A multi-layer hybrid coating with a biomimetic cell membrane structure and a preparation method thereof

Abstract

The present invention provides a multi-layer hybrid coating with a biomimetic cell membrane structure and a preparation method thereof. For the first time, by mimicking the cell membrane structure, the hydrophobicity and bioactivity are organically combined in the same coating, solving the problems of poor tissue integration and osteogenesis performance of traditional organic hydrophobic coatings. Moreover, a transition layer mainly composed of MgF2 is used to enhance the interfacial bonding force between the coating and the magnesium substrate, improving the mechanical stability of the coating and the reliability during long-term use. And through the verification of subcutaneous implantation experiments in rats, it is proved that the coating has excellent in vivo corrosion resistance and biocompatibility, can effectively inhibit the in vivo corrosion of magnesium-based materials, and has good tissue binding potential. In summary, the multi-layer hybrid coating with a biomimetic cell membrane structure and the preparation method thereof proposed by the present invention can not only inhibit the biological corrosion of magnesium, but also have the potential to promote bone bonding and generation, especially in the fields of orthopedics and dental restoration, etc., and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of medical implantable devices such as oral, orthopedic, and cardiac stents, and particularly relates to a multi-layer hybrid coating with a biomimetic cell membrane structure and a preparation method thereof. Background Art

[0002] Magnesium metal and its alloy materials have extremely similar density, mechanical properties, good biocompatibility and biodegradable characteristics in vivo to human bone, and are widely regarded as the next-generation breakthrough implant materials, having broad application prospects in medical devices such as dentistry, orthopedics, and cardiac stents. However, due to its low reduction potential and active nature, magnesium metal corrodes too fast and uncontrollably after being implanted into the human body, causing problems such as high pH value, hydrogen production, and loss of mechanical properties around the implant, affecting the tissue compatibility of magnesium metal and the bone tissue repair effect, and even bringing potential safety hazards of tissue necrosis, which greatly limits its further clinical application transformation.

[0003] Up to now, the surface coating technology is still considered to be a direct and effective means to delay the in vivo corrosion of magnesium-based metals. Moreover, when a bioactive material is selected as the coating, while solving the corrosion problem, it will also endow the magnesium-based material with certain biological functions to achieve clinical multifunctionality. CN102286767A discloses a composite coating on the surface of a magnesium alloy biological implant material and a preparation method thereof. It uses the micro-arc oxidation technology to in-situ form an oxide porous film on the surface layer of the magnesium alloy matrix as a transition layer, and then a DLC thin film layer is set on the transition layer to reduce electrochemical corrosion. However, the above structure does not isolate the contact between the magnesium-based metal and the corrosion medium, so it will still be continuously affected by in vivo corrosion. Therefore, using a hydrophobic modified material (such as a polymer, surfactant or micro / nano structured material) as a physical barrier to isolate the contact between the magnesium-based metal and the corrosion medium is a more effective anti-corrosion means.

[0004] However, hydrophobicity often inhibits the biological activities such as cell adhesion, proliferation and differentiation on the surface of implant devices, thereby affecting subsequent tissue integration and bone regeneration effects. And directly incorporating bioactive substances into the skeleton of hydrophobic materials without structural design may damage the tight structure of the hydrophobic coating and reduce its anti-corrosion effect. In addition, the interface compatibility between the hydrophobic coating and the magnesium-based metal substrate is poor, resulting in the shedding and long-term instability of the coating. Therefore, there is an urgent need to develop a next-generation coating technology that can effectively combine good anti-corrosion performance, biocompatibility and excellent interface bonding to obtain high-performance magnesium-based metal implant devices. Summary of the Invention

[0005] In order to overcome the technical problems existing in the above-mentioned prior art, the present invention provides a multi-layer hybrid coating with a biomimetic cell membrane structure and a preparation method thereof.

[0006] Specifically, the present invention proposes a multi-layer hybrid coating imitating the structure of biological cell membranes, including a transition layer formed on the surface of a magnesium-based metal and a coating imitating the structure of biological cell membranes located on the transition layer. The coating imitating the structure of biological cell membranes is an alternately arranged calcium phosphate layer and an alkyl chain bilayer, and the main component of the calcium phosphate layer is octacalcium phosphate.

[0007] Furthermore, the transition layer is a porous coral-like structure, and the main component is MgF2.

[0008] Furthermore, the coating imitating the structure of biological cell membranes presents a flaky nanostructure.

[0009] Furthermore, the coating imitating the structure of biological cell membranes has hydrophobicity.

[0010] Furthermore, the coating imitating the structure of biological cell membranes and the transition layer are in an interlocking structure.

[0011] Correspondingly, the present invention also proposes a preparation method for a multi-layer hybrid coating with a structure of biological cell membranes. The specific process is as follows:

[0012] (1) Preparation of the transition layer: Use the micro-arc fluorination MAF process to prepare a transition layer (hereinafter simply referred to as the MAF layer) on the magnesium surface;

[0013] (2) Preparation of the fluorapatite coating (hereinafter simply referred to as the FAP layer): Form a fluorapatite coating through biomimetic mineralization induced by nano-hydroxyapatite NanoHAP;

[0014] (3) Preparation of the coating imitating the structure of biological cell membranes: React the fluorapatite coating with a dodecyl phosphate surfactant, and the fluorapatite coating is transformed into a flaky coating imitating the structure of biological cell membranes.

[0015] Among them, the preparation method of the transition layer specifically further includes:

[0016] 1) Cut the magnesium sheet into disc-shaped samples with a laser;

[0017] 2) Gradually polish through sandpaper in ethanol to obtain a smooth surface;

[0018] 3) During the micro-arc fluorination MAF process, the magnesium sample serves as the anode and the graphite rod serves as the cathode. Immerse them in a PTFE container containing 46 wt.% hydrogen fluoride. Apply a constant voltage of 185 - 190 V through a DC power supply in a constant voltage mode. The current is 0.145 - 0.155 A, and the current is 0.045 A. The reaction continues until the current drops below 0.01 A, indicating that the reaction is completed;

[0019] 4) The treated samples are rinsed three times with distilled water and air-dried at room temperature.

[0020] Among them, the preparation method of the fluorapatite coating specifically further includes:

[0021] 1) The sample after the transition layer treatment is immersed in a 2 wt.% NanoHAP seed suspension and left standing for 30 minutes;

[0022] 2) Air-dry at room temperature for 2 hours;

[0023] 3) The sample including NanoHAP seeds is immersed in a mineralization solution and incubated at 37 °C for 3 hours to induce the remineralization of NanoHAP seeds to form a fluorapatite coating;

[0024] 4) Air-dry at room temperature.

[0025] Among them, the preparation method of the biomimetic cell membrane structure coating specifically further includes:

[0026] 1) After the second air-drying, the sample is immersed in a 2 wt.% dodecyl phosphate surfactant solution (pH value: 10 ± 0.5, adjusted with 20% NaOH) and left standing for 2 hours;

[0027] 2) The obtained biomimetic cell membrane structure coating is thoroughly rinsed with deionized water and air-dried at room temperature.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] The present invention first proposes a multi-layer hybrid coating with a biomimetic cell membrane structure. For the first time, by mimicking the biological cell membrane structure, the hydrophobicity and bioactivity are organically combined in the same coating, solving the problem that traditional hydrophobic coatings are difficult to promote tissue integration and bone formation. And a transition layer mainly composed of MgF2 is used to enhance the interfacial bonding force between the coating and the magnesium substrate, improving the mechanical stability of the coating and the reliability during long-term use. And through the verification of the rat subcutaneous implantation experiment, it is proved that the coating has excellent in vivo corrosion resistance and biocompatibility, can effectively inhibit the corrosion of magnesium-based materials, and has good tissue bonding potential. In summary, the multi-layer hybrid coating with a biomimetic cell membrane structure proposed by the present invention can not only inhibit the biological corrosion of magnesium, but also has the potential to promote bone bonding and bone formation, especially in the fields of bone repair in orthopedics and stomatology, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the function, internal structure and preparation process of the multi-layer hybrid coating with a biomimetic cell membrane structure on magnesium metal in the present invention. Figure 1In: a is a schematic diagram of the rapid corrosion of pure magnesium; b is a schematic diagram of the dual functions of the OCP-SH coating; c is a schematic diagram of the multi-layer hybrid structure of the OCP-SH coating; d is a schematic diagram of the preparation process of the OCP-SH coating.

[0031] Figure 2 It is the characterization of the magnesium metal coating in the present invention. Figure 2 In: The SEM images in a-c respectively show the surface morphologies of the MAF, FAP, and OCP-SH coatings; d is the small-angle XRD pattern of the magnesium metal coating; e is the FTIR spectrum of the magnesium metal coating; f is the water contact angle test result of the magnesium metal coating.

[0032] Figure 3 It is the experimental results of the mechanical properties and interfacial bonding strength of the coating in the present invention. Figure 3 In: The SEM images in a-c respectively show the porous MAF layer (Figure a), and the chimeric interfaces between the FAP layer and the MAF layer (Figure b) and the OCP-SH layer and the MAF layer (Figure c), and the interfaces are marked by yellow dotted lines; d-f are respectively the optical surface image (Figure d), the displacement-scratch depth curve (Figure e), and the critical interfacial fracture force (Figure f) of the coating scratch test.

[0033] Figure 4 It is the experimental results of the corrosion resistance of the coating in the present invention. Figure 4 In: a and b are the electrochemical test results in simulated body fluid (SBF), including the EIS spectra (Figure a) and potentiodynamic polarization curves (PDP) (Figure b) of pure magnesium and samples coated with MAF, FAP / MAF, and OCP-SH / MAF coatings; c is the hydrogen release test result for 912 hours in SBF, and the inset shows the digital photo of the 8-mm-diameter sample after the test; d is the magnesium ion release data of the 28-day immersion test in α-MEM containing 10 wt.% fetal bovine serum protein (FBS).

[0034] Figure 5 It is the experimental results of the in vitro cell compatibility and osteogenic ability of the coating in the present invention. Figure 5In the figure: a shows the confocal fluorescence images of MC3T3-E1 cells and human gingival fibroblasts (HGFs) on pure magnesium and coated magnesium, respectively showing the cell adhesion morphology after culturing for 4 hours, 24 hours, and 72 hours. Among them, the cell nuclei are stained blue, the actin filaments are stained red, and the vinculin is stained green. The scale bars of the fluorescence images are all 20 μm; b and c respectively show the biocompatibility of HGFs and MC3T3-E1 cells evaluated by the CCK-8 experiment, testing the cell proliferation after culturing for 24 hours, 48 hours, and 72 hours; d shows the alkaline phosphatase (ALP) activity of human bone marrow mesenchymal stem cells (hBMSCs) under osteogenic induction conditions after culturing on different substrates for 7 days.

[0035] Figure 6 It is a comparison chart of the in vivo anti-corrosion performance and biocompatibility of the coating in the present invention. Figure 6 In the figure: a-d are the cross-sectional morphologies and corresponding element distribution maps of magnesium substrate (Figure a), MAF-coated magnesium (Figure b), FAP-coated magnesium (Figure c), and OCP-SH / MAF-coated magnesium (Figure d) after implantation for 4 weeks, respectively. Among them, Figure a is under a 30-μm scale bar, and Figures b-d are under a 9-μm scale bar; Figure e is an optical micrograph of H&E staining of the subcutaneous tissue around the implant after implantation for 1 week and 4 weeks under a 100-μm scale bar, where "*" represents the insert, and the blue-stained area is inflammatory cells. Detailed implementation manners

[0036] Combined with the accompanying drawings in the examples of the present invention below, a multi-layer hybrid coating with a biomimetic cell membrane structure and its preparation method in the present invention will be further described. The following examples are only used to illustrate the present invention and are not intended to limit the present invention.

[0037] As Figure 1 shown in Figure a, magnesium-based implants have problems of rapid biological corrosion and insufficient bone integration. Based on such technical problems, the present invention discovers that in nature, the structure of the biological cell membrane cleverly combines hydrophobic isolation regions with hydrophilic bioactive regions, and can further closely bind to other cells or substrates through special linking structures such as glycoprotein anchor connection structures.

[0038] The layered calcium phosphate material, octacalcium phosphate (OCP), has the advantages of both calcium phosphate materials and layered materials. First, OCP has good biocompatibility, strong bone tissue affinity, and osteogenic bioactivity. Second, the layered structure of OCP consists of apatite layers and hydrated layers. And different guest molecules, including cations, dicarboxylic acids, amino triols, and silicon and other organic molecules, can be introduced into the hydrated layer through substitution. Based on this structural feature, hydrophobic organic materials can be introduced into the inorganic OCP bioactive material to construct an organic / inorganic hybrid material.

[0039] Therefore, in order to highly mimic the structure of biological cell membranes, the present invention selects a phospholipid-like dodecyl phosphate (MDP) surfactant, specifically sodium dodecyl phosphate, as the guest molecule and inserts it into the hydrated layer of the OCP host structure. In this way, a hybrid coating of alternating bioactive OCP layers and hydrophobic alkyl chain bilayers is formed, effectively combining hydrophobicity and bioactivity. At the same time, this coating can inhibit the biological corrosion of magnesium and promote bone formation, showing great potential in the application of magnesium-based implants.

[0040] Specifically, the present invention proposes a multi-layer hybrid coating with a bio-mimetic cell membrane structure, which includes a transition layer formed on the magnesium-based metal surface by micro-arc fluorination (MAF) process, and a bio-mimetic cell membrane structure coating (hereinafter referred to as OCP-SH coating) located on the transition layer. The bio-mimetic cell membrane structure coating is an alternating arrangement of calcium phosphate layers and alkyl chain bilayers. Among them, the main component of the calcium phosphate layer is octacalcium phosphate. The meaning of the main component is that it occupies a large proportion. For example, in the present invention, the component content > 50%, and it can also be preferably 60%, 70%, 80% or more than 90%, and it is the core of the calcium phosphate layer, determining the basic physical, chemical and biological properties of the calcium phosphate layer. This coating combines alternating bioactive OCP layers and hydrophobic alkyl chain bilayers, enhancing both the anti-corrosion performance and promoting bioactivity (such as Figure 1 in b and c). The hydrophobic alkyl chain bilayer structure of phospholipid-like in the coating can effectively prevent the penetration of corrosive biological fluids, while the surface bioactive OCP layer helps cell integration. To achieve this coating, the present invention adopts a step-by-step in-situ growth method (such as Figure 1 in d).

[0041] The material characterization of the multi-layer hybrid coating proposed by the present invention is as follows:

[0042] The morphology of the coating was observed using a scanning electron microscope (SEM), showing that the MAF layer has a highly porous coral-like structure (thickness about 2 - 7 μm), while the OCP-SH layer presents a flaky nanostructure (coating thickness about 1 μm) (such as Figure 2 in a - c). The OCP-SH coating and the MAF layer are in an interlocking structure. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) indicate that the OCP-SH coating has a highly ordered layered structure (such as Figure 2 in d and e). In addition, the water contact angle test of the coating shows that the OCP-SH coating has significant hydrophobicity (contact angle 148.5°) (such as Figure 2 f). These results prove the successful preparation of the OCP-SH coating.

[0043] The present invention also provides a method for preparing a multi-layer hybrid coating with a biomimetic cell membrane structure:

[0044] (1) Preparation of the MAF layer: The micro-arc fluorination (MAF) process is used to prepare a transition layer (hereinafter referred to as the MAF layer) on the magnesium surface. This layer provides preliminary corrosion resistance and supports the deposition of subsequent coatings and enhances interfacial bonding through its porous structure, effectively mechanically interlocking the OCP-SH coating with the magnesium substrate, ultimately ensuring the in-situ formation of the OCP-SH coating and improving the adhesion and interfacial bonding stability of the coating.

[0045] (2) Preparation of the fluorapatite coating (FAP layer): The fluorapatite coating is formed through biomimetic mineralization induced by nano-hydroxyapatite (NanoHAP).

[0046] (3) Preparation of the OCP-SH coating: The FAP layer is reacted with a dodecyl phosphate (MDP) surfactant for 2 hours to transform it into a flaky OCP-SH coating. By means of an in-situ chemical reaction on the surface of the FAP layer with the MDP surfactant, the MDP surfactant is intercalated into the OCP matrix to form an alternately arranged bioactive calcium phosphate layer (OCP layer) and a hydrophobic alkyl chain bilayer, thereby achieving an organic combination of hydrophobicity and bioactivity and forming a coating with dual functions.

[0047] The preparation method further includes the following steps for preparing the nano-HAP seed suspension and the mineralization solution:

[0048] 1) Preparation of the nano-HAP seed suspension: A nano-HAP seed suspension is prepared by mixing 2 wt.% of NanoHAP particles with 1 wt.% of gelatin binder in ultrapure water and performing ultrasonic treatment for 40 minutes. This suspension serves as the precursor for the subsequent NanoHAP seed adsorption process.

[0049] 2) Preparation of the mineralization solution: The mineralization solution (artificial saliva) is prepared by adding 5 ppm fluoride ions to a NaF reagent and is formulated with 1 mM CaCl2·2H2O, 4 mM KH2PO4, 20 mM HEPES, 16 mM KCl, 4.5 mM NH4Cl, and 0.2 mM MgCl2·6H2O, and the pH value is adjusted to 7.0.

[0050] The preparation method of the MAF layer specifically further includes:

[0051] 1) The magnesium sheet is cut into discs by laser. The magnesium sheet is a commercial magnesium sheet, specifically, the commercial magnesium sheet produced by Dongguan Feitai Metal Products Co., Ltd. can be selected. The size of the disc can be specifically Ø16×1.5 mm, Ø8×0.5 mm, or other sizes. In this invention, a disc sample with a diameter of 16×1.5 mm is taken as an example;

[0052] 2) Gradually polish with 1000# and 2000# SiC sandpaper in ethanol to obtain a smooth surface;

[0053] 3) During the micro-arc fluorination (MAF) process, the magnesium sample serves as the anode and the graphite rod serves as the cathode, and they are immersed in a PTFE container containing 46 wt.% hydrogen fluoride. Through the constant voltage mode of a DC power supply, a constant voltage of 185 - 190 V is applied, and the current is 0.145 - 0.155 A. The reaction continues until the current drops below 0.01 A, indicating the completion of the reaction.

[0054] 4) The treated samples are rinsed three times with distilled water and air-dried at room temperature.

[0055] The preparation method of the FAP layer specifically further includes:

[0056] 1) The samples after MAF layer treatment are immersed in a 2 wt.% nano-HAP seed suspension, and left standing for 30 minutes to allow the seed crystals to adsorb onto the porous surface;

[0057] 2) Air-dry at room temperature;

[0058] 3) The samples including NanoHAP seeds are immersed in a mineralization solution and incubated at 37 °C for 3 hours to induce the remineralization of NanoHAP seeds to form a fluorapatite (FAP) coating;

[0059] 4) Air-dry at room temperature.

[0060] The preparation method of the OCP-SH coating specifically further includes:

[0061] 1) After the second air-drying, the samples are immersed in a 2 wt.% MDP solution (pH value: 10 ± 0.5, adjusted with 20% NaOH), and left standing for 2 hours to promote the in-situ reaction between the FAP layer and the MDP surfactant;

[0062] 2) The obtained OCP-SH coating samples are thoroughly rinsed with deionized water and air-dried at room temperature.

[0063] The experimental results analysis of the multi-layer hybrid coating with a biomimetic cell membrane structure proposed by the present invention is as follows:

[0064] 1. The mechanical properties are analyzed as follows:

[0065] The mechanical properties of the coating are crucial for its practicality. In the present invention, a nano-scratch tester is used to test the adhesion strength of the coating. A diamond indenter is used, and the lateral force is gradually increased to a maximum of 5 N, and a lateral movement of 30 μm is performed.

[0066] To enhance the interfacial adhesion of the OCP-SH coating, the MAF layer provides a porous structure, enhancing the physical interlocking effect (as shown in Figure 3 a-c in Figure 3 ). Nano-indentation scratch tests show that its adhesion strength is significantly improved, reaching 4.57 ± 0.02 N / cm (as shown in

[0067] d-f in 5 ). The corrosion resistance analysis of the coating proposed in the present invention is as follows:

[0068] The present invention uses an electrochemical workstation to conduct corrosion performance tests. A classical three-electrode cell is adopted, with the working electrode being a sample with an exposed area of 1 cm², the reference electrode being a saturated potassium chloride electrode, and the auxiliary electrode being a platinum electrode. At room temperature, the sample is immersed in 300 ml of simulated body fluid (SBF) for 30 minutes to reach a stable open-circuit potential. Electrochemical impedance spectroscopy (EIS) tests are performed, with the frequency range from 0.1 Hz to 10

[0069] Hz and the amplitude of 20 mV; and potentiodynamic polarization curve (PDP) tests are conducted, with the scanning range from -250 mV to +250 mV (relative to the open-circuit potential) and the scanning rate of 0.225 mV / s. 6 Ω·cm², which is 13 times that of the MAF and FAP / MAF coatings (as shown in Figure 4 a and b in Figure 4 ). The 912-hour hydrogen release experiment shows that the hydrogen release amount of the OCP-SH coating is reduced by 87%, indicating its excellent long-term corrosion resistance (as shown in Figure 4 c in

[0070] ). The 28-day magnesium ion release experiment further verifies the stability of the coating (as shown in

[0071] d in

[0072] The immersion medium was α-MEM containing glucose, amino acids, and 10 wt.% fetal bovine serum (FBS) to simulate complex biological fluids. In the in vitro immersion method, coated and uncoated magnesium samples (n = 3, Ø16×1.5 mm) were placed in 24-well plates, 2 ml of immersion medium was added, and it was changed every 24 hours. The supernatants of three parallel samples were analyzed by inductively coupled plasma spectrometry to quantify the magnesium ion leaching at different time points.

[0073] (2)In vitro cell proliferation assay

[0074] After sterilizing the samples by ultraviolet irradiation for 2 hours, the four groups of samples, Mg, MAF, FAP / MAF, and OCP-SH / MAF, were immersed in α-MEM overnight. Then, mouse pre-osteoblasts (MC3T3-E1) and human gingival fibroblasts (HGFs) were seeded onto the material surfaces at a density of 3×10 4 cells / sample, and cultured in α-MEM and DMEM containing 10% FBS and 1% penicillin-streptomycin, respectively, in an environment of 37°C and 5% CO2. Cell proliferation was measured at 24-hour intervals after seeding using the CCK-8 method.

[0075] (3)In vitro cell adhesion observation

[0076] To observe the adhesion morphology of cells at 4 hours, 24 hours, and 72 hours, the samples were first fixed with 4% paraformaldehyde. Subsequently, the cytoskeletal protein F-actin was stained red with fluorescein-labeled phalloidin-rhodamine, and the nucleus was stained blue with DAPI. At 24 hours, focal adhesion protein was further stained green with fibronectin / FN1. Finally, three-dimensional images were taken using a confocal laser scanning microscope.

[0077] (4)In vitro cell differentiation experiment

[0078] Human bone marrow mesenchymal stem cells were seeded onto the material surfaces at a density of 1×10 5 cells / sample. After two days of culture, osteogenic induction medium was used to promote the osteogenic differentiation of hBMSCs. This medium included α-MEM medium, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1×10⁻ 8 mol / L dexamethasone, 2.8×10⁻ 46.5×10⁻³ mol / L β-glycerophosphate and 6.5×10⁻³ mol / L ascorbic acid, and the culture medium was changed every two days. After 7 days of induction culture, alkaline phosphatase (ALP) staining and quantitative analysis were performed using a BCIP / NBT alkaline phosphatase colorimetric kit, which quantifies ALP activity by measuring the conversion of p-nitrophenyl phosphate (pNPP) to colored p-nitrophenol. The total protein concentration in the cells was determined using a protein assay kit, and the ALP activity was normalized by calculating the ratio of the absorbance of p-nitrophenol to the total protein concentration.

