Method for in situ construction of anti-corrosion / osteopromotion bifunctional composite coating on magnesium-based implant surface

Abstract

The application discloses a method for constructing a corrosion-resistant / bone-repair-promoting bifunctional composite coating on a magnesium-based implant surface in situ, which comprises the following steps: (1) removing impurities from the surface of a magnesium-based material substrate, cleaning the magnesium-based material substrate, and drying the magnesium-based material substrate for standby use; (2) treating the magnesium-based material substrate with a sodium phytate solution to form a magnesium phytate passivation layer on the surface of the magnesium-based material substrate in situ; and (3) coating a coating liquid containing ethyl cellulose and oleoyl serine on the surface of the magnesium-based material substrate, and drying the magnesium-based material substrate to form a polymer functional layer. The magnesium-based implant surface has both corrosion resistance and bone-repair-promoting functions, the problem of too fast corrosion of magnesium and magnesium alloys is solved, and the biocompatibility and biological functions are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical functional coating processing, in particular to a method for constructing an anti-corrosion / bone repair promoting dual functional composite coating on a magnesium-based implant surface in situ. BACKGROUND

[0002] Magnesium and its alloys have attracted extensive attention in the field of orthopedic implant materials in recent years due to their good biocompatibility, bioactivity and biodegradability. The density (1.7-2.0 g / cm³) of magnesium and its alloys is very close to that of human bone (1.7-2.1 g / cm³), and the elastic modulus (40-45 GPa) is also better than that of traditional metal materials such as stainless steel (180-210 GPa), titanium alloy (110-120 GPa) and cobalt-chromium alloy (200-250 GPa), which can effectively alleviate the "stress shielding effect", reduce the risk of bone density loss and secondary fracture after implantation. In addition, Mg 2+ ions released after the degradation of magnesium in the body can participate in the bone regeneration process, further improving its application potential.

[0003] However, magnesium and its alloys still face the following challenges in practical application at present:

[0004] (1) The degradation rate is too fast or uneven, which causes rapid decay of mechanical properties and cannot meet the support time required for damaged tissue repair;

[0005] (2) Excessive hydrogen and Mg 2+ release may cause local tissue irritation, pH increase and imbalance of biological environment, affecting the healing effect;

[0006] (3) Lack of surface functional regulation ability, making it difficult to achieve the dual functions of anti-corrosion and bone repair promotion, limiting its clinical translation.

[0007] Therefore, it is urgent to develop a simple, efficient surface modification strategy with anti-corrosion and bone repair promotion functions to improve the comprehensive performance and clinical applicability of magnesium-based materials in the field of biomedical materials, especially in the field of orthopedic implants. SUMMARY

[0008] The purpose of the present application is to provide a method for constructing an anti-corrosion / bone repair promoting dual functional composite coating on a magnesium-based implant surface in situ, which has anti-corrosion and bone repair promotion functions, overcomes the problem of too fast corrosion of magnesium and magnesium alloys, and improves biocompatibility and biological function.

[0009] The technical scheme adopted by the present application to solve its technical problems is:

[0010] A method for constructing an anti-corrosion / bone repair promoting dual functional composite coating on a magnesium-based implant surface in situ, comprising the following steps:

[0011] (1) Clean the surface of the magnesium-based material substrate to remove impurities, wash and dry for later use; the surface can be cleaned by mechanical polishing to remove the oxide layer and improve the surface flatness, and then ultrasonic cleaning is performed to remove surface contaminants;

[0012] (2) The magnesium-based material substrate is treated with a sodium phytate solution to form a magnesium phytate passivation layer in situ on the surface thereof;

[0013] (3) A coating liquid containing ethyl cellulose and oleoyl serine is coated on the surface of the magnesium-based material substrate treated in step (2), and after drying, a polymer functional layer is formed; the magnesium phytate passivation layer and the polymer functional layer form a composite functional coating with corrosion resistance and bone repair promoting function.

[0014] The surface of pure magnesium or magnesium alloy is relatively smooth and has no active groups, and cannot form a strong binding force with the polymer functional layer. By pretreating the magnesium or magnesium alloy with sodium phytate, microcracks and a rough surface and phosphoric acid groups are formed, thereby significantly improving the binding strength and surface reactivity of the subsequent coating and substrate.

