Coatings for Implanting Metal Surfaces to Modulate Bone Microenvironment and Promote Osteogenesis and Their Preparation Method

Abstract

This invention discloses a coating for implanting on a metal surface to regulate the bone microenvironment and promote osteoogenesis, and its preparation method. The coating uses a metal-based biomaterial as a matrix and comprises, from the inside out, an oxide nanotube layer, a polydopamine layer, and a carboxymethyl chitosan / alendronate composite layer. The preparation method involves: preparing a regular oxide nanotube layer on the surface of the metal-based biomaterial using an electrochemical oxidation method; then subjecting it to hydrothermal treatment in a solution containing metal ions, followed by annealing; and then sequentially undergoing self-polymerization and covalent reactions in a dopamine solution and a carboxymethyl chitosan / alendronate mixed solution. The coating of this invention exhibits excellent biocompatibility and bioactivity, controllable release of metal ions and alendronate, promotes early integration of the metal-based biomaterial with the bone tissue interface, ensures the functional expression of normal osteoblasts, and the preparation method is simple and easy to implement, resulting in a coating with good stability.

Description

Technical Field

[0001] This invention relates to the field of orthopedic implantable biomedical materials, specifically to an implantable metal surface coating that modulates the bone microenvironment to promote osteogenic formation and its preparation method. Background Technology

[0002] Metals, as one of the commonly used materials for manufacturing orthopedic implants, have many advantages. First, they possess high mechanical strength and rigidity, providing stable support and load-bearing capabilities. Second, metals are highly machinable, allowing for processing and manufacturing through forging, casting, powder metallurgy, and other techniques to meet the needs of implants of different shapes, sizes, and structures. Furthermore, metals such as titanium, zirconium, tantalum, cobalt, and nickel, and their alloys, commonly used in orthopedic implants, exhibit good corrosion resistance, maintaining long-term stability in bodily fluids. However, the bioinertness exhibited by metal surfaces also leads to a relatively slow integration rate with bone, which is one of the main reasons for early implant failure.

[0003] Divalent metal ions play various roles in bone growth and repair. Strontium, a trace element found primarily in bones, stimulates osteoblast proliferation and differentiation, promotes bone matrix deposition, and increases bone density and strength. Calcium is a major component of bone tissue, and adequate calcium ions contribute to bone mineralization and increased bone density. Zinc is a cofactor for many enzymes; in bone tissue, zinc ions regulate osteoblast and osteoclast metabolism, as well as growth factor expression. Copper ions participate in the synthesis of bone matrix proteins, and trace amounts contribute to bone tissue formation. Magnesium ions can promote osteoblast activity and differentiation by activating the PI3K / Akt signaling pathway, thus promoting osteoblast activity and differentiation.

[0004] Alendronate is one of the most commonly used drugs for the prevention and treatment of osteoporosis. It reduces the risk of fractures by inhibiting bone resorption, promoting bone formation, and increasing bone density. However, alendronate has limited oral absorption and bioavailability, and its oral administration is accompanied by serious side effects, including osteonecrosis of the jaw; gastrointestinal irritation; nausea; musculoskeletal pain; and cardiovascular risks. Summary of the Invention

[0005] Purpose of the invention: In order to solve the technical problems existing in the prior art, the present invention aims to provide a coating for implanting on the metal surface to regulate the bone microenvironment and promote osteoogenesis, and provides a method for preparing the coating.

[0006] Technical solution: The coating for regulating bone microenvironment and promoting osteoogenesis on implantable metal surfaces described in this invention uses metal-based biomaterials as the matrix and includes, from the inside out, an oxide nanotube layer, a polydopamine layer, and a carboxymethyl chitosan / alendronate composite layer. The oxide nanotube layer is formed by metal ions carried in the oxide nanotube layer.

[0007] Furthermore, in the polydopamine layer and the carboxymethyl chitosan / alendronate composite layer, the molar ratio of the carboxyl group in the carboxymethyl chitosan to the amino group in the alendronate is >1, the relative molecular mass of the carboxymethyl chitosan is 4000-500000, and the alendronate is sodium alendronate.

