Nacre mother structure fibronectin-like coating modified magnesium alloy material and preparation method and application thereof

Abstract

The invention relates to a mother-of-pearl structure fibronectin-like coating modified magnesium alloy material and a preparation method and application thereof.The preparation method of the mother-of-pearl structure fibronectin-like coating modified magnesium alloy material comprises the following steps that a calcium-phosphorus particle suspension and a bionic polypeptide solution are prepared, and bionic polypeptide is of a repeated RGD structure and has biological viscosity; and sequentially adhering the calcium-phosphorus particle suspension and the bionic polypeptide solution to the surface of magnesium alloy, and repeatedly freezing and thawing for a plurality of times to obtain the mother-of-pearl structure fibronectin coating modified magnesium alloy material. The nacre structure fibronectin-like coating modified magnesium alloy material disclosed by the invention not only shows excellent biocompatibility, but also has a good degradation characteristic, and an important material basis is provided for further improving the osseointegration effect under the osteoporosis condition.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a nacreous structure-like fibronectin-coated modified magnesium alloy material, a preparation method thereof, and an application thereof. Background Art

[0002] Osteoporosis is a common chronic bone metabolic disease. According to statistics, there are about 140 million people with osteoporosis in China, and nearly 10 million people suffer from fractures due to osteoporosis every year. With the aggravation of population aging, the number of patients with osteoporosis and osteoporotic fractures is increasing. At present, the pathological mechanism and drug treatment research of osteoporosis have achieved remarkable results, but there is still no exact and effective treatment plan for osteoporotic fractures. The bone repair ability of osteoporosis patients is insufficient, and delayed fracture healing often occurs, which brings great challenges to orthopedic surgery. Traditional orthopedic implants are often made of inert metals (such as titanium, cobalt, chromium, molybdenum, etc.), which have good biocompatibility, but the biological inertness determines that the material cannot be fully combined with bone tissue, and it is easy to cause loosening; on the other hand, the elastic modulus of these metal implants is relatively high and they cannot be degraded and absorbed in the human body, and stress shielding and periprosthetic fractures are likely to occur in osteoporosis patients. These implants may require secondary surgery to remove, bringing unnecessary pain and economic burden to patients. Therefore, the research and application of bioactive and degradable implants are of great significance for bone repair in osteoporosis patients.

[0003] Magnesium (Mg) is a bioactive and biodegradable material with osteogenic activity and has become a research hotspot in biomaterials. Magnesium metal has good ductility, plasticity, degradability and osteogenic activity, and can promote fracture healing and avoid secondary removal surgery, so it is considered to be a good substitute for traditional orthopedic implants. In recent years, a large number of studies have confirmed that magnesium metal can promote fracture healing and bone tissue repair. However, for osteoporosis patients, due to insufficient osteogenesis and excessive osteoclastogenesis, the bone regeneration rate often cannot match the degradation rate of magnesium metal, resulting in unstable binding at the implant-bone interface. On the other hand, magnesium metal often induces an inflammatory response after being implanted into the body, which also leads to poor bone integration and further causes implant failure. Therefore, achieving the dynamic adaptation of magnesium metal degradation and bone regeneration, improving the bone integration performance of the binding interface, and further promoting bone repair under osteoporosis conditions have become extremely urgent clinical needs. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a nacreous structure-like fibronectin-coated modified magnesium alloy material, a preparation method thereof, and an application thereof. The nacreous structure-like fibronectin-coated modified magnesium alloy material not only exhibits excellent biocompatibility, but also has good degradation characteristics, providing an important material basis for further improving the bone integration effect under osteoporosis conditions.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The present invention first provides a preparation method of a nacre-structure-like fibronectin-coated modified magnesium alloy material, which includes the following steps: preparing a calcium phosphate particle suspension and a biomimetic polypeptide solution, where the biomimetic polypeptide has a repetitive RGD structure and has biological adhesiveness; sequentially adhering the calcium phosphate particle suspension and the biomimetic polypeptide solution to the surface of the magnesium alloy, and after repeated freezing and thawing for several times, the nacre-structure-like fibronectin-coated modified magnesium alloy material is obtained.

[0006] As a further improvement of the above solution of the present invention, the magnesium alloy is prepared by the following method: adding magnesium, magnesium-calcium alloy and magnesium-lanthanum alloy into a high-purity graphite crucible, heating and melting to obtain a melt; pouring the melt into a preheated steel mold to obtain an ingot, and subjecting the ingot to heat treatment and then extruding it into a predetermined shape. The present invention alloyizes by adding rare earth elements to magnesium metal to adjust its elastic modulus, slow down the degradation rate and improve osteogenic activity.

