In vivo mineralization self - growth method of a double - layer protective coating, corresponding double - layer protective coating and its preparation method

Abstract

The present invention provides an in-vivo mineralization self-growth method for a double-layer protective coating, as well as a corresponding double-layer protective coating and a preparation method thereof, which can be used in the technical field of the preparation of medical implantable medical devices such as oral cavity, orthopedics, and cardiac stents. By continuously releasing bio-mineralization promoting factors into body fluids, bio-mineralization is promoted, and then the abundant "calcium source" and "phosphorus source" in body fluids are dynamically and in-situ transformed into a mineral protective layer on the magnesium metal surface under the action of in-vivo bio-mineralization. Through reasonable structural design, newly formed minerals will continuously deposit inside the coating, repair the internal defects of the coating, and maintain the denseness and integrity of the coating. Since the newly formed minerals are spontaneously generated in the body fluid environment, they can better adapt to the body fluid corrosion environment and maintain good stability in body fluids, thereby achieving persistent and self-adaptive protection for magnesium-based metals in complex body fluid corrosion environments and having good biocompatibility and bioactivity.

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 method for in vivo mineralization self-growth of a double-layer protective coating, a double-layer protective coating, and a preparation method thereof. Background Art

[0002] Based on mechanical properties similar to bone tissue, good biocompatibility, and biodegradable characteristics, magnesium and its alloys have shown broad application prospects in the field of medical implantable devices such as dentistry, orthopedics, and cardiac stents. However, due to the relatively low reduction potential and active nature of magnesium-based metals, after implantation into the human body, they are corroded by body fluids, resulting in an excessively fast and uncontrollable degradation rate, further causing problems such as local high pH value, hydrogen production, and premature loss of mechanical properties, affecting the tissue compatibility and bone repair effect of magnesium-based metals, and even posing a safety hazard of tissue necrosis, which greatly limits their further clinical application transformation. Up to now, surface coating technology is still considered a direct and effective means to control the degradation rate of magnesium-based metals. And when a bioactive material is selected as the coating material, while controlling the degradation rate, it also endows the magnesium-based material with certain biological functions, realizing greater clinical application value.

[0003] However, traditional organic coatings (such as polylactic acid-based, polycaprolactone-based, etc.) have inherent defects such as poor ability to promote bone tissue regeneration and weak interfacial bonding with magnesium metal, and are prone to produce acidic substances after degradation in vivo, further exacerbating the corrosion of magnesium metal. Therefore, traditional organic coatings are not an ideal choice for magnesium-based metal coating technology.

[0004] In the prior art DE10143410A1, a method for forming synthetic apatite by self-deposition of hydroxyapatite particles on the metal surface is disclosed, which can improve sufficient adhesion strength and incorporate hydroxyapatite into the organic phase in a bio-mineralized characteristic form. The prior art CN111973812A discloses a degradable magnesium-based bone implant with a bioactive and hierarchical hydroxyapatite coating on its surface and a preparation method thereof. The coating includes an inner layer fluorinated film and an outer layer hydroxyapatite conversion coating. This method first immerses magnesium and its alloys in hydrofluoric acid to generate a chemical conversion film MgF2, and then immerses them in a calcium phosphate salt solution, and uses chemical deposition to generate a brushite coating; finally, a hydrothermal conversion method is used to in-situ convert the brushite coating into a thicker hydroxyapatite coating with a micro-nano hierarchical structure composite surface morphology under high temperature and pressure, which can improve the corrosion resistance and biological activity of the magnesium matrix. However, although traditional inorganic coatings have advantages in bone bonding performance and mechanical properties, due to their high brittleness, they are prone to internal defects such as cracks during surgical procedures such as screw implantation and bending, which in turn causes the infiltration of corrosive body fluids and triggers rapid corrosion of the magnesium metal substrate. Therefore, they cannot provide long-term and effective protection.

[0005] To extend the protection time of the coating, the strategy of "self-healing anti-corrosion coating" has also been proposed. This strategy involves loading anti-corrosion (or self-healing) agents into the coating skeleton and releasing them in a responsive manner after the coating is damaged to prevent further corrosion. However, due to the fact that the loaded anti-corrosion agents (or self-healing agents) are usually consumed over time and cannot be effectively replenished in the body, and there are also problems such as potential toxicity, the long-term reliability and biosafety of this strategy still face huge challenges. Summary of the Invention

[0006] In order to overcome the technical problems existing in the above-mentioned prior art, based on the innovative strategy of "in vivo mineralization self-growing coating", the present invention proposes an in vivo mineralization self-growing method for a double-layer protective coating, as well as a corresponding product of the double-layer protective coating and its preparation method.

[0007] Specifically, the present invention first provides an in vivo mineralization self-growing method for a double-layer protective coating, which includes a double-layer protective coating based on magnesium-based metal. The first layer of the double-layer protective coating is a mineralization-promoting factor release layer, and the second layer is a mineral growth-promoting layer. After the double-layer protective coating is implanted into the human body, the mineralization-promoting factors in the mineralization-promoting factor release layer are released into the body fluid environment, promoting the biomineralization of calcium and phosphorus in the body fluid environment and continuously depositing and mineralizing in the mineral growth-promoting layer, converting into new minerals. The new minerals continuously fill the defects in the second layer, and finally form a stable and dense coating in the corrosive body fluid, providing lasting protection for the magnesium-based metal.

[0008] Furthermore, the mineralization-promoting factor in the present invention can be fluoride ions. The mineralization-promoting factor release layer of the first layer can be a porous MAF layer formed on the surface of the magnesium-based metal by the micro-arc fluorination (MAF) process, and the mineral growth-promoting layer of the second layer can be a fluorapatite (FAP) layer.

[0009] Correspondingly, the present invention also proposes a double-layer protective coating, in which the first layer is a mineralization-promoting factor release layer and the second layer is a mineral growth-promoting layer.

[0010] Furthermore, the mineralization-promoting factor release layer is a porous MAF layer formed on the surface of the magnesium-based metal by the micro-arc fluorination (MAF) process, and the mineral growth-promoting layer can be a fluorapatite (FAP) layer.

[0011] Furthermore, the MAF layer has a coral-like porous structure, with a thickness of 0.5 to 9 micrometers, preferably 3 to 5 micrometers, and the main component is MgF2.

[0012] Further, in the fluorohydroxyapatite FAP layer, the FAP nanorods can be assembled into FAP-P substantially parallel to the surface of the MAF layer, or the FAP nanorods can be assembled into FAP-V substantially perpendicular to the MAF surface, and the FAP layer and the MAF layer form an interpenetrating structure.

[0013] Among them, due to certain errors in the generation in the vertical or horizontal directions, the meanings of substantially parallel and substantially perpendicular are to roughly present a vertical or parallel state, but there may be certain errors or slight deviations.

[0014] Correspondingly, the present invention also proposes a preparation method for a double-layer protective coating, and its specific process is as follows:

[0015] (1) Preparation of the MAF layer: Through the MAF process, a porous MAF layer is formed on the magnesium-based metal surface, and the main component of the porous MAF layer is MgF2;

[0016] (2) Preparation of the FAP layer: Adsorb nano-hydroxyapatite NanoHAP seed crystals on the surface of the MAF layer and place them in a mineralization solution, and control the adsorption amount of the seed crystals, the temperature and time of the mineralization reaction, so as to controllably form the FAP layer microstructure on the MAF layer.

[0017] Further, the preparation method of the MAF layer further includes:

[0018] (1) Cut the magnesium sheet into a disk with a handle by laser, and the size of the disk can specifically be selected as Ø16×1.5mm, Ø8×0.5mm, Ø6×0.5mm, and other sizes;

[0019] (2) And gradually polish the disk with #1000 and #2000 silicon carbide papers, polish it, and clean it with absolute ethanol, and dry it at room temperature in air.

[0020] (3) After polishing, the magnesium disk with a holding tail is used as the anode, and the graphite rod is used as the cathode, and it is immersed in a polytetrafluoroethylene PTFE container filled with 46% hydrofluoric acid, and micro-arc fluorination is carried out in the direct current constant voltage mode;

[0021] (4) After the MAF process is completed, the sample is washed with deionized water and ethanol in turn and dried at room temperature.

[0022] Preferably, the conditions of the micro-arc fluorination MAF process are: when the sample is a disk with a size of Ø16×1.5mm, in the direct current constant voltage mode: the voltage is 180~210V, preferably 185~195V; the current is 150~165mA, and it changes with the diameter of the sample, the voltage remains unchanged, and the current decreases in proportion to the contact area.

[0023] Further, the preparation method of the FAP layer further includes:

[0024] (1) Immerse the MAF layer into the suspension of NanoHAP.

[0025] (2) Take out the MAF sample after soaking it in the NanoHAP dispersion for 5 - 40 minutes. After the surface level volatilizes, immerse it into the mineralization solution for mineralization.

[0026] Among them, the concentration of the NanoHAP suspension can be determined according to the amount of crystal seed layer to be adsorbed and deposited as needed, and is further preferably a 2% NanoHAP deionized water dispersion.

[0027] Further, 1% gelatin can be added or not added as a dispersant.

[0028] Further, in the fluorohydroxyapatite FAP layer, FAP nanorods can be assembled into FAP-P basically parallel to the surface of the MAF layer, or FAP nanorods can be assembled into FAP-V basically perpendicular to the MAF surface.

[0029] Among them, the synthesis conditions for assembling FAP-P with FAP nanorods basically parallel to the surface of the MAF layer are: after the deposition of NanoHAP seeds, grow in the mineralization solution at 12 - 17 °C, preferably at 15 °C, for 2 - 8 hours, preferably for 3 hours, then wash with deionized water and dry at room temperature.

[0030] Among them, the synthesis conditions for assembling FAP-V with FAP nanorods basically perpendicular to the MAF surface are: deposit NanoHAP seeds, grow in the mineralization solution at 37 °C for 2 - 8 hours, preferably for 3 hours, then wash with deionized water and dry at room temperature.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects:

[0032] By proposing an in-vivo mineralization self-growth method for a double-layer protective coating, the present invention continuously releases mineralization-promoting factors into the body fluid to promote biomineralization, and then dynamically and in-situ converts the abundant "calcium source" and "phosphorus source" in the body fluid into a mineral protective layer on the magnesium metal surface under the action of in-vivo biomineralization. Through reasonable structural design, new minerals will continuously deposit inside the coating, repair the internal defects of the coating, and maintain the compactness and integrity of the coating. Since the new minerals are spontaneously generated in the body fluid environment, they can better adapt to the body fluid corrosion environment and maintain good stability in the body fluid, thereby achieving the persistent and self-adaptive protection of magnesium-based metals in a complex body fluid corrosion environment. Similarly, since the self-growth protective layer is formed in the in-vivo environment, the self-growth coating has good biocompatibility and bioactivity. Description of the Drawings

[0033] Figure 1It is the design concept diagram and mineralization self-growth schematic diagram of the in-vivo mineralization self-growth method of the double-layer protective coating proposed by the present invention.

[0034] Figure 2 It is the characterization diagram of the MAF coating in the present invention. Figure 2 Among them: A is the XRD diagram; B is the cross-section SEM and EDS diagrams; C and D are the SEM cross-section diagrams.

[0035] Figure 3 It is the schematic diagram of the FAP / MAF double-layer coating in the present invention. Figure 3 Among them: A is the schematic diagram of the synthesis preparation process; B are the SEM photos of the surface morphologies of the MAF, FAP-P / MAF, and FAP-V / MAF coatings; C is the SEM cross-section photo of the surface morphology.

[0036] Figure 4 It is the characterization diagram of the FAP / MAF double-layer coating in the present invention. Figure 4 Among them: A is the atomic number ratio of Ca element and P element in the FAP-P and FAP-V coatings; B are the XRD curves of the FAP-P / MAF and HAP-V / MAF coatings; C are the ATR-FTIR curves of the FAP-P / MAF and FAP-V / MAF coatings.

[0037] Figure 5 It is the characterization diagram of the ordinary MgF2 coating in the comparative example of the present invention at different magnifications after 3 h of mineralization growth.

[0038] Figure 6 It is the test result diagram of the bonding performance of the double-layer coating in the present invention. Figure 6 Among them: A is the result of the scratch test; B is the statistical chart of the coating interface bonding force.

[0039] Figure 7 It is the statistical chart of the release of F ions from the MAF coating in the present invention.

[0040] Figure 8 It is the effect comparison diagram of the performance of F ions promoting the regrowth and densification of the FAP-P layer in the present invention. Figure 8 Among them: A is the SEM surface morphology after soaking the FAP-P coating deposited on a glass slide in an α-MEM culture medium without fluorine for 3 days; B is the SEM surface morphology after soaking in an α-MEM culture medium containing 10 ppm fluorine for 3 days.

[0041] Figure 9 It is the effect comparison diagram of the in-vitro mineralization regrowth and densification of the FAP-P / MAF layer in the present invention.

[0042] Figure 10 It is the characterization diagram of the in-vitro mineralization regrowth and composition change of the FAP-P / MAF coating in the present invention. Figure 10In the figure: A is the XRD pattern of the coating of the FAP-P / MAF sample after 1 week, 4 weeks, and 8 weeks of subcutaneous implantation; B is the TEM-EDS elemental distribution of FAP particles of the FAP-P / MAF sample after 4 weeks of subcutaneous implantation.

[0043] Figure 11 It is a comparison diagram of the effects of in vitro mineralization regrowth and densification of the FAP-V / MAF layer in the present invention.

[0044] Figure 12 It is a characterization diagram of in vitro mineralization regrowth and composition change of the FAP-V / MAF coating in the present invention. Figure 12 In the figure: A is the XRD pattern of the coating of the FAP-V / MAF sample after 1 week, 4 weeks, and 8 weeks of subcutaneous implantation. B is the TEM-EDS elemental distribution of FAP particles of the FAP-V / MAF sample after 4 weeks of subcutaneous implantation.

[0045] Figure 13 It is a comparison diagram of the in vitro anti-corrosion performance of the FAP / MAF coating in the present invention. Figure 13 In the figure: A is the Taffel curve of pure magnesium and magnesium metal modified with different coatings in simulated body fluid; B is the impedance curve of pure magnesium and magnesium metal modified with different coatings in simulated body fluid; C is the hydrogen evolution curve of magnesium metal modified with different coatings in simulated body fluid; D is the Mg ion release curve of magnesium metal modified with different coatings in α-MEM medium (containing protein).

[0046] Figure 14 It is a characterization of the in vivo mineralization self-growth performance of the FAP-P / MAF coating in the present invention. Figure 14 In the figure: A is a schematic diagram of the in vivo subcutaneous implantation model; B is the SEM comparison of the cross-sectional morphology of HAP-P before and after 4 weeks of implantation; C-E are the FIB-TEM characterizations of the internal structure of FAP-P / MAF after 4 weeks of in vivo implantation; F is the TEM-EDS characterization of the changes in the Ca / P ratio and F / Ca ratio of the FAP-P coating before and after 4 weeks of in vivo implantation.

[0047] Figure 15 It is a comparison diagram of the in vivo mineralization self-growth morphology of different implanted samples after 4 weeks of implantation in the present invention.

[0048] Figure 16 It is a diagram of the measurement results of the in vivo anti-corrosion performance of the FAP / MAF coating in the present invention. Figure 16 In the figure: A is the corrosion depth of pure magnesium metal at different implantation times; B is the change of the corrosion depth of magnesium metal modified with different coatings over time. Detailed implementation manners

[0049] The following further describes the in - vivo mineralization self - growth protective coating and its preparation method in the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. The following embodiments are only used to illustrate the present invention and not to limit the present invention.

[0050] As Figure 1 shown, the present invention first provides a method for in - vivo mineralization self - growth of a double - layer protective coating, which includes a double - layer protective coating based on magnesium - based metal. The first layer of the double - layer protective coating is a mineralization - promoting factor release layer, and the second layer is a mineral growth - promoting layer. After the double - layer protective coating is implanted into the human body, the mineralization - promoting factors in the mineralization - promoting factor release layer are released into the body fluid environment, promoting the biomineralization of calcium and phosphorus in the body fluid environment, and continuously depositing and mineralizing in the mineral growth - promoting layer, transforming into new minerals. The new minerals continuously fill the defects in the second layer, and finally form a stable and dense coating in the corrosive body fluid, providing lasting protection for the magnesium - based metal.

[0051] Furthermore, the mineralization - promoting factor in the present invention can be fluoride ions. The first mineralization - promoting factor release layer can be a porous MAF layer formed on the surface of magnesium - based metal by micro - arc fluoridation (MAF) process, and the second mineral growth - promoting layer can be a fluoridated hydroxyapatite (FAP) layer.

[0052] Correspondingly, the present invention also proposes a double - layer in - vivo mineralization self - growth protective coating, which has a double - layer structure, where the first layer is a mineralization - promoting factor release layer and the second layer is a mineral growth - promoting layer.

[0053] Furthermore, the mineralization - promoting factor release layer can be a porous MAF layer formed on the surface of magnesium - based metal by micro - arc fluoridation (MAF) process, and the mineral growth - promoting layer can be a fluoridated hydroxyapatite (FAP) layer.

[0054] Furthermore, the MAF layer has a coral - like porous structure, with a thickness of 0.5 - 9 microns, preferably 3 - 5 microns, and the main component is MgF2.

[0055] Furthermore, in the fluoridated hydroxyapatite (FAP) layer, the FAP nanorods can be assembled into FAP - P basically parallel to the surface of the MAF layer, or the FAP nanorods can be assembled into FAP - V basically perpendicular to the surface of the MAF layer.

[0056] Among them, due to certain errors in the generation in the vertical or horizontal direction during formation, the meanings of "basically parallel" and "basically perpendicular" are to roughly present a vertical or parallel state, but there may be certain errors or slight deviations.

[0057] Correspondingly, the present invention also proposes a preparation method for a double - layer in - vivo mineralization self - growth protective coating, and its specific process is as follows:

[0058] (1)Preparation of the MAF layer: Through the MAF process, a porous MAF layer is formed on the surface of the magnesium-based metal. The component of the porous MAF layer is MgF2;

[0059] (2)Preparation of the FAP layer: Adsorb nano-hydroxyapatite NanoHAP seed crystals on the surface of the MAF layer and place it in the mineralization solution. Control the temperature and time of the mineralization reaction to form the FAP layer on the MAF layer;

[0060] Furthermore, in the preparation process, by controlling different reaction temperatures, FAP layers with different morphologies can be formed. By regulating the reaction time, the microstructure such as the thickness and compactness of the FAP layer can be controlled.

[0061] Furthermore, the preparation method of the MAF layer also includes:

[0062] (1)Cut the magnesium sheet into a disk with a handle by laser. Among them, 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 disk is specifically Ø16×1.5mm, Ø8×0.5mm and Ø6×0.5mm. Other sizes can also be selected.

[0063] (2)And polish the disk step by step with #1000 and #2000 silicon carbide papers, polish it, and clean it with anhydrous ethanol, and dry it at room temperature in the air;

[0064] (3)The polished magnesium disk (with a holding tail) is used as the anode, and the graphite rod is used as the cathode. It is immersed in a polytetrafluoroethylene PTFE container containing 46% hydrofluoric acid to form a closed circuit with a DC constant voltage power supply device, and start micro-arc fluorination, that is, carry out micro-arc fluorination in the DC constant voltage mode;

[0065] (4)After the MAF process is completed, the sample is washed with deionized water and ethanol in turn and dried at room temperature.

[0066] Preferably, the conditions of the micro-arc fluorination process are: when the size of the sample disk is Ø16×1.5mm, in the DC constant voltage mode: the voltage is 185~210V; further preferably 185~195V, the current is 150~165mA, and as the sample diameter changes, the voltage remains unchanged, and the current decreases proportionally with the contact area.

[0067] Figure 2 Characterization of the MAF layer: Atomic force microscope photos, XRD diffraction patterns, SEM scanning electron microscope photos and element distribution photos of the prepared MAF samples. The characterization shows that the MAF coating is a coral-like porous structure with a thickness of 2~7 microns, and the main component is MgF2.

[0068] For other samples, MAF coatings of other samples can also be obtained by the same method, which are also coral-shaped porous structures with a thickness between 0.5 and 9 microns and mainly composed of MgF2. When the preparation process is controlled under the most optimal conditions, the optimal thickness of the MAF coating is between 3 and 5 microns.

[0069] Furthermore, the preparation method of the FAP layer further includes:

[0070] (1) Immerse the MAF layer into the suspension of NanoHAP, and the concentration of the suspension can be determined according to the amount of crystal seed layer to be adsorbed and deposited as needed.

[0071] Specifically, it can be preferably 2% NanoHAP deionized water dispersion, and a dispersant of 1% gelatin can be added.

[0072] (2) Take out the MAF sample after soaking it in the NanoHAP dispersion for 5 - 60 minutes, and after the surface level volatilizes, immerse it into the mineralization solution for mineralization. The soaking time can be determined according to the concentration of the suspension and the amount of crystal seed layer to be adsorbed and deposited.

[0073] The FAP morphology in the present invention can be adjusted according to the composition of the mineralization solution, the mineralization time, and the mineralization temperature. Specifically, two morphologies of the FAP layer are prepared on the MAF layer in the present invention:

[0074] One of them is that the FAP nanorods are basically parallel to the surface of the MAF layer to assemble FAP-P. The synthesis conditions are as follows: After the deposition of NanoHAP seeds, grow in the mineralization solution at 12 - 17 °C for 2 - 8 hours, then wash with deionized water and dry at room temperature. Preferably, the deposition of NanoHAP seeds is repeated 3 times, the immersion time for each deposition is 15 minutes, the temperature is preferably 15 °C, and the growth time in the mineralization solution is 3 hours.

[0075] The formula of the mineralization solution is shown in Table 1, containing 5 ppm fluorine (added through sodium fluoride reagent). The prepared double-layer coating of FAP and MAF is denoted as: FAP-P / MAF.

[0076] Another morphology is that the FAP nanorods are basically perpendicular to the MAF surface to assemble FAP-V. The synthesis conditions are as follows: After the deposition of NanoHAP seeds, grow in the mineralization solution at 20 - 90 °C for 2 - 8 hours, then wash with deionized water and dry at room temperature. Preferably, the NanoHAP seeds are deposited 1 time, the immersion time for deposition is 30 minutes, the temperature is preferably 37 °C, and the growth time in the mineralization solution is 3 hours.

[0077] Table 1 Components and ratios of the mineralization solution

[0078] Component mM Mass (g / L) <![CDATA[CaCl2•2H2O]]> 1 0.147 <![CDATA[KH2PO4]]> 4 0.540 KCl 16 1.200 <![CDATA[NH4Cl]]> 4.5 0.241 HEPES 20 4.766 <![CDATA[MgCl2•6H2O]]> 0.2 0.04

[0079] The mineralization solution formula is shown in Table 1 and contains 5 ppm fluorine (added through sodium fluoride reagent). The prepared double-layer coating of FAP and MAF is denoted as: FAP-V / MAF.

[0080] As Figure 3 and Figure 4 shown in the characterization of the prepared FAP-P / MAF and FAP-V / MAF coatings, SEM, XRD, and FTIR show that two morphologies of the FAP layer are synthesized, and the coating forms an interpenetrating structure with the porous MAF layer.

[0081] 1. Comparative analysis of the importance of the MAF process

[0082] Comparative example: Ordinary immersion fluorination process

[0083] In traditional processes, the formation of a general fluorinated layer usually adopts the ordinary immersion fluorination process. For example, in CN111973812A, it is disclosed that magnesium and magnesium alloys are immersed in hydrofluoric acid to generate a chemical conversion film MgF2. In this invention, the ordinary immersion fluorination process is used instead of the MAF process as a comparative example. After the pure magnesium metal wafers are treated by the same polishing process, they are immersed in a 46% hydrofluoric acid solution for 24 h for fluorination treatment, then a traditional MgF2 coating is formed. On top of the traditional MgF2 coating, 1 time of NanoHAP seed deposition is carried out under the same process conditions, the immersion time is also 30 min, and at a temperature of 37 °C, it grows in the same mineralization solution (see Table 1: containing 5 ppm fluorine) for 3 hours. The results show that no FAP-V layer is formed (see Figure 5 ).

[0084] That is to say, if only the ordinary immersion fluorination process is adopted, a double-layer coating that can mineralize and self-grow in vivo cannot be formed. That is, the MAF process for preparing a porous MgF2 coating is crucial for the formation of the double-layer self-growing coating.

[0085] In addition, as Figure 6 shown, according to the surface scratch resistance test, it can be proved that since the FAP coating forms an interpenetrating structure with the porous MAF layer, the interfacial bonding strength is further improved, laying a foundation for the stability of the clinical application of the self-growing coating.

[0086] 2. The in vitro mineralization self-growth performance of the FAP / MAF coating proposed in this invention is as follows:

[0087] Release of fluoride ions in the MAF coating: In order to simulate the complex body fluid components in vitro, an α-MEM medium containing sugar, amino acids, 10% fetal bovine serum protein, and calcium and phosphorus ion sources is selected as the immersion solution. By immersing the magnesium metal samples covered with the MAF coating in α-MEM, the release of fluoride ions at different times is investigated ( Figure 7). The results showed that the MAF coating could continuously release fluoride ions into the surrounding culture medium, and the amount of fluoride ion release tended to increase with time.

[0088] The specific test method was as follows: A magnesium metal disc (diameter: 1.6 cm, thickness: 0.5 cm) with a double-sided MAF coating was placed in 2 ml of α-MEM medium, and the α-MEM medium was changed every 24 hours. The fluoride ion content in the α-MEM medium at different times was detected by ion chromatography.

[0089] Promotion of fluoride ions on the mineralization self-growth of the FAP layer: To verify the promotion of fluoride on the mineralization growth of the FAP-P coating in body fluid, we first deposited the FAP-P layer on a blank glass slide and immersed it in α-MEM culture medium. Then, 0.5 mM NaF was added or not added to the α-MEM culture medium to confirm the promotion of fluoride ions on the mineralization growth of FAP-P.

[0090] As Figure 8 shown, after adding 0.5 mM NaF, the FAP-P coating grew and became a dense coating, while the morphology of the coating without fluoride addition showed no obvious change, indicating that trace fluoride ions could promote the growth and densification of the FAP-P coating.

[0091] 3. The in vitro self-growth and densification properties of the FAP / MAF coating based on magnesium-based metal are as follows:

[0092] (1) For the FAP-P / MAF coating:

[0093] The magnesium metal FAP-P / MAF coating was immersed in α-MEM medium containing sugar, amino acids, and 10% fetal bovine serum protein. The morphological growth change process was as Figure 9 shown. As time extended, the gaps in the coating were gradually filled with newly grown minerals, forming a dense morphology.

[0094] In addition, with the mineralization self-growth densification, the FAP in the coating gradually matured, and more characteristic peaks of FAP appeared in the XRD characterization ( Figure 10 A), and the fluoride ions released by the MAF layer were internalized into FAP-P during mineralization growth.

[0095] (2) For the FAP-V / MAF coating:

[0096] The magnesium metal FAP-V / MAF coating was immersed in α-MEM medium containing sugar, amino acids, and 10% fetal bovine serum protein. The morphological growth change process was as Figure 11As shown. As time goes by, the area near MAF in the FAP-V coating is gradually filled with newly grown minerals, forming a dense morphology at the bottom of FAP-V (the part marked by the yellow dotted line).

[0097] In addition, with the densification of mineralization self-growth, the FAP in the coating gradually matures, and more characteristic peaks of FAP appear in the XRD characterization ( Figure 12 A), and the fluoride ions released from the MAF layer are internalized into FAP-V with the growth of mineralization.

[0098] 4. In vitro corrosion resistance of FAP / MAF coating

[0099] Electrochemical corrosion test methods such as Tafel curves and impedance curves have proved that the first-layer MAF coating improves the corrosion resistance of pure magnesium ( Figure 13 A and B in). Compared with the MAF coating, the corrosion resistance of the FAP-P / MAF and FAP-V / MAF coatings is further slightly improved ( Figure 13 A and B in). And the room-temperature hydrogen evolution experiment with long-term immersion proves that the corrosion resistance of the FAP-P / MAF and FAP-V / MAF coatings is greatly improved compared with the traditional MAF coating ( Figure 13 C in). And the Mg ion release curve tested by long-term immersion in α-MEM medium (containing protein) further proves the great corrosion resistance advantage of the FAP / MAF coating. Among them, the FAP-P / MAF coating with higher self-growth and self-densification degree shows the best corrosion resistance ( Figure 13 D in). The above long-term comparative tests show that the strategy of self-growth and self-densification in body fluid helps to improve the long-term corrosion resistance of the coating.

[0100] 5. In vivo mineralization self-growth and corrosion resistance of FAP / MAF coating

[0101] (1) In vivo self-growth performance:

[0102] First, a mouse subcutaneous implantation model was established. After nurturing in the in vivo environment for 4 weeks, different analysis methods were used to investigate the mineralization self-growth and in vivo corrosion resistance of the coating ( Figure 14 A in).

[0103] The cross-sectional SEM test results show that after 4 weeks of implantation, the initially highly porous FAP-P layer of the FAP-P / MAF coating transforms into a dense structure, and the internal pores are filled with newly formed minerals ( Figure 14 B in).

[0104] The experimental results are similar to the in vitro self-growth and self-consolidation processes. Both low-magnification and high-magnification bright-field FIB-TEM images show an extremely dense structure inside the FAP-P layer after 4 weeks of mineralized self-growth and a tight interfacial connection between the FAP-P layer and the MAF layer ( Figure 14 C and D in which the interface is marked with a yellow dashed line).

[0105] The experimental results further investigated the in vivo self-growth and self-consolidation capabilities of the designed coatings, as well as the interfacial bonding stability between the FAP-P / MAF layers. Further TEM-EDS characterization showed that the regrown FAP-P layer in vivo contained magnesium and fluoride ions. TEM-EDS semi-quantitative analysis indicated that the F / Ca ratio increased during the self-growth process compared to the FAP-P layer before implantation, while the Ca / P ratio decreased. The experimental results demonstrated that fluoride ions participated in the self-growth process of the coating ( Figure 14 F in which).

[0106] In addition, similar to the in vitro test results, after 4 weeks of implantation, a partially dense structure appeared in the FAP-V / MAF layer, showing certain self-growth performance, further demonstrating the rationality of the design of the magnesium fluoride-hydroxyapatite double-coating self-growth system and the necessity of designing the FAP morphology. ( Figure 15 ).

[0107] (2)In vivo corrosion resistance

[0108] In addition, after 1, 2, and 4 weeks of implantation, the in vivo corrosion resistance was evaluated by measuring the average corrosion depth of the corrosion layer shown by SEM-EDS using ImageJ software ( Figure 16 A, B).

[0109] The test results showed that the corrosion layer thickness of pure magnesium and magnesium metal modified with the MAF coating gradually increased over time. After 4 weeks of in vivo implantation, the corrosion thickness of pure magnesium reached 40 μm. The traditional MAF coating gradually failed over time, and the depth of the corrosion layer reached 6.8 μm after 4 weeks of implantation, even exceeding the thickness of the MAF layer itself.

[0110] The FAP-P / MAF coating with self-growth and self-consolidation capabilities exhibited the most powerful corrosion resistance, with an average corrosion depth of 0.73 μm after 4 weeks of implantation. Under the protection of the FAP-P layer, only minor local corrosion occurred in the MAF layer.

[0111] The anti-corrosion performance of the FAP-V / MAF layer with a partially dense structure is also higher than that of the MAF coating, reaching a corrosion layer depth of 2.71 μm. SEM-EDS characterization of the corrosion layer shows that the corrosion layer compositions of each group are similar, with relatively high contents of P and O elements. These experimental results indicate that the in vivo mineralization self-growth method of the double-layer protective coating proposed in the present invention can achieve satisfactory corrosion resistance in the complex in vivo environment.

[0112] 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 implementation scope of the present invention. All equivalent changes and improvements made according to the scope of the present invention application should still fall within the scope covered by the patent of the present invention.

Claims

1. A double-layer protective coating, characterized in that, The first layer is a mineralization factor releasing layer, and the mineralization factor releasing layer is a porous micro-arc fluorination layer formed on the surface of a magnesium-based metal by a micro-arc fluorination process. The second layer is a mineral growth promoting layer, and the mineral growth promoting layer is a fluorohydroxyapatite layer. In the fluorohydroxyapatite layer, fluorohydroxyapatite nanorods are assembled parallel to the surface of the micro-arc fluorination layer, and the fluorohydroxyapatite layer and the micro-arc fluorination layer form an inter-embedded structure.

2. The double-layer protective coating according to claim 1, wherein: The micro-arc fluorination layer is a coral-like porous structure with a thickness of 0.5 - 9 microns, and its main component is MgF2.

3. The double-layer protective coating according to claim 2, wherein: The thickness of the micro-arc fluorination layer is 3 - 5 microns.

4. A preparation method of the double-layer protection coating according to any one of claims 1 - 3, and its specific process is as follows: (1) Preparation of the micro-arc fluorination layer: A porous micro-arc fluorination layer is formed on the surface of a magnesium-based metal by a micro-arc fluorination process, and the main component of the porous micro-arc fluorination layer is MgF2; (2) Preparation of the fluorohydroxyapatite layer: Adsorb nano-apatite seed crystals on the surface of the micro-arc fluorination layer, and place it in a mineralization solution, and control the adsorption amount of the seed crystals, the temperature and time of the mineralization reaction, so as to controllably form the microstructure of the fluorohydroxyapatite layer on the micro-arc fluorination layer.

5. The preparation method of the double-layer protective coating according to claim 4, wherein: The preparation method of the micro-arc fluorination layer also includes: (1) Cut the magnesium sheet into a disk with a handle by laser; (2) Grind and polish the disk step by step, wash it with absolute ethanol, and dry it at room temperature in air; (3) Use the polished magnesium disk with a clamping tail as the anode and a graphite rod as the cathode, and immerse it in a polytetrafluoroethylene container filled with hydrofluoric acid, and carry out micro-arc fluorination in the direct current constant voltage mode; (4) After the micro-arc fluorination process is completed, the sample is washed with deionized water and ethanol in sequence and dried at room temperature.

6. The preparation method of the double-layer protective coating according to claim 5, characterized in that: The preparation method of the fluorohydroxyapatite layer also includes: (1) Immerse the micro-arc fluorination layer into the suspension of nano-apatite; (2) Immerse the micro-arc fluorination sample in the nano-apatite dispersion for 5 - 60 min and then take it out. After the surface moisture evaporates, immerse it in the mineralization solution for mineralization.

7. The preparation method of the double-layer protective coating according to any one of claims 4-6, characterized in that: After the nano-apatite seed is deposited, it grows in the mineralization solution at 12 - 17 °C for 2 - 8 hours, and then is washed with deionized water and dried at room temperature.

8. The preparation method of the double-layer protective coating according to claim 7, characterized in that: It grows in the mineralization solution at 15 °C for 3 hours.

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