[0079] The experimental results showed that the OCP-SH coating exhibited excellent performance in promoting cell adhesion, proliferation, and differentiation. In in vitro experiments, human gingival fibroblasts (HGFs) and mouse pre-osteoblasts (MC3T3-E1) showed significantly enhanced adhesion and spreading abilities on the surface of the OCP-SH coating (as shown in Figure 5 a). Compared with uncoated magnesium, the OCP-SH coating not only promoted the extension of filopodia between cells but also formed stronger cell-cell network connections. In addition, cell proliferation experiments showed that the proliferation ability of cells on the coated surface was significantly better than that of other groups (as shown in Figure 5 b and c). On human bone marrow mesenchymal stem cells (hBMSCs), the OCP-SH coating showed high alkaline phosphatase (ALP) activity, indicating its osteogenic potential (as shown in Figure 5 d). These performance improvements can be attributed to the nanosheet structure and unique chemical properties of the coating, especially the bioactive OCP layer with a bone-like mineral structure and the stable hydrophobic alkyl chain bilayer, which together provide a favorable microenvironment for cells.

[0080] 3. The in vivo anti-corrosion performance and biocompatibility experiments of the coating proposed in the present invention are as follows:

[0081] (1) Establishment of a rat subcutaneous implantation model

[0082] All animal surgeries in this study were approved by the Animal Ethics Committee of Peking University, approval number PUIRB-LA2023439. Eighty-week-old male Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Before implantation, the dorsal area of each rat was disinfected with iodophor. Subsequently, the skin was incised bilaterally along the midline of the back to create subcutaneous pockets, with three pockets set on each side with the spine as the central axis. Samples from four groups, including Mg, MAF-Mg, FAP / MAF-Mg, and OCP-SH / MAF (sample size: Ø8×0.5 mm), were randomly inserted into each pocket. After the surgery, antibiotics were used to prevent infection in the animals to ensure their health throughout the study.

[0083] (2)In vivo corrosion analysis

[0084] Four weeks after implantation, the animals were euthanized and three samples were taken from each group. The samples were fixed in 10% neutral formalin for 48 hours to maintain their structural integrity. Subsequently, the surface tissue was digested with 0.25% trypsin-EDTA to expose the implant. The removed samples were embedded in epoxy resin and sequentially polished with 1000#, 2000#, and 5000# silicon carbide (SiC) sandpaper, and then polished with a silk cloth in an ethanol environment. To enhance the surface conductivity, a carbon layer was deposited on the exposed cross-section using a high-vacuum ion sputtering instrument. The morphology and elemental composition of the corrosion layer were analyzed by scanning electron microscopy (SEM) and energy-dispersive spectroscopy (SEM-EDS).

[0085] (3)In vivo biocompatibility analysis

[0086] To evaluate tissue compatibility, the animals were euthanized at 1 week and 4 weeks after implantation, respectively. The implant and the surrounding tissue were carefully excised and fixed in 10% neutral formalin for 48 hours. The tissue was then washed, dehydrated in 70% ethanol, and processed for paraffin embedding for histological analysis. The paraffin-embedded samples were cut into 5-μm thick sections and stained with hematoxylin and eosin (H&E) to evaluate cell infiltration and the formation of fibrous capsule. Panoramic images of the stained sections were captured using a high-resolution digital scanner to record and analyze the tissue reaction.

[0087] The experimental results showed that after implanting the samples subcutaneously in SD rats for 4 weeks, the OCP-SH coating exhibited significant in vivo anti-corrosion performance, with a corrosion depth of only 1.1 μm, much lower than the MAF and FAP coatings (as shown in Figure 6 a-d). Histological analysis showed that all samples induced an inflammatory response at the initial stage of implantation, but the fibrous capsule thickness of the OCP-SH coating was only 65.2 μm, significantly lower than that of other coating groups and uncoated magnesium, and maintained the lowest inflammatory response after 4 weeks (as shown in Figure 6 e). These results indicate that the OCP-SH coating has excellent in vivo biocompatibility and long-term anti-corrosion performance, providing reliable support for the clinical application of magnesium-based implants.

[0088] The above embodiments have described the present invention in detail, but the described content is only the preferred embodiment of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of the application of the present invention shall still fall within the scope covered by the patent of the present invention.

Claims

1. A multi-layer hybrid coating with a biomimetic cell membrane structure, comprising a transition layer formed on the surface of a magnesium-based metal and a biomimetic cell membrane structure coating located on the transition layer. The biomimetic cell membrane structure coating is an alternately arranged calcium phosphate layer and alkyl chain bilayer, where the main component of the calcium phosphate layer is octacalcium phosphate, and the coating has in vivo corrosion resistance.

2. The multi-layer hybrid coating with a biomimetic cell membrane structure according to claim 1, characterized in that: The transition layer is a porous coral-like structure with a main component of MgF2.

3. The multilayer hybrid coating with a biomimetic cell membrane structure according to claim 1, characterized in that: The biomimetic cell membrane structure coating presents a flaky nanostructure.

4. The multi-layer hybrid coating with a biomimetic cell membrane structure according to claim 1, characterized in that: The biomimetic cell membrane structure coating has hydrophobicity.

5. The multi-layer hybrid coating with a biomimetic cell membrane structure according to claim 1, wherein: The biomimetic cell membrane structure coating and the transition layer are in an interlocking structure.

6. A preparation method of a multi-layer hybrid coating with a biomimetic cell membrane structure according to any one of claims 1-5, and its specific process is as follows: (1) Preparation of the transition layer: Use the micro-arc fluorination (MAF) process to prepare the transition layer on the magnesium surface; (2) Preparation of the fluorapatite coating: Form a fluorapatite coating through biomimetic mineralization induced by nano-hydroxyapatite (NanoHAP); (3) Preparation of the biomimetic cell membrane structure coating: React the fluorapatite coating with a dodecyl phosphate surfactant, and the fluorapatite coating is transformed into a flaky biomimetic cell membrane structure coating.

7. The preparation method of the multi-layer hybrid coating with a biomimetic cell membrane structure according to claim 6, characterized in that The preparation method of the transition layer specifically further includes: 1) Cut the magnesium sheet into disc-shaped samples with a laser; 2) Gradually polish with sandpaper to obtain a smooth surface; 3) During the micro-arc fluorination (MAF) process, the magnesium sample is used as the anode and the graphite rod is used as the cathode, immersed in a PTFE container containing 46 wt.% hydrogen fluoride, and a constant voltage of 185-190V is applied through a DC power supply in a constant voltage mode. The reaction continues until the current drops below 0.01A, indicating the completion of the reaction; 4) The treated samples are rinsed three times with distilled water and air-dried at room temperature.

8. The preparation method of the multi-layer hybrid coating with a biomimetic cell membrane structure according to claim 6, characterized in that, The preparation method of the fluorapatite coating specifically further includes: 1) The samples after the transition layer treatment are immersed in a 2 wt.% nano-hydroxyapatite (NanoHAP) seed suspension and left standing for 30 minutes; 2) Air-dry at room temperature; 3) The samples including nano-hydroxyapatite (NanoHAP) seeds are immersed in a mineralization solution and incubated at 37 °C for 3 hours to induce the remineralization of nano-hydroxyapatite (NanoHAP) seeds to form a fluorapatite coating; 4) Air-dry at room temperature.

9. The preparation method of the multi-layer hybrid coating with a biomimetic cell membrane structure according to claim 6, characterized in that, The preparation method of the biomimetic cell membrane structure coating specifically further includes: 1) After the second air-drying, the samples are immersed in a 2 wt.% dodecyl phosphate surfactant solution and left standing for 2 hours; 2) The obtained biomimetic cell membrane structure coating is thoroughly rinsed with deionized water and air-dried at room temperature.

10. The preparation method of the multi-layer hybrid coating with a biomimetic cell membrane structure according to claim 9, characterized in that: The pH value of the dodecyl phosphate surfactant solution is 10 ± 0.5, adjusted with 20% NaOH.

Images