[0007] As a further improvement of the above solution of the present invention, in the magnesium alloy, the content of magnesium element is 85-97wt%, the content of calcium element is 0.1-1.5wt%, and the content of lanthanum element is 0.2-2.0wt%; and / or, the purity of magnesium is 99.99%, the content of calcium element in the magnesium-calcium alloy is 30wt%, and the content of lanthanum element in the magnesium-lanthanum alloy is 30wt%.

[0008] As a further improvement of the above solution of the present invention, the temperature of the heating and melting is 700-800°C; and / or, the temperature of the preheated steel mold is 250-320°C; and / or, the heat treatment is heating at 450-500°C for 4-8h.

[0009] As a further improvement of the above solution of the present invention, the preparation method of the calcium phosphate particle suspension is: mixing a calcium nitrate solution and a sodium dihydrogen phosphate solution, and adding sodium bicarbonate to adjust the pH to precipitate calcium phosphate salt, thus obtaining the calcium phosphate particle suspension.

[0010] As a further improvement of the above solution of the present invention, the concentration of the calcium nitrate solution is 12 - 15 mM, and the concentration of the sodium dihydrogen phosphate solution is 8 - 9 mM; adjusting the pH means adjusting the pH of the solution to 6.8 - 7.2. When the pH value is adjusted to an appropriate level, calcium ions and phosphate ions will react to form calcium phosphate salts, and these salts will precipitate out of the solution to form a suspension. In the pH range of 6.8 - 7.2, stable hydroxyapatite (the main target precipitate) can be formed, avoiding the formation of other unnecessary calcium phosphate salts (such as calcium hydrogen phosphate or calcium dihydrogen phosphate). The pH value in this range is close to neutral, which can maintain the stability of calcium and phosphate ions, ensure the uniformity and dispersibility of the particles, so that they can be uniformly coated on the surface of the magnesium alloy subsequently. And the slightly alkaline environment helps the binding of calcium phosphate particles and fibronectin-like proteins, while reducing the excessive corrosion of the magnesium alloy surface.

[0011] As a further improvement of the above solution of the present invention, the molar ratio of calcium element to phosphorus element in the calcium phosphate particle suspension is 1.0 - 2.0:1, preferably 1.67:1, simulating the calcium-phosphorus ratio in human bones; and / or, the concentration of the calcium phosphate particle suspension is 0.08 - 0.12 mg / mL, and the concentration of the biomimetic polypeptide solution is 0.008 - 0.015 mg / mL.

[0012] As a further improvement of the above solution of the present invention, the sequence of the biomimetic polypeptide is: MPA-PEG-Glu{c[Arg-Gly-Asp-D-Phe-Lys(Ahx)]}-{c[Arg-Gly-Asp-D-Phe-Lys(Ahx)]}.

[0013] The present invention also provides a nacreous structure fibronectin-like protein-coated modified magnesium alloy material prepared by the preparation method as described above.

[0014] The present invention also provides an application of the nacreous structure fibronectin-like protein-coated modified magnesium alloy material as described above as an orthopedic implant.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention forms a calcium phosphate coating and a fibronectin-like coating on the surface of a magnesium alloy by sequentially adhering a calcium phosphate particle suspension and a biomimetic polypeptide solution to the surface of the magnesium alloy, and finally obtains a nacre-structure fibronectin-like coating modified magnesium alloy material. Among them, the calcium phosphate coating is similar to the inorganic components of human bones, so it has high biocompatibility, can reduce the immune response and inflammation caused by implant materials, can provide raw materials for bone tissue regeneration, accelerate bone calcification and bone growth, and can also activate senescent macrophages, realize the immune regulation of the phenotypic transformation of macrophages from M1 to M2, enhance the bioactivity and bone integration ability of the magnesium alloy material. At the same time, the calcium phosphate coating can slow down the degradation rate of the magnesium alloy, make the magnesium alloy closer to the speed of bone tissue repair, and avoid the failure of the implant caused by excessive degradation. The calcium phosphate coating can promote the adhesion and proliferation of bone cells, improve the bonding strength between the implant and the bone tissue, and reduce postoperative complications. In addition, the introduction of the fibronectin-like coating uses biomimetic polypeptides to tightly adhere the calcium phosphate coating to the alloy surface to obtain good mechanical properties and increase adhesion, uses the biomimetic polypeptide coating to relieve the inflammatory reaction after the material is implanted in the body, promotes better bone integration, delays the release rate of magnesium ions, and shows a more stable degradation behavior. By using biomimetic active polypeptide macromolecules to replace natural heterologous proteins, the immunogenicity is effectively reduced and the controllability of the whole system is improved, providing an important material basis for further improving the bone integration effect under osteoporosis conditions.