[0008] Furthermore, the metal ion is one or more of strontium ions, calcium ions, zinc ions, copper ions, and magnesium ions; the implanted metal is titanium, zirconium, tantalum, cobalt, nickel, or their alloys; the oxide is an oxide of titanium, zirconium, tantalum, cobalt, nickel, or their alloys; and the inner diameter of the oxide nanotube layer is 30-120 nm, and the tube length is 0.1-5 μm.

[0009] The method for preparing the coating on the implantable metal surface that modulates the bone microenvironment and promotes osteogenic formation according to the present invention includes the following steps:

[0010] (1) Using metal-based biomaterials as anodes, regular oxide nanotube layers are prepared on their surface by electrochemical oxidation.

[0011] (2) The material obtained in step (1) is subjected to hydrothermal treatment in a solution containing metal ions;

[0012] (3) Anneal the material obtained in step (2) to obtain an oxide nanotube layer with a crystal structure;

[0013] (4) The material obtained in step (3) is placed in a dopamine solution to carry out a self-polymerization deposition reaction;

[0014] (5) The material obtained in step (4) is placed in a carboxymethyl chitosan / alendronate mixed solution, shaken to react, and then washed and dried to obtain a coating that can be implanted on a metal surface to regulate the bone microenvironment and promote osteoogenesis.

[0015] Further, in step (1), the conditions for electrochemical oxidation are: oxidation voltage of 10-200V, oxidation time of 1-120min, reaction temperature controlled at 0℃-50℃, and distance between anode and cathode controlled at 1-5cm.

[0016] Furthermore, in step (2), the concentration of the metal ion-containing solution is 0.002-2.5 mol / L, the hydrothermal treatment temperature is 150-200℃, and the hydrothermal treatment time is 1-5h.

[0017] Furthermore, in step (3), the annealing temperature is 200-900℃, the annealing time is 1-5h, and the temperature increases from room temperature to the maximum temperature by 5-15℃ per minute.

[0018] Further, in step (4), the solvent of the dopamine solution is Tris solution or physiological saline, the final solution pH is controlled between 7 and 9, and the dopamine concentration is 0.5 mg to 10 mg / ml; the conditions for the self-polymerization deposition reaction are: deposition at room temperature for 12 to 24 hours.

[0019] Further, in step (5), the preparation method of the carboxymethyl chitosan / alendronate mixed solution is as follows: after dissolving carboxymethyl chitosan in a solvent, add alendronate, stir thoroughly, then add an activator for activation, and obtain the solution after sufficient reaction; the solvent is a weakly acidic aqueous solution or an alcohol solution with pH = 4-7; the weakly acidic aqueous solution is a Tris buffer solution, which is dissolved in a weakly acidic aqueous solution at 2-8℃ for 2-24 hours; the alcohol solution includes ethanol or isopropanol, which requires ultrasonic treatment or long-term (1-3 hours) stirring when dissolved in an alcohol solution.

[0020] Further, the activator is a substance with activating effect of carboxyl groups. Preferably, the activator is one or more of N,N-dimethylaminopropyltrimethoxysilane, N,N'-diisopropylcarbodiimide, trifluoroacetic anhydride N-acetyl-N'-(dimethylamino)pyrrolidine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide. The molar ratio of the activator to the carboxyl groups in carboxymethyl chitosan is ≥1, and the activation time is 10-30 min. The shaking reaction time is 2-24 h.