[0015] In addition, the magnesium phytate passivation layer has good physical shielding effect and can effectively block the penetration of corrosive media, and has certain preliminary biological activity, providing a stable interface for the subsequent polymer functional layer. As a basic corrosion-resistant barrier, the magnesium phytate passivation layer has good surface affinity and stability.

[0016] In the polymer functional layer, ethyl cellulose (EC) is a film-forming material that can form a continuous and dense covering film on the surface of the substrate, thereby enhancing the corrosion resistance, biocompatibility and controllable degradation (blocking magnesium ion release) of the material; oleoyl serine is an active ingredient with bone-promoting function, which can achieve stable release of bone-promoting factors, thereby improving the bone induction and bone integration ability of the implant. Oleoyl serine (OS) is a molecule with the functions of promoting M2-type macrophage polarization and anti-inflammatory function, achieving slow release control. Ethyl cellulose and oleoyl serine form a dense anti-corrosion coating layer with bone-promoting biological function on the surface of magnesium and magnesium alloy through layer-by-layer self-assembly, achieving stable combination and slow release control of bone-promoting factors in the coating layer.

[0017] The thickness of the polymer functional layer is controlled to be between 0.1 and 200 microns.

[0018] Preferably, the concentration of sodium phytate in the sodium phytate solution is 5wt% to 20wt%.

[0019] Preferably, the treatment temperature of the sodium phytate solution treatment is 60°C to 90°C, and the treatment time is 6 to 24 hours.

[0020] Preferably, the concentration of ethyl cellulose in the coating solution is 0.1wt%-10wt%, and the concentration of oleoyl serine is 0.01wt%-1wt%.

[0021] Preferably, the coating solution is obtained by dissolving ethyl cellulose and oleoyl serine in anhydrous ethanol.

[0022] Preferably, the coating and drying operations of step (3) are repeated to form polymer functional layers of different thicknesses.

[0023] Preferably, in step (3), the coating solution is coated on the surface treated in step (2) by immersing the magnesium-based material substrate treated in step (2) in the coating solution at room temperature, and standing for 0.5-2 hours in a vacuum environment.

[0024] Preferably, the magnesium-based material is magnesium or magnesium alloy.

[0025] The beneficial effects of the present application are:

[0026] 1. The present application effectively overcomes the problem of strong corrosion of magnesium-based materials and serious substrate loss in traditional hydrothermal methods, avoids the defects of high pollution emission and high energy consumption caused by chemical conversion film method, and also makes up for the low coating formation efficiency and poor adhesion between coating and substrate of surface treatment methods such as anodic oxidation and ion implantation.

[0027] 2. The functional solution formed by compounding cellulose derivatives (ethyl cellulose) and oleoyl serine can quickly self-assemble a dense, uniform and high biocompatibility and excellent corrosion resistance organic (polymer functional layer)-inorganic (magnesium phytate passivation layer) composite coating on the surface of magnesium and magnesium alloy. The coating can effectively reduce the corrosion rate of the substrate in the body fluid environment, alleviate the release of hydrogen gas and the formation of alkaline microenvironment, and significantly improve the biological fusion and bone repair ability of the material and the surrounding tissue.

[0028] 3. The coating prepared by the present application has good optical transparency, which is conducive to real-time observation and research of the microstructure, degradation behavior and mechanism of the coating in the physiological environment, providing visual support for subsequent clinical evaluation and mechanism exploration, and has important application research value and clinical transformation potential. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a process flow diagram of the present application;

[0030] Figure 2 is an optical diagram of the magnesium-based material surface after different treatments;

[0031] Figure 3 is a SEM diagram of the magnesium-based material surface after different treatments;

[0032] Figure 4 Element distribution mapping analysis chart of magnesium-based material surface after different treatments;

[0033] Figure 5 Atomic force microscope chart of magnesium-based material surface after different treatments;

[0034] Figure 6 Micron scratch chart of magnesium-based material surface after different treatments;

[0035] Figure 7 Normal pressure load-friction force-friction coefficient performance characterization chart of magnesium-based material surface after different treatments;

[0036] Figure 8 In vitro biocompatibility comparison chart of magnesium-based material surface after different treatments;

[0037] Figure 9 Corrosion resistance evaluation chart of magnesium-based material surface after different treatments;

[0038] Figure 10 In vitro bone repair promotion evaluation chart of magnesium-based material surface after different treatments;

[0039] Figure 11 In vitro anti-inflammatory ability evaluation chart of magnesium-based material surface after different treatments;

[0040] Figure 12 In rat in vivo bone repair promotion schematic diagram of magnesium-based material surface after different treatments. DETAILED DESCRIPTION

[0041] The technical solutions of the present application are further specifically described below through specific examples.