[0016] 2. The present invention highly biomimetically utilizes the layered structure of nacre, combines functionalized calcium phosphate inorganic substances with fibronectin organic substances, and exhibits multifunctional, efficient and stable characteristics; the dense layered coating not only further controls the degradation of the metal, but also enhances the cell adhesion ability, and at the same time regulates the polarization reaction of macrophages in the immune microenvironment, plays an immune regulation role in the early stage of implantation, promotes the osteogenic process, and finally significantly improves the bone integration effect on the surface of the internal implant under osteoporosis pathological conditions; the nacre-structure fibronectin-like coating of the present invention combines the characteristics of inorganic and organic modules, has extremely high stability, is not easy to peel off or lose, and can meet the application requirements under various complex surgical environments.

[0017] 3. The present invention constructs a hierarchical coating structure with good adhesion by the layer-by-layer method. The biomimetic coating constructed in this way combines the characteristics of inorganic and organic modules, and at the same time meets the requirements of mechanical conditions and complex biology, providing a new idea for the material surface modification method. The present invention builds a modified coating with a "brick-mortar" like microstructure on the surface of the magnesium alloy. While further reducing the corrosion rate of the magnesium alloy and controlling the release of ions, it quickly converts the immune activation state (represented by macrophage M1 polarization) induced by surgical operations and magnesium degradation into an immune repair state (represented by macrophage M2 polarization), and promotes the adaptation of internal metal degradation and new bone formation and the integration effect of the implant-bone surface. Description of the Drawings

[0018] Figure 1 It is the synthesis schematic diagram of the bionic polypeptide in the embodiment; Figure 2 It is the scanning electron microscope image of the modified magnesium alloy obtained in step S3 of the embodiment; Figure 3 It is the flowchart of step S4 of the embodiment; Figure 4 It is the scanning electron microscope image of the modified magnesium alloy with a calcium phosphate coating obtained in the embodiment; Figure 5 It is the scanning electron microscope image (cross-section) of the nacreous structure-like fibronectin-coated modified magnesium alloy material obtained in the embodiment; Figure 6 It is the scanning electron microscope image (longitudinal section) of the nacreous structure-like fibronectin-coated modified magnesium alloy material obtained in the embodiment; Figure 7 It is the XPS spectrum of the nacreous structure-like fibronectin-coated modified magnesium alloy material obtained in the embodiment; Figure 8 It is the FTIR spectrum of the nacreous structure-like fibronectin-coated modified magnesium alloy material obtained in the embodiment; Figure 9 It is the electrochemical corrosion test result graph in Test Example 1; Figure 10 It is the ion release kinetics test result graph in Test Example 1; Figure 11 It is the Live / Dead cell staining detection result graph in Test Example 2; Figure 12 It is the CCK-8 detection result graph of BMMs in Test Example 2; Figure 13 It is the CCK-8 detection result graph of BMSCs in Test Example 2; Figure 14 It is the Transwell plate staining result graph in Test Example 3; Figure 15 It is the fluorescence staining, alkaline phosphatase staining and alizarin red staining result graph in Test Example 3; Figure 16 It is the H&E staining, Masson staining, immunofluorescence and immunohistochemistry detection result graph 2 weeks after operation in Test Example 3; Figure 17 It is the Micro-CT scan graph 8 weeks after operation in Test Example 3; Figure 18 It is the bone density analysis result graph in Test Example 3; Figure 19 It is the BV / TV analysis result graph in Test Example 3. Detailed implementation manners

[0019] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the implementation manners described herein. On the contrary, the purpose of providing these implementation manners is to make the disclosure of the present invention more thorough and comprehensive.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific implementation manners and are not intended to limit the present invention.

[0021] Embodiment This embodiment provides a nacre-structure-like fibronectin-coated modified magnesium alloy material, and its preparation method includes the following steps: S1. Prepare a calcium phosphate particle suspension: Dissolve Ca(NO 3 ) 2 ·4H 2 O in deionized water to prepare a calcium nitrate solution with a concentration of 14 mM; dissolve NaH 2 PO 4 ·2H 2 O in deionized water to prepare a sodium dihydrogen phosphate solution with a concentration of 8.4 mM; mix the calcium nitrate solution and the sodium dihydrogen phosphate solution; then add sodium bicarbonate with a concentration of 4 mM to the mixed solution to adjust the pH of the mixed solution to promote the precipitation of calcium phosphate salts, thereby obtaining a 0.1 mg / mL calcium phosphate particle suspension. The molar ratio of calcium element to phosphorus element in the mixed solution prepared in this embodiment is 1.67:1.

[0022] S2. Prepare a biomimetic polypeptide solution: Dissolve the biomimetic polypeptide powder in PBS buffer to prepare a 0.01 mg / mL biomimetic polypeptide solution.

[0023] The biomimetic polypeptide in this embodiment has a repetitive RGD structure and has biological adhesiveness, and its sequence is: MPA-PEG-Glu{c[Arg-Gly-Asp-D-Phe-Lys(Ahx)]}-{c[Arg-Gly-Asp-D-Phe-Lys(Ahx)]}, where MPA and PEG are used as stabilizers, which can reduce the degradation of the polypeptide and are beneficial to its modification and grafting on the MgO skeleton. Combining Figure 1 , the biomimetic polypeptide is synthesized by the Fmoc solid-phase synthesis method in this embodiment. The biomimetic polypeptide is specifically: Graft the compound shown by formula a onto the resin, then add the compound shown by formula b and piperidine, use N,N-dimethylformamide (DMF) as the solvent, react at room temperature for 4 hours, wash the resin with DMF, then add 1-hydroxybenzotriazole (HOBT) and HBTU, and use dichloromethane (CH 2 Cl 2 )as the solvent and react at room temperature for 6 hours to obtain the compound shown by formula c through an esterification reaction. On this basis, add the compound shown by formula d, use DMF as the solvent, add piperidine and react at room temperature for 4 hours, then add 1-hydroxybenzotriazole (HOBT) and HBTU, and use dichloromethane (CH 2 Cl 2 )as the solvent and react at room temperature for 6 hours to obtain the compound shown by formula e. Graft the compound shown by formula e onto the dendrimer, add the compound shown by formula f, dissolve it with DMF, add piperidine and react at room temperature for 4 hours, then add 1-hydroxybenzotriazole (HOBT) and HBTU, and use dichloromethane (CH 2 Cl 2 )as the solvent and react at room temperature for 6 hours to obtain the compound shown by formula g. Graft the compound shown by formula g onto the dendrimer, add the compound shown by formula h, dissolve it with DMF, add piperidine and react at room temperature for 4 hours to deprotect, then add 1-hydroxybenzotriazole (HOBT) and HBTU, and use dichloromethane (CH 2 Cl 2 )as the solvent and react at room temperature for 6 hours to obtain the compound shown by formula i. On this basis, add PdO and react at room temperature for 8 h to obtain the compound shown by formula j, graft the compound shown by formula j onto the resin, add piperidine, and use DMF as the solvent, react at room temperature for 5 hours to deprotect to obtain the compound shown by formula k. Add N,N-diisopropylethylamine (EtN(Pr-i) 2 ), HOBT and HBTU, prepare the compound shown by formula l through an intramolecular amidation reaction, then in the presence of hydrazine hydrate, use DMF as the solvent to remove the amino protecting group to obtain the compound shown by formula m with the amino group exposed, and further prepare the compound shown by formula o through an amidation reaction with the carboxyl group of the compound shown by formula n. Finally, the compound shown by formula o reacts with functionalized PEG-maleimide to prepare the target product, a fibronectin-like polypeptide shown by formula p.

[0024] S3. Preparation of modified magnesium alloy: Under argon protection, Mg (99.99%) alloy, Mg-30%Ca alloy and Mg-30%La alloy are added into a high-purity graphite crucible and melted at 750 °C to obtain a melt; the melt is poured into a steel mold preheated to 300 °C to obtain an ingot. After heating the ingot at 500 °C for 6 hours, the ingot is extruded into a metal rod at an extrusion ratio of 32:1 at 400 °C, thus obtaining the modified magnesium alloy, wherein the content of magnesium element is 85-97 wt%, the content of calcium element is 0.1-1.5 wt%, and the content of lanthanum element is 0.2-2.0 wt%. Figure 2 This is the scanning electron microscope image of the modified magnesium alloy prepared in this example. From Figure 2 it can be seen that the surface of the matrix of the modified magnesium alloy in this example is relatively smooth, but there are tiny holes and cracks.

[0025] S4. The calcium phosphate particle suspension obtained in step S1 and the biomimetic polypeptide solution obtained in step S2 are successively adhered to the surface of the modified magnesium alloy prepared in step S3. After repeated freeze-thaw cycles for physical cross-linking and curing, a nacre structure-like fibronectin (Fn) coating modified magnesium alloy material with the characteristics of firm adhesion, rough surface and high porosity is obtained; for the specific process, please refer to Figure 3 .

[0026] Figure 4 In this example, this is the scanning electron microscope image of the calcium phosphate coating obtained by adhering the calcium phosphate particle suspension to the surface of the modified magnesium alloy. From Figure 4 it can be seen that a uniformly distributed spherical or flaky microcrystalline structure is formed on the surface of the calcium phosphate coating, showing three-dimensional porous characteristics, simulating the morphology of natural bone minerals. This morphology can enable the subsequent coating to better fill the gaps between calcium phosphate particles and form a stable bonding interface; the three-dimensional network porous structure not only enhances the mechanical interlocking force between the subsequent coating and the calcium phosphate "bricks", but also provides abundant chemical bonding sites, thus significantly improving the overall stability and functional performance of the coating.

[0027] Figure 5 This is the scanning electron microscope image (cross-section) of the nacre structure-like fibronectin coating modified magnesium alloy material prepared in this example, Figure 6 This is the scanning electron microscope image (longitudinal section) of the nacre structure-like fibronectin coating modified magnesium alloy material prepared in this example. From Figure 5 a、 Figure 6 it can be seen that the fibronectin-like coating forms a uniform and tightly bound nacreous layered structure on the surface of the magnesium alloy, and no obvious cracks or peeling are seen, proving that the coating is successfully prepared; among them, the calcium phosphate particles ("bricks") are uniformly distributed ( Figure 5 b), embedded in the fibrous network matrix formed by the fibronectin-like polypeptide (fibronectin-like, "mud") coating, and covering the surface of the magnesium oxide (MgO) skeleton ( Figure 5c); The Fn-like coating tightly fills the gaps between the calcium phosphate particles, forming a stable bonding interface with the calcium phosphate particles and the MgO framework. At the same time, the surface has appropriate roughness and micron-scale pore characteristics.

[0028] Figure 7 This is the XPS spectrum of the nacreous structure-like fibronectin-coated modified magnesium alloy material prepared in this example. From Figure 7 It can be seen that through X-ray photoelectron spectroscopy (XPS) experiments, the composition of this material was further verified, the existence of calcium phosphate particles and the magnesium oxide framework and their chemical bonding relationship were confirmed, providing strong evidence support for the "nacreous structure-like Fn coating" morphology observed by SEM.

[0029] Comparative Example 1 This comparative example provides a modified magnesium alloy material, and its preparation method is as follows: Under argon protection, Mg (99.99%), Mg-30%Ca alloy and Mg-30%La alloy are added to a high-purity graphite crucible and melted at 750 °C to obtain a melt; the melt is poured into a steel mold preheated to 300 °C to obtain an ingot. After heating the ingot at 500 °C for 6 hours, the ingot is extruded into a metal rod at an extrusion ratio of 32:1 at 400 °C, and the modified magnesium alloy material is obtained.

[0030] Comparative Example 2 This comparative example provides a modified magnesium alloy material, and its preparation method is as follows: S1. Prepare a calcium phosphate particle suspension: Dissolve Ca(NO 3 ) 2 ·4H 2 O in deionized water to prepare a calcium nitrate solution with a concentration of 14 mM; dissolve NaH 2 PO 4 ·2H 2 O in deionized water to prepare a sodium dihydrogen phosphate solution with a concentration of 8.4 mM; mix the calcium nitrate solution and the sodium dihydrogen phosphate solution, and the molar ratio of calcium element to phosphorus element in the mixed solution is 1.67:1; then add sodium bicarbonate with a concentration of 4 mM to the mixed solution to adjust the pH of the mixed solution to promote the precipitation of calcium phosphate salts, and a 0.1 mg / mL calcium phosphate particle suspension is obtained.

[0031] S2. Prepare a modified magnesium alloy: Under argon protection, Mg (99.99%) alloy, Mg-30%Ca alloy and Mg-30%La alloy are added to a high-purity graphite crucible and melted at 750 °C to obtain a melt; the melt is poured into a steel mold preheated to 300 °C to obtain an ingot. After heating the ingot at 500 °C for 6 hours, the ingot is extruded into a metal rod at an extrusion ratio of 32:1 at 400 °C, and the modified magnesium alloy material is obtained.

[0032] S3. Adhere the calcium phosphate particle suspension obtained in step S1 to the surface of the modified magnesium alloy prepared in step S3. After performing physical cross-linking and curing by repeated freezing and thawing for several times, the modified magnesium alloy material is obtained.

[0033] Comparative Example 3 This comparative example provides a modified magnesium alloy material, and its preparation method is as follows: S1. Prepare the biomimetic polypeptide solution: Dissolve the biomimetic polypeptide powder in PBS buffer to obtain a 0.01 mg / mL biomimetic polypeptide solution. The biomimetic polypeptide in this comparative example is the same as that in the example.

[0034] S2. Prepare the modified magnesium alloy: Under argon protection, add Mg (99.99%) alloy, Mg-30%Ca alloy, and Mg-30%La alloy into a high-purity graphite crucible, and melt them at 750 °C to obtain a melt; pour the melt into a steel mold preheated to 300 °C to obtain an ingot. After heating the ingot at 500 °C for 6 hours, extrude the ingot into a metal rod at an extrusion ratio of 32:1 at 400 °C, and the modified magnesium alloy material is obtained.

[0035] S3. Adhere the biomimetic polypeptide solution obtained in step S1 to the surface of the modified magnesium alloy prepared in step S2. After performing physical cross-linking and curing by repeated freezing and thawing for several times, the modified magnesium alloy material is obtained.

[0036] Figure 8 This is the FTIR spectra of the modified magnesium alloy materials and pure magnesium materials of this example and Comparative Examples 1-2. From Figure 8 It can be seen that characteristic absorption peaks of typical phosphate groups (PO 4 3- ) are detected in the calcium phosphate coating, indicating the successful deposition of the calcium phosphate coating; characteristic peaks such as amino (-NH 2 ), carbonyl (C=O), and amide (-CONH-) are detected in the fibronectin-like polypeptide coating, further confirming the effective introduction of polypeptide molecules into the coating.

[0037] Next, the performance of the nacreous structure fibronectin-like coating modified magnesium alloy material prepared in this example is further illustrated by experiments. It should be noted that the experiments adopted in the following test examples are all conventional technical means in the art.

[0038] Test Example 1 This test example tests the electrochemical characteristics and ion release kinetics changes of the nacreous structure fibronectin-like coating modified magnesium alloy material prepared in the example.

[0039] (1) Electrochemical corrosion test: According to the ASTM-G1-03 standard, immerse the modified magnesium alloy materials prepared in the example and Comparative Examples 1-4 into boiling CrO 3In a solution (with a concentration of 150 g / L) for 1 minute to remove surface corrosion products, the change trend of the open circuit potential (OCP) values of each modified magnesium alloy material was measured using an electrochemical working platform (Autolab, Metrohm, Switzerland). According to the Tafel extrapolation method, the corrosion potential ( E corr ), corrosion current ( I corr ), and corrosion rate ( CR PDP ) of these alloys were obtained; the Nyquist plots of the alloys and the corresponding equivalent circuit diagrams were analyzed, and a double time-constant equivalent circuit (R s (Q 1 (R 1 (Q 2 R 2 ))) was used to simulate the EIS results. The results are as shown in Figure 9 . The results show that the magnesium alloy matrix exhibits corrosion instability, with a relatively negative corrosion potential E corr , a relatively high corrosion current density I corr , and relatively low charge transfer resistance (Rct) and low-frequency impedance values; the calcium phosphate coating significantly improves E corr , reduces I corr , and increases the Rct and low-frequency impedance values, showing good corrosion resistance and interfacial stability; the fibronectin-like polypeptide coating further enhances the protection effect, with a greater shift of its E corr in the positive direction, I corr significantly reduced, and the Rct and low-frequency impedance values reaching the highest levels throughout the frequency range, demonstrating excellent anti-corrosion performance and interfacial stability.

[0040] (2) Ion release kinetics test: 10 g of the modified magnesium alloy materials prepared in the examples and Comparative Examples 1-4 and pure magnesium materials were respectively immersed in PBS buffer solution (8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4 , 0.24 g of KH 2 PO 4 dissolved in 800 mL of distilled water to prepare the PBS buffer solution), and stored in an incubator at 37 °C for 5 days. The concentration of Mg 2+ in the PBS was measured by inductively coupled plasma optical emission spectrometry (ICP-OES, Optima 2100DV, Perkin Elmer, USA). The results are as shown in Figure 10As shown, the results indicate that the magnesium ion release rate of pure magnesium and magnesium alloy substrates is relatively high, showing the characteristic of rapid degradation; the calcium phosphate coating significantly reduces the initial magnesium ion release amount, and the polypeptide coating further delays the magnesium ion release rate, showing a more stable degradation behavior; this indicates that the calcium phosphate coating and polypeptide coating can effectively regulate the slow release of magnesium ions, and at the same time further verifies the protective effect and biological functionality of the nacreous structure-like fibronectin coating on the magnesium alloy substrate.

[0041] Test Example 2 This test example tests the biocompatibility of the nacreous structure-like fibronectin coating-modified magnesium alloy material prepared in the test example.

[0042] (1)Target cell culture Cell source and culture: Bone marrow mesenchymal stem cells (BMSCs) and bone marrow-derived macrophages (BMMs) were respectively inoculated at a density of 20,000 cells / well in a 24-well culture plate, and DMEM medium was added and cultured for 7 days.

[0043] (2)Biocompatibility of the nacreous structure-like fibronectin coating-modified magnesium alloy material Bone marrow mesenchymal stem cells (BMSCs) were respectively inoculated onto the modified magnesium alloys prepared in the example, Comparative Example 1, and Comparative Example 2, and PBS extracts with a final magnesium ion concentration of 700 μg / mL, 900 μg / mL, and 800 μg / mL were added respectively (the preparation method of the PBS extract is: put the prepared modified magnesium alloy material into PBS buffer, soak for 1 day, and take the solution), and the cell viability was detected by Live / Dead cell staining and CCK-8; Bone marrow-derived macrophages (BMMs) were respectively inoculated onto the modified magnesium alloys prepared in the example, Comparative Example 1, and Comparative Example 2, and PBS extracts with a final magnesium ion concentration of 700 μg / mL, 900 μg / mL, and 800 μg / mL were added respectively (the preparation method of the PBS extract is: put the prepared modified magnesium alloy material into PBS buffer, soak for 1 day, and take the solution), and the cell viability was detected by Live / Dead cell staining and CCK-8; The results are as Figures 11 - 13 shown. It can be seen that the CCK-8 results show that the viability of bone marrow mesenchymal stem cells (BMSCs) and bone marrow-derived macrophages (BMMs) is relatively high, and the live / dead staining mainly shows green surviving cells, reflecting that the cell activity is not significantly affected, indicating that the nacreous structure-like fibronectin coating-modified magnesium alloy material prepared in this example has good biocompatibility.

[0044] Test Example 3 This test example examines the effect of the nacre - like fibronectin - coated modified magnesium alloy material prepared in the example on bone integration under osteoporosis conditions.

[0045] (1)Detect the effect of the nacre - like fibronectin - coated modified magnesium alloy material prepared in this example on the osteogenic differentiation of BMSCs.

[0046] Bone marrow mesenchymal stem cells (BMSCs) were respectively seeded onto the surfaces of the modified magnesium alloys prepared in the example, comparative example 1, comparative example 2, and comparative example 3, and PBS extracts with final magnesium ion concentrations of 700 μg / mL, 900 μg / mL, and 800 μg / mL were added respectively (the preparation method of the PBS extract is: put the prepared modified magnesium alloy material into PBS buffer, soak for 1 day, and take the solution); then: ① Use Transwell plates and fluorescence staining to evaluate the effect of the modified magnesium alloy material - induced macrophage polarization on the chemotaxis, morphology, and proliferation of BMSCs; ② Alkaline phosphatase (ALP) staining and alizarin red (ARS) staining were used to detect calcium nodules. The results are as Figures 14 - 15 shown. The results illustrate that the nacre - like fibronectin - coated modified magnesium alloy material prepared in this example can induce macrophage polarization to promote the osteogenesis of BMSCs; it can be seen from the immunofluorescence staining and the results of ALP and ARS that the nacre - like fibronectin - coated modified magnesium alloy material prepared in this example can effectively promote the osteogenic differentiation of BMSCs.

[0047] (2)The nacre - like fibronectin - coated modified magnesium alloy material improves the bone integration effect at the magnesium alloy - bone interface under osteoporosis conditions.

[0048] Rat osteoporosis and supracondylar femoral bone defect models were established, and the modified magnesium alloy materials prepared in the example, the modified magnesium alloy materials prepared in comparative example 1, the modified magnesium alloy materials prepared in comparative example 3, and pure magnesium materials were respectively implanted to repair the supracondylar femoral bone defect. Samples were collected at 2 weeks and 8 weeks respectively, and the following were carried out: HE staining and Masson staining were used to observe the inflammatory conditions around the magnesium alloy; immunofluorescence and immunohistochemistry were used to observe the infiltration of different phenotypic macrophages around the magnesium alloy. The results are as Figure 16 shown.

[0049] From Figure 16As can be seen from the results, after 2 weeks of implantation, the femoral samples of rats were stained with HE to examine the tissues around the bone defect. The HE images and semi - quantitative evaluation of new bone formation showed that the Fn - MgCaP group (Example) exhibited a milder inflammatory response and presented a more complete bone structure around the bone defect. To further analyze the immune response in the defect area, we performed immunofluorescence staining at 2 weeks after implantation to evaluate the phenotypic transformation of macrophages. The Fn - MgCaP group showed more Arg - 1+ cells (M2 phenotype) and fewer iNOS - positive cells (M1 phenotype). In addition, the expression of the anti - inflammatory cytokine IL - 10 was significantly enhanced, while the TNF - expression was significantly reduced. These experimental results further confirmed the good immunomodulatory activity in vivo of the nacre - structured fibronectin - coated modified magnesium alloy material prepared in this example and effectively improved the inflammatory response in the bone defect microenvironment.

[0050] The magnesium alloy - bone interface was scanned by Micro - CT to analyze the bone density and BV / TV around the metal. The results are as Figures 17 - 19 shown. The results showed that at 8 weeks, obvious osteogenesis was shown in the bone defect areas of all groups, and the defect area of the Fn - MgCaP group (Example) was almost completely filled with new bone tissue. The quantitative analysis of bone tissue parameters by Micro - CT scanning further confirmed these results. The Fn - MgCaP group showed the highest bone mineral density (BMD) and bone volume to tissue volume percentage (BV / TV), as well as better characteristics of the new bone trabecular structure.

[0051] In summary, it was proved that the nacre - structured fibronectin - coated modified magnesium alloy material prepared in this example not only has excellent biocompatibility and immunomodulatory activity, but also can effectively promote bone repair and integration. These results provide a potential treatment strategy for the treatment of osteoporosis and other bone defects.

[0052] The technical features of the above - described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above - described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0053] The above - described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A method for preparing a magnesium alloy material modified by a nacre-like fibronectin coating, characterized in that: It includes the following steps: A calcium-phosphorus particle suspension and a biomimetic polypeptide solution are prepared, wherein the biomimetic polypeptide has a repeated RGD structure and has biological viscosity; the calcium-phosphorus particle suspension and the biomimetic polypeptide solution are sequentially adhered to the surface of the magnesium alloy, and after repeated freezing and thawing for several times, a magnesium alloy material modified by a fibronectin coating of a mother-of-pearl structure is obtained.

2. The method for preparing the magnesium alloy material modified by the nacreous fibronectin coating according to claim 1, characterized in that: The magnesium alloy is prepared by the following method: magnesium, magnesium-calcium alloy and magnesium-lanthanum alloy are added into a high-purity graphite crucible, heated and melted to obtain a melt; the melt is poured into a preheated steel mold to obtain an ingot, and the ingot is extruded into a predetermined shape after heat treatment.

3. The method for preparing the magnesium alloy material modified by the nacreous fibronectin coating according to claim 2, characterized in that: In the magnesium alloy, the content of magnesium is 85-97wt%, the content of calcium is 0.1-1.5wt%, and the content of lanthanum is 0.2-2.0wt%; and / or the purity of magnesium is 99.99%, the content of calcium in the magnesium-calcium alloy is 30wt%, and the content of lanthanum in the magnesium-lanthanum alloy is 30wt%.

4. The method for preparing the magnesium alloy material modified by the nacreous fibronectin coating according to claim 2, characterized in that: The heating and melting temperature is 700-800°C; and / or, the temperature of the preheated steel mold is 250-320°C; and / or, the heat treatment is heating at 450-500°C for 4-8h.

5. The method for preparing the magnesium alloy material modified by the nacreous fibronectin coating according to claim 1, characterized in that: The preparation method of the calcium-phosphorus particle suspension is as follows: after mixing a calcium nitrate solution and a sodium dihydrogen phosphate solution, sodium bicarbonate is added to adjust the pH to precipitate calcium phosphate, thereby obtaining the calcium-phosphorus particle suspension.

6. The method for preparing the magnesium alloy material modified by the nacreous fibronectin coating according to claim 5, characterized in that: The concentration of the calcium nitrate solution is 12-15 mM, the concentration of the sodium dihydrogen phosphate solution is 8-9 mM, and the pH adjustment is to adjust the solution pH to 6.8-7.

2.

7. The method for preparing the magnesium alloy material modified by the nacreous fibronectin coating according to claim 1, characterized in that: The molar ratio of calcium to phosphorus in the calcium-phosphorus particle suspension is 1.0-2.0:1; and / or the concentration of the calcium-phosphorus particle suspension is 0.08-0.12 mg / mL, and the concentration of the bionic polypeptide solution is 0.008-0.015 mg / mL.

8. The method for preparing the magnesium alloy material modified by the nacreous fibronectin coating according to claim 1, characterized in that: The sequence of the biomimetic polypeptide is: MPA-PEG-Glu{c[Arg-Gly-Asp-D-Phe-Lys(Ahx)]}-{c[Arg-Gly-Asp-D-Phe-Lys(Ahx)]}.

9. A magnesium alloy material modified by a pearl-like fibronectin coating, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the magnesium alloy material modified by the nacreous fibronectin coating as claimed in claim 9 as an orthopedic implant.

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