only increases the amount of new bone formation, but also promotes the structural reconstruction of bone tissue (such as...). Figure 16 As shown in D, Figure 16 D represents the quantitative parameter analysis of micro-CT three-dimensional reconstruction. Quantitative analysis showed that bone mineral density (BMD) in the DFO@MOF-ZMCS group significantly increased at both 6 and 12 weeks. Simultaneously, bone volume fraction (BV / TV), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.Th) all significantly increased, while trabecular bone spacing (Tb.Sp) significantly decreased. Overall, it exhibited high-quality bone reconstruction characteristics of "increased number, thickened structure, and reduced spacing" (e.g., Figure 16 As shown in E–I, Figure 16E–I represents quantitative parameter analysis for micro-CT.

[0079] In summary, the DFO@MOF-ZMCS composite coating significantly promotes callus formation and bridging in an osteoporotic fracture model, and simultaneously improves bone microstructure parameters, suggesting that it can effectively reconstruct a microenvironment and mechanical structure conducive to bone regeneration in vivo. Combined with the aforementioned in vitro results of promoting angiogenesis, osteogenic activity, and inhibiting osteoclastogenesis, this composite coating is expected to restore the dynamic balance of bone remodeling by improving local blood supply, enhancing osteogenic activity, and inhibiting excessive bone resorption, thereby achieving sustained benefits to the osteoporotic fracture repair process.

[0080] Therefore, the organometallic framework composite coating loaded with active molecules on the zinc alloy surface is degradable and controllable and safe. By constructing a ZnO micro-nano transition layer and a metal-organic framework coating on the zinc alloy surface containing Mg, Ca, and Sr, the degradation rate of the zinc alloy matrix containing Mg, Ca, and Sr can be effectively slowed down, allowing zinc ions to be released stably and slowly. The zinc alloy substrate containing Mg, Ca, and Sr does not exhibit localized corrosion and will not cause toxicity to cells and tissues around the implantation site. Furthermore, due to the micro-nano structure of the metal-organic framework coating, the loaded DFO is released slowly, which can significantly accelerate the regeneration of blood supply to the fracture / bone defect site, promote angiogenesis and reconstruction, and at the same time, the coating regulates the release of active molecules and metal ions, which can maintain a dynamic balance between osteogenic and osteoclastogenic processes.

[0081] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0082] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface, characterized in that, Includes the following steps: S1. Preparation of ZnO nanostructure transition layer: Zinc alloy samples containing Mg, Ca, and Sr were cleaned, polished, and dried with sandpaper before use. The cleaned and dried zinc alloy samples containing Mg, Ca, and Sr were placed in a polytetrafluoroethylene-lined reactor. A hydrothermal reaction solution to induce the growth of a ZnO nanostructure transition layer was added to completely immerse the zinc alloy samples containing Mg, Ca, and Sr. The reactor was then sealed and tightened. The reactor was placed in a constant temperature oven for treatment to construct a ZnO nanostructure transition layer on the surface of the zinc alloy containing Mg, Ca, and Sr. The samples were then removed, gently rinsed with deionized water, and dried before use. S2, 2,5-Dihydroxyterephthalic acid induces in-situ formation of metal-organic framework coatings: A zinc alloy containing Mg, Ca, and Sr with a ZnO nanostructure transition layer on its surface is immersed in a reaction solution containing organic ligands to carry out an in-situ coordination reaction to generate a metal-organic framework coating. After immersion, the coating is gently rinsed with deionized water, dried, and then placed in a vacuum drying oven for later use. S3. Preparation of metal-organic framework composite coating loaded with deferroamine; Zinc alloy samples containing Mg, Ca, and Sr with a metal-organic framework coating were immersed in a deferoxamine solution to allow deferoxamine molecules in the solution to fully diffuse into the tiny pores of the metal-organic framework to complete drug loading. After loading, the samples were gently rinsed with deionized water, dried, and stored in a vacuum drying oven for later use.

2. The method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface according to claim 1, characterized in that, In S1, the hydrothermal reaction solution is a solution of 0.02M Zn(NO3)2·6H2O and 0.5M NaOH after thorough stirring, and the reaction vessel is placed in a constant temperature oven for hydrothermal treatment for 4 hours.

3. The method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface according to claim 2, characterized in that, In S3, the deferoxamine solution is a solution in which 9.43 mg / mL of Mg(NO3)2·6H2O, 0.19 mg / mL of Zn(NO3)2·6H2O, 4.51 mg / mL of 2,5-dihydroxyterephthalic acid, and 1.00 mg / mL of deferoxamine are fully dissolved.

4. The method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface according to claim 3, characterized in that, In step S1, the zinc alloy sample containing Mg, Ca, and Sr is mechanically polished using sandpaper with a grit size of 400-800 mesh to remove the oxide layer and impurities on the surface of the zinc alloy sample containing Mg, Ca, and Sr. The polished zinc alloy sample containing Mg, Ca, and Sr is then placed in an ultrasonic cleaner and cleaned sequentially with anhydrous ethanol and deionized water for 10 minutes each time. Anhydrous ethanol is used to remove oil stains from the surface of the zinc alloy sample containing Mg, Ca, and Sr, while deionized water is used to remove residual ethanol and other water-soluble impurities. The cleaned zinc alloy sample containing Mg, Ca, and Sr is then dried with nitrogen to remove any residual liquid from the surface.

5. The method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface according to claim 4, characterized in that, In step S1, the drying conditions of the drying oven are set to a temperature of 80°C, a vacuum degree of less than 0.1 MPa, and a duration of 2 hours, so that the moisture on the surface and inside of the zinc alloy sample containing Mg, Ca, and Sr is completely evaporated.

6. The method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface according to claim 5, characterized in that, In step S1, after the hydrothermal treatment is completed, the zinc alloy sample containing Mg, Ca, and Sr is taken out of the reaction vessel and gently rinsed with deionized water to remove the residual reaction solution and unreacted solutes on the surface. The rinsed zinc alloy sample containing Mg, Ca, and Sr is placed in a vacuum drying oven and dried at 80°C for 1 hour to remove the adsorbed moisture on the surface.

7. The method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface according to claim 6, characterized in that, In step S2, the reaction solution containing organic ligands is a 2,5-dihydroxyterephthalic acid ligand reaction solution with a concentration of 4.51 mg / mL. When the ZnO nanostructure transition layer undergoes a coordination reaction with the 2,5-dihydroxyterephthalic acid ligand and gradually transforms in situ to form a metal-organic framework crystal coating, a magnetic stirrer is used to continuously stir at a speed of 200 rpm to ensure that the 2,5-dihydroxyterephthalic acid molecules uniformly contact the surface of the zinc alloy sample containing Mg, Ca, and Sr. After the coordination reaction, the coordination bonds between the 2,5-dihydroxyterephthalic acid ligands on the ZnO surface and the Zn²⁺ ions are further extended, forming a three-dimensional network structure metal-organic framework coating.

8. The method for preparing a metal-organic framework composite coating with active molecules loaded on a zinc alloy surface according to claim 7, characterized in that, In step S2, after the in-situ conversion is completed, the sample is gently rinsed with deionized water to remove the residual 2,5-dihydroxyterephthalic acid solution and unreacted solutes on the surface. The rinsed zinc alloy sample containing Mg, Ca, and Sr is placed in a vacuum drying oven and dried at 80°C for 1 hour to remove the adsorbed moisture on the surface.

9. The application of the metal-organic framework composite coating prepared by the method for preparing a zinc alloy surface-loaded active molecule-based metal-organic framework composite coating as described in claim 8, characterized in that, The application of organometallic framework composite coatings in angiogenesis, bone repair, and fracture healing.

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