[0042] In the present application, unless specified, the raw materials and equipment used can be purchased from the market or commonly used in the art. The methods in the following examples are conventional methods in the art, unless otherwise specified.

[0043] Example 1:

[0044] A method for constructing an anti-corrosion / bone repair promotion dual-functional composite coating on the surface of a magnesium-based implant in situ (as shown in the figure), comprising the following steps: Figure 1 The method comprises the following steps:

[0045] (1) The pure magnesium (99.98%) substrate was mechanically polished with 500 mesh, 1000 mesh, and 2000 mesh sandpaper, respectively, to remove surface oxides and impurities. The polished sample was first washed thoroughly with deionized water, then placed in anhydrous ethanol in an ultrasonic cleaner for 10 minutes to further remove residual particles and organic contaminants. Then it was taken out and dried in a vacuum drying oven at 40°C for 30 minutes.

[0046] (2) The sample treated in step (1) was immersed in a 10% sodium phytate aqueous solution and reacted at 80°C for 12 hours. After the reaction of sodium phytate with the magnesium substrate, a dense and uniform magnesium phytate passivation layer was formed in situ on the surface. After treatment, it was washed with deionized water and air dried for use.

[0047] (3) Preparation of coating solution containing ethyl cellulose and oleoyl serine:

[0048] Under the condition of 50°C water bath, oleoyl serine was added to anhydrous ethanol and magnetically stirred for 6 hours to fully dissolve, obtaining a 0.5% oleoyl serine solution. Then ethyl cellulose was added to the solution to make the final mass fraction of ethyl cellulose 5%, and the stirring was continued at 50°C for 12 hours to obtain the coating solution. The obtained coating solution was filtered through a 0.45 μm polytetrafluoroethylene filter membrane and treated by centrifugation at 10000 rpm for 10 minutes to remove insoluble particles and impurities. After standing at room temperature for 6 hours to promote the stability of the solution and remove some bubbles.

[0049] The sample treated in step (2) was immersed in the coating solution, and after 1 hour of standing in a 10 Pa vacuum environment, the sample was extracted from the coating solution at a constant pulling rate (about 5 mm / min) to form a uniform liquid film. The sample was placed in a vacuum drying oven and dried at 30°C for 12 hours to control the solvent evaporation rate slowly, so that the coating layer was dense and formed a film (thickness about 1 micron).

[0050] Example 2:

[0051] The difference between this example and Example 1 is that magnesium alloy replaces pure magnesium as the substrate material.

[0052] Example 3:

[0053] The difference between this example and Example 1 is that in step (2), the sample treated in step (1) is immersed in a 5% sodium phytate aqueous solution and reacted at 60°C for 24 hours.

[0054] Example 4:

[0055] The difference between this example and Example 1 is that in step (2), the sample treated in step (1) is soaked in a 20% by mass aqueous sodium phytate solution and reacted at 90°C for 6 hours.

[0056] Example 5:

[0057] The difference between this example and Example 1 is that in step (2), the sample treated in step (1) is soaked in a 20% by mass aqueous sodium phytate solution and reacted at 90°C for 6 hours.

[0058] In step (3), oleoyl serine is added to anhydrous ethanol under a 50°C water bath, magnetically stirred for 6 hours, and fully dissolved to prepare a 0.01% by mass oleoyl serine solution. Subsequently, ethyl cellulose is added to the solution to make the final mass fraction of ethyl cellulose 0.1%, and magnetically stirred at 50°C for 12 hours to obtain the coating liquid.

[0059] Example 6:

[0060] The difference between this example and Example 1 is that in step (2), the sample treated in step (1) is soaked in a 20% by mass aqueous sodium phytate solution and reacted at 90°C for 6 hours.

[0061] In step (3), oleoyl serine is added to anhydrous ethanol under a 50°C water bath, magnetically stirred for 6 hours, and fully dissolved to prepare a 1% by mass oleoyl serine solution. Subsequently, ethyl cellulose is added to the solution to make the final mass fraction of ethyl cellulose 10%, and magnetically stirred at 50°C for 12 hours to obtain the coating liquid.

[0062] Example 7:

[0063] The difference between this example and Example 1 is that in step (2), the sample treated in step (1) is soaked in a 20% by mass aqueous sodium phytate solution and reacted at 90°C for 6 hours.

[0064] In step (3), oleoyl serine is added to anhydrous ethanol under a 50°C water bath, magnetically stirred for 6 hours, and fully dissolved to prepare a 0.1% by mass oleoyl serine solution. Subsequently, ethyl cellulose is added to the solution to make the final mass fraction of ethyl cellulose 1%, and magnetically stirred at 50°C for 12 hours to obtain the coating liquid.

[0065] Example 8:

[0066] The difference between this example and Example 1 is that in step (2), the sample treated in step (1) is soaked in a 20% by mass aqueous sodium phytate solution and reacted at 90°C for 6 hours.

[0067] The coating-drying process of step (3) is repeated twice.

[0068] Example 9:

[0069] The difference between this example and Example 1 is that in step (2), the sample treated in step (1) is soaked in a 20% by mass aqueous sodium phytate solution and reacted at 90°C for 6 hours.

[0070] The coating-drying process of step (3) is repeated three times.

[0071] Example 10:

[0072] The difference between this embodiment and embodiment 1 is that:

[0073] The coating-drying process of step (3) is repeated 5 times.

[0074] Example 11:

[0075] The difference between this embodiment and embodiment 1 is that:

[0076] In step (3), the sample treated in step (2) is immersed in the coating solution, and is left to stand for 0.5 hours under a vacuum of 10 Pa, and then is extracted from the coating solution at a constant pulling rate (about 5 mm / min) to form a uniform liquid film.

[0077] Example 12:

[0078] The difference between this embodiment and embodiment 1 is that:

[0079] In step (3), the sample treated in step (2) is immersed in the coating solution, and is left to stand for 2 hours under a vacuum of 10 Pa, and then is extracted from the coating solution at a constant pulling rate (about 5 mm / min) to form a uniform liquid film.

[0080] The polymer functional layer structure formed by the present application is dense and has strong adhesion, and is firmly combined with the passivated surface through hydrogen bonding, van der Waals force and mechanical interlocking, significantly improving the corrosion resistance of the material. In in vitro cell experiments, the coating can achieve slow release of oleoyl serine, induce osteoblast proliferation and differentiation, and inhibit osteoclast activity, thereby contributing to the bone integration process.

[0081] Test Example 1

[0082] The pure magnesium (99.98%) substrate was mechanically polished with 500 mesh, 1000 mesh and 2000 mesh sandpaper, respectively, to remove surface oxides and impurities. The polished sample was first washed thoroughly with deionized water, and then ultrasonically treated in ethanol in an ultrasonic cleaner for 10 minutes to further remove residual particles and organic contaminants. Then it was taken out and dried in a vacuum drying oven at 40°C for 30 minutes to obtain the Mg group.

[0083] The Mg group was soaked in an 80°C 5wt% sodium phytate aqueous solution overnight. After the reaction of sodium phytate (SP) and the magnesium substrate, a dense and uniform magnesium phytate passivation layer was formed in situ on the surface. After treatment, it was washed with deionized water and air dried for use. The magnesium + sodium phytate group (Mg-SP group) was obtained.

[0084] Test Example 2

[0085] The Mg-SP group was immersed in 4wt% ethyl cellulose ethanol solution overnight to form a dense polymer layer on the sample. The sample was air-dried at room temperature to obtain the magnesium + sodium phytate + ethyl cellulose group (Mg-SP-EC group).

[0086] Test Example 3

[0087] The Mg-SP group was immersed in 10ml 4wt% ethyl cellulose and 0.3wt% oleoyl serine ethanol solution to form a polymer layer on the sample. The sample was air-dried at room temperature to obtain the magnesium + sodium phytate + ethyl cellulose + oleoyl serine group (Mg-SP-EC-OS group).

[0088] Test characterization

[0089] The surface morphology and structure of the magnesium implant were observed by light microscopy (LM) (Fig. 1), scanning electron microscopy (SEM) (Fig. 2) and atomic force microscopy (AFM) (Fig. 3). Figure 2 Figure 3 Figure 5 The binding strength between the substrate and its coating was measured by a nanoscratch tester (Fig. 4). Figures 6-7

[0090] Figure 2 The surface morphology of the four groups of samples Mg, Mg-SP, Mg-SP-EC, Mg-SP-EC-OS was sequentially displayed. From the optical image, it can be seen that the surface of the bare Mg sample has scratches after polishing and polishing, and there is no obvious structure, and after SP treatment (Mg-SP), it presents a rough and porous morphology, showing the formation of the sodium phytate passivation layer. The EC and OS loaded coating (Mg-SP-EC, Mg-SP-EC-OS) forms a uniform and continuous polymer film layer on the surface, has good coverage and density, and effectively seals the magnesium substrate.

[0091] Figure 3 SEM and Figure 4 elemental distribution mapping analysis. The SEM image combined with the energy dispersive elemental mapping results further verifies the structural continuity and elemental composition distribution of the coating: only magnesium element is observed in the Mg sample; P and O are significantly observed in the Mg-SP sample, confirming the formation of the SP layer; the Mg-SP-EC group increases the distribution of dense carbon (C) element, which is derived from ethyl cellulose; the Mg-SP-EC-OS sample also shows the characteristic element distribution of oleoyl serine (N element), indicating the success of drug loading.

[0092] The elements in the elemental distribution mapping image are uniformly distributed, indicating that the coating is continuous and complete on a microscale, and has good interfacial bonding (Fig. 4). Figure 4

[0093] Figure 5 ​​​​The microstructure of the sample surface of bare Mg, Mg-SP, Mg-SP-EC and Mg-SP-EC-OS was exhibited by atomic force microscope contact scanning sample surface. The sample surface of Mg showed regular grinding marks. The surface of Mg-SP was rough and showed granular accumulation, which was derived from the SP deposition layer. The surface of Mg-SP-EC was relatively flat and uniform in thickness. The surface of Mg-SP-EC-OS showed regular nano-recess structure, which indicated that the OS molecules might form phase separation structure in the EC network. The line scanning graph further revealed the surface height variation in different directions. The fluctuation amplitude of Mg-SP sample was the largest, while the EC coating significantly reduced the surface roughness. The surface of Mg-SP-EC-OS sample was regular and moderately increased the roughness, which was conducive to cell adhesion.

[0094] Figures 6-7 The adhesion strength between the substrate and the coating was measured by an automatic scratch tester. The main analysis parameters included friction force (Ft), normal pressure load and friction coefficient (u), etc. The Mg group showed the lowest load and friction change, indicating that there was no obvious film layer; the Mg-SP group was slightly enhanced, which was attributed to the complex coordination formed between the SP passivation layer and the magnesium surface; the Mg-SP-EC group showed significantly enhanced binding force, which was speculated to be the formation of strong intermolecular interaction between the EC molecules and the SP intermediate layer; the Mg-SP-EC-OS group showed the best binding force index, indicating that the OS doping did not weaken but promoted the integration between the coating layers, which might be related to the intermolecular van der Waals force and the fatty chain structure. The above results verified that the composite coating had excellent mechanical stability and was suitable for long-term implantation needs.

[0095] Test Example 4

[0096] After the Mg, Mg-SP, Mg-SP-EC and Mg-SP-EC-OS were sterilized by ultraviolet irradiation, they were immersed in a-MEM complete culture medium (containing 10% FBS) for 1 day to obtain the leaching solution (37°C, 5% CO2). The ratio of sample area to solution volume was 1.25 cm 2 / ml, which was determined by ISO 10993-5. The leaching solution obtained from each sample was then used for in vitro cell experiments. MC3T3-E1 was inoculated in a 24-well plate at a density of 5×10 4 Each well was divided into Blank, Mg, Mg-SP, Mg-SP-EC and Mg-SP-EC-OS. After 1 day of culture, the complete culture medium was replaced with the leaching solution of each group. On the 3rd day of culture, 250 μL of Calcein AM / PI detection working solution (Bi Yun Tian Biological Technology Co., Ltd.) was added to each well, and the cells were incubated in the incubator for 30 min. The fluorescent images of the cells were taken using an inverted fluorescence microscope.

[0097] The in vitro biocompatibility of the samples was determined by LIVE / DEAD dual fluorescence staining. Live cells were stained green by Calcein AM, and dead cells were stained red by PI. Figure 8 It was found that the Mg group had significantly more dead cells than the other groups, and it had an inhibitory effect on the proliferation of MC3T3-E1 cells. This is related to the high concentration of Mg released during Mg degradation. 2+ Related to high pH, ​​the Mg-SP-EC and Mg-SP-EC-OS groups showed a higher cell count compared to the Blank group, indicating a certain proliferative effect. Furthermore, the Mg-SP-EC-OS group promoted MC3T3-E1 cell proliferation more significantly than the other groups. The Mg-SP-EC-OS coating samples, due to their excellent corrosion resistance and the slow-release of the small biomolecule OS, significantly improved cell viability, suggesting that Mg... 2+ The combination of Mg-SP-EC-OS and small biomolecules may synergistically promote the proliferation of MC3T3-E1 cells. Overall, the Mg-SP-EC-OS we developed exhibits high biocompatibility, making it suitable for use in biological environments.

[0098] Experimental Example 5

[0099] The corrosion resistance of Mg, Mg-SP, Mg-SP-EC, and Mg-SP-EC-OS in a body fluid environment was evaluated by immersion in SBF solution (Beijing Solarbio Science & Technology Co., Ltd.). The corrosion resistance was assessed using light microscopy. Figure 9 (a) Observe the surface morphology of the magnesium implant on day 15 and day 30. The pH value of the solution at the selected time points was measured using a pH meter on days 1, 3, and 7. Figure 9 (b) Detection of Mg in solution by inductively coupled plasma optical emission spectrometry (ICP-OES) 2+ concentration( Figure 9 (c in the text)

[0100] Each group of samples was immersed in simulated body fluid for 15 and 30 days, and then... Figure 9 The optical images clearly show that the sodium phytate passivation layer has a certain anti-corrosion effect compared to pure magnesium, but it is insufficient to maintain the entire implantation cycle. Therefore, further coverage with an ethyl cellulose coating can effectively achieve anti-corrosion. Subsequently, pH and Mg levels were measured on days 1, 3, and 7. 2+ Concentrations, pH and Mg of Mg-SP, Mg-SP-EC and Mg-SP-EC-OS 2+ The concentrations were all lower than those in the Mg group, with Mg-SP-EC and Mg-SP-EC-OS showing significant effects, further confirming the above viewpoint. It is well known that higher pH and Mg concentrations... 2+ It has an inhibitory effect on cell activity, while appropriate pH and Mg 2+Concentration has osteogenesis and anti-inflammatory effects.

[0101] Test Example 6

[0102] After sterilization of Mg, Mg-SP, Mg-SP-EC, Mg-SP-EC-OS by ultraviolet irradiation, the leaching solution was obtained by immersing in osteogenic induction medium (Haisheng Biotechnology Co., Ltd.) for 1 day (37°C, 5% CO2). The ratio of sample area to solution volume was 1.25 cm2 / ml, which was determined by ISO 10993-5. The leaching solution obtained from each sample was then used for the following in vitro cell experiments. 2

[0103] MC3T3-E1 cells (Haisheng Biotechnology Co., Ltd.) were seeded at 5×10 4 cells per well in a 24-well plate, and each group of leaching solution containing osteogenic induction factors was co-cultured. After 7 days of co-culture, alkaline phosphatase (ALP) staining and ALP activity determination were performed. The stained samples were observed under a microscope, and the ALP activity was determined by an enzyme marker.

[0104] After sterilization of Mg, Mg-SP, Mg-SP-EC, Mg-SP-EC-OS by ultraviolet irradiation, the leaching solution was obtained by immersing in α-MEM complete medium (containing 10% FBS) for 1 day (37°C, 5% CO2). The 8-week-old C57BL / 6J mice were provided by the Mouse Animal Experiment Center of Ningbo University. The bone marrow cavity was repeatedly flushed with a 1 mL syringe to isolate the bone marrow cells. The cells were resuspended in α-MEM + 10% FBS complete medium and transferred to a culture dish, which was then incubated in a cell incubator (37°C, 5% CO2). After 24 h, the supernatant medium was aspirated and centrifuged at 2000 rpm for 5 min, and then the supernatant was aspirated again. The cells were resuspended in α-MEM complete medium (containing 10% FBS) containing 40 ng / mL M-CSF (Amizona Biotechnology Co., Ltd.) and plated in a well plate for further incubation and expansion. Subsequently, BMMs cells were seeded at 5×10 4 cells per well in a 24-well plate and co-cultured with each group of leaching solution, with the addition of 40 ng / mL M-CSF and 20 ng / mL RANKL (Amizona Biotechnology Co., Ltd.) per well. The medium was changed every 2 days, and after 6 days of co-culture, TRAP staining and TRAP activity determination were performed. The samples were observed under a microscope, and the TRAP activity was determined by an enzyme marker.

[0105] To evaluate the osteogenic potential, we studied the early osteogenic differentiation ability of MC3T3-E1 cells by ALP staining and ALP activity determination experiments. After 7 days of culture, a significant blue deposit was observed in the Mg-SP-EC-OS group, indicating that it has the effect of promoting osteogenesis (​Figure 10 ALP activity. Subsequently, quantification by ALP activity assay experiment showed that the ALP activity of Mg-SP-EC and Mg-SP-EC-OS groups were higher than the rest at day 7, and the Mg-SP-EC-OS group had higher ALP activity, which indicated that the ethyl cellulose coating slowly released Mg 2+ can promote osteogenic differentiation of MC3T3-E1 cells, and OS can synergize with slowly released Mg 2+ to achieve stronger osteogenic promotion ability. Figure 10 c).

[0106] Osteoclasts, as the only cells with bone resorption function in vivo, maintain the homeostasis of bone tissue by secreting various acids and lysosomal enzymes to absorb bone. To evaluate its effect on BMMs cell osteoclast differentiation, we verified it by TRAP staining and Trap activity assay experiment. By Figure 10 b and Figure 10 d, we found that the number and size of osteoclasts in Mg group, Mg-SP group, Mg-SP-EC group and Mg-SP-EC-OS group were significantly reduced, all had the ability to inhibit osteoclasts, and Mg-SP-EC-OS group was the most significant. Mg-SP-EC and Mg-SP-EC-OS had the right amount of Mg 2+ had the ability to inhibit BMMs cell osteoclast differentiation, and OS synergized with Mg 2+ to enhance the effect of inhibiting osteoclasts. Bone has a certain self-repairing ability at the damaged site by regulating anabolic (bone formation) and catabolic (bone resorption) processes. Therefore, the Mg-SP-EC-OS group prepared by the application can promote bone repair by differentially regulating the response of osteoblasts and osteoclasts.

[0107] Test Example 7

[0108] After the Mg, Mg-SP, Mg-SP-EC, Mg-SP-EC-OS were sterilized by ultraviolet irradiation, they were immersed in DMEM complete culture medium (containing 10% FBS) for 1 day to obtain the leaching solution (37℃, 5% CO2). The ratio of sample area to solution volume was 1.25 cm 2 / ml, which was determined by ISO 10993-5. The leaching solution obtained from each sample was then used for the following in vitro cell experiments.

[0109] An inflammatory model was established by stimulating RAW264.7 macrophage cells (Haixing Biological Co., Ltd.) with lipopolysaccharide (LPS), and the anti-inflammatory ability of the samples was evaluated by fluorescence intensity. Cells were seeded at 1×10 3Each well was seeded with 24 samples, and 500 μl of DMEM complete medium (containing 10% FBS) with 0.2 mg / ml LPS was added to each well for incubation. After 24 h, the medium was discarded, and 1 ml of each group's extract was added for further incubation. After 14 days of induction, the levels of DAPI, CD86, and CD206 were determined by immunofluorescence analysis, and the mean fluorescence intensity of CD86 and CD206 was analyzed using ImageJ software.

[0110] The immune system plays a crucial role in bone formation and remodeling. During trauma-related bone regeneration and repair, a large number of immune cells are enriched at bone defect sites. We used immunofluorescence to investigate changes in gene expression and transcriptional regulation associated with macrophage phenotype switching. Currently, two main macrophage phenotypes have been characterized: classically activated (M1) macrophages, involved in pro-inflammatory responses and microbial defense, and alternatively activated (M2) macrophages, involved in anti-inflammatory responses and tissue repair. Identifying these phenotypes relies on specific markers; upregulation of CD86 is a marker of M1 polarization, indicating their involvement in pro-inflammatory activity and antimicrobial responses, while CD206 is a traditional marker for M2 macrophages, suggesting their role in anti-inflammatory processes and healing. Through immunofluorescence staining and mean fluorescence intensity measurements, we found that Mg-SP-EC and Mg-SP-EC-OS have the ability to promote the transition of macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype, with the Mg-SP-EC-OS group being significantly stronger than the Mg-SP-EC group. This indicates that OS and Mg... 2+ It has a synergistic promoting effect. This is also the first time that OS has been shown to have anti-inflammatory capabilities. Figure 11 ).

[0111] Experimental Example 8

[0112] A distal femoral defect model was established in SD rats, and magnesium screws (Mg, Mg-SP, Mg-SP-EC, and Mg-SP-EC-OS) were implanted. All animals were euthanized 4 and 8 weeks post-surgery. The femurs were fixed in 4% paraformaldehyde and evaluated using micro-CT. Images were acquired at 18-micron resolution using a tube potential of 40 kV, an intensity of 250 uA, and an integration time of 240 ms. Reconstructed images were used to determine the percentage of new bone formation. Regions of interest (ROIs) were selected from 2D imaging, with a normalized threshold (>220) for mineralized tissue and a threshold (>184) for the implant, depending on the specific circumstances. CTAn was used to analyze the micro-CT images to determine bone structure parameters such as bone volume / tissue volume (BV / TV) and trabecular bone number (Tb). In the final step, CTVox was used to reconstruct 3D images of the samples. Mean and standard deviation were calculated using at least three replicates.

[0113] As orthopedic implants, the rapid degradation of magnesium causes Mg 2+ Enrichment, local hydrogen accumulation and high alkaline environment formation, activate osteoclast activity and inhibit osteoblast adhesion and proliferation in the later bone reconstruction stage, have a significant inhibitory effect on the mineralization and maturation of bone tissue, leading to the fact that the bone regeneration and healing at the implant site cannot keep up with the degradation rate of magnesium-based materials, and it is easy to form osteolytic cavities, leading to fixation failure. In order to evaluate the in vivo corrosion resistance and bone defect repair effect, we established a distal femoral defect model in SD rats and implanted the prepared Mg, Mg-SP, Mg-SP-EC and Mg-SP-EC-OS nails. After 4 weeks, 8 weeks of operation, the rats were euthanized, and the degree of degradation of magnesium-based implants was immediately evaluated by Micro-CT, and the healing of the surrounding bone tissue was examined. By Figure 12 In a we can find that the pure magnesium group degrades faster in vivo, and larger cavities appear around the implant, which is related to the rapid degradation of magnesium nails and the release of a large amount of H2. While the implant around the Mg-SP-EC-OS group has no obvious cavity, and the quantitative results of Micro-CT analysis also show that compared with the previous groups, the trabecular bone BV / TV, thickness, mineral density and separation of the Mg-SP-EC-OS group have been significantly improved, indicating that the Mg-SP-EC-OS group promotes bone repair in the rat femoral defect model, and the fixation effect is more significant (b in Figure 12 ).

[0114] The above-described embodiments are only a preferred scheme of the present application, and do not limit the present application in any form. There are other variants and modifications without exceeding the technical solutions recorded in the claims.

Claims

1. A method for in-situ construction of a dual functional composite coating of corrosion resistance / osteopromotion on a magnesium-based implant surface, characterized in that, The method comprises the following steps: (1) removing impurities from the surface of a magnesium-based material substrate, cleaning, drying, and reserving; (2) treating the magnesium-based material substrate with a sodium phytate solution to form a magnesium phytate passivation layer in situ on the surface thereof; (3) coating a coating liquid containing ethyl cellulose and oleoyl serine on the surface of the magnesium-based material substrate treated in step (2), and drying to form a polymer functional layer; the magnesium phytate passivation layer and the polymer functional layer form a composite functional coating with corrosion resistance and bone repair promotion functions.

2. The method of claim 1, wherein, The concentration of sodium phytate in the sodium phytate solution is 5wt%-20wt%.

3. The method of claim 1, wherein, The treatment temperature of the sodium phytate solution treatment is 60°C-90°C, and the treatment time is 6-24 hours.

4. The method of claim 1, wherein, The concentration of ethyl cellulose in the coating liquid is 0.1wt%-10wt%, and the concentration of oleoyl serine is 0.01wt%-1wt%.

5. The method of claim 1, wherein, The coating liquid is obtained by dissolving ethyl cellulose and oleoyl serine in anhydrous ethanol.

6. The method of claim 1, wherein, The coating and drying operations of step (3) are repeated to form polymer functional layers with different thicknesses.

7. The method of claim 1, wherein, In step (3), the coating liquid is coated on the surface treated in step (2) as follows: the magnesium-based material substrate treated in step (2) is immersed in the coating liquid at room temperature, and is left to stand in a vacuum environment for 0.5-2 hours.

8. The method of claim 1, wherein, The magnesium-based material is magnesium or a magnesium alloy.

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