[0021] Invention Principle: This invention utilizes anodized metal oxide nanotube arrays with an inner diameter of 30-120 nm and a length of 0.1-5 μm. Cell adhesion, growth, and the expression of normal functions are closely related to the surface topology of materials. Therefore, cell behavior can be regulated by adjusting the nanotube size, accelerating early cell adhesion and promoting cell functional expression. Through a hydrothermal method, active metal ions such as Sr, Ca, Zn, Cu, and Mg are doped into the nanotubes. The release of appropriate concentrations of these ions at the implantation interface stimulates osteoblast proliferation and differentiation, promotes normal functional expression, and exhibits anti-inflammatory effects. Annealing treatment alters the nanotube crystal structure, further improving cell adhesion behavior. Simultaneously, it allows the active metal ions to form ionic bonds with the metal oxide nanotubes, generating metal salts with a tighter binding. After implantation, the active metal ions can be slowly and controllably released into the body fluids, avoiding the cytotoxicity caused by a sudden release of large amounts of metal ions. Furthermore, depositing a polydopamine layer on the surface of the metal ion-loaded nanotube array can act as an intermediate layer for the fixation of alendronate, while also controlling the release of internal metal ions. Alendronate has the effects of inhibiting bone resorption and promoting bone formation, and can be used to treat osteoporosis. At the same time, the bisphosphate groups of alendronate have significant bone targeting and bone affinity, and can specifically adsorb hydroxyapatite and calcium ions in bone tissue, making it suitable as a bone-finding material. Carboxymethyl chitosan is a polysaccharide structure. Covalent bonding is achieved by the amide reaction between the carboxyl groups of carboxymethyl chitosan and the amino groups of alendronate. Excess carboxymethyl chitosan allows the remaining carboxyl groups after the reaction to covalently bond with the amino groups in the polydopamine layer through the amide reaction, thereby fixing alendronate and carboxymethyl chitosan on the material surface and slowing down the release of alendronate through covalent bonds, avoiding the cytotoxicity caused by burst release.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0023] (1) This invention increases the surface activity and hydrophilicity of the metal material by preparing a regular array of oxide nanotubes on the implanted metal surface, providing good sites for the subsequent loading of active ions and biomolecules; Sr, Ca, Zn, Cu, Mg active metal ions and alendronate molecules have good ability to regulate the surface microenvironment of the implant material and promote bone function; carboxymethyl chitosan has good biocompatibility and antibacterial properties. Therefore, this invention can achieve a variety of physiological functions as an implant material and can be applied in orthopedic implant materials;

[0024] (2) This invention achieves the loading of active metal ions through hydrothermal treatment and annealing, and changes the crystal structure of nanotubes; the salt formed by metal ions and nanotubes and the polydopamine coating deposited on the surface effectively control the sudden release of metal ions; through amide reaction, alendronate is covalently bound to carboxymethyl chitosan and polydopamine layer, avoiding the sudden release of alendronate and avoiding cytotoxicity;

[0025] (3) The preparation method of the present invention requires mild conditions, does not require special equipment, and has a simple process and low cost. Attached Figure Description

[0026] Figure 1 The images shown are electron microscope images of the coatings prepared in Example 2 of the present invention. (a) and (c) are TiO2 nanotube layers anodized on the surface of titanium alloy substrate, and (b) and (d) are coatings that regulate the bone microenvironment on the implanted metal surface to promote osteoogenesis.

[0027] Figure 2 The water contact angle morphology of the coating obtained in Example 2 of the present invention is shown in (a) the water contact angle formed by the titanium alloy base layer: 69.8°±3.1°, and (b) the water contact angle of the coating formed by the titanium alloy base layer and the coating being a coating that regulates the bone microenvironment on the implanted metal surface to promote osteogenic formation: 35.2°±1.7°.

[0028] Figure 3 The Sr ion release of the intermediate sample TNT+Sr (a) and the coating surface (b) obtained in Example 2 of this invention are shown.

[0029] Figure 4 The release of sodium alendronate (ALN) from the comparative sample TNT+ALN (a) and the coating surface (b) prepared in Example 2 of this invention is shown.

[0030] Figure 5 The adhesion of osteoblasts to the titanium alloy base layer (a) and coating surface (b) prepared in Example 2 of this invention after 1 day, 3 days and 7 days of culture.

[0031] Figure 6 The ALP expression of cells on the surface of the titanium alloy base layer and coating prepared in Example 2 of this invention after 1 day, 3 days and 7 days of cell culture.

[0032] Figure 7 The OCN release amount is the amount of cells cultured on the surface of the titanium alloy base layer and coating prepared in Example 2 of the present invention for 1 day, 3 days and 7 days.

[0033] Figure 8 The coating surface release of ALN obtained in Examples 1-5 of this invention is shown. Detailed Implementation

[0034] The present invention will now be further described in conjunction with specific embodiments and accompanying drawings.

[0035] Example 1: The coating for implanting metal surfaces to regulate the bone microenvironment and promote osteoogenesis provided in this example uses TC4 alloy as the base layer and includes anatase TiO2 nanotubes, oxide nanotube layers carrying Sr ions, polydopamine layers, and carboxymethyl chitosan / alendronate sodium composite layers.

[0036] The preparation method of the above coating is as follows: TC4 alloy is made into a sample of 70mm×50mm×2.5mm. The surface is polished by sanding with 320, 400, 600, 800 and 1000# sandpaper, then polished, electropolished for 5s with a mixed solution of HF+HNO3, washed with water and dried; anodic oxidation is performed in an electrolyte of ethylene glycol + 0.5wt% ammonium fluoride + 2vol.% deionized water, with the voltage maintained at 50V, the distance between the anode and cathode controlled at 4cm, and oxidation for 60min. After oxidation, the sample is ultrasonically cleaned with water, ethylene glycol and ethanol for 5min each, and dried; then, the sample is placed in a reaction vessel, 0.1M Sr(OH)2 is added, and the reaction is carried out at 200℃ for 2h. After that, the sample is taken out, washed with water 3 times, and air-dried; then, it is annealed at 450℃ for 3.5h; a dopamine solution of 1mg / ml is prepared using Tris buffer, and the pH of the solution is adjusted to 8.0. The prepared TiO2 nanotubes loaded with metal ions were placed in the above-mentioned dopamine solution and reacted at room temperature for 12 h. They were then washed with water and dried. 0.05 M carboxymethyl chitosan and 0.02 M sodium alendronate were added to Tris buffer and stirred. The mixture was then placed in an environment of 2-8 °C for 10 h. The mixture was then removed and activated for 20 min with (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDC) and N-hydroxysuccinimide (NHS) (wt(EDC):(NHS)=2.6853g:0.5165g, 50mL). Subsequently, the regular array of metal ions loaded with metal ions was placed in the mixed solution and reacted fully for 8 h to obtain the final product.

[0037] Example 2: The coating for implanting metal surfaces to regulate the bone microenvironment and promote osteoogenesis provided in this example uses TA2 alloy as the base layer and includes anatase TiO2 nanotubes and Sr-loaded oxide nanotube layers, a polydopamine layer and a carboxymethyl chitosan / alendronate sodium composite layer.

[0038] The preparation method of the above coating is as follows: TA2 alloy is made into a sample of 70mm×50mm×2.5mm. The surface is polished with 320, 400, 600, 800, and 1000# sandpaper, then polished, electropolished for 5s with a mixed solution of HF+HNO3, washed with water, and dried. Anodizing is performed in an electrolyte of ethylene glycol + 0.5wt% ammonium fluoride + 2vol.% deionized water, with the voltage maintained at 45V, the distance between the anode and cathode controlled at 3cm, and oxidation for 60min. After oxidation, the sample is ultrasonically cleaned with water, ethylene glycol, and ethanol for 5min each, and then dried (TNT). Subsequently, the sample is placed in a reaction vessel, 0.02M Sr(OH)2 is added, and the reaction is carried out at 200℃ for 1h. After that, the sample is taken out, washed with water 3 times, and air-dried (TNT+Sr). Then, it is annealed at 450℃ for 3.5h. A dopamine solution of 1mg / ml is prepared using Tris buffer, and the pH of the solution is adjusted to 8.0. The prepared TiO2 nanotubes loaded with metal ions were placed in the above-mentioned dopamine solution and reacted at room temperature for 12 h. They were then washed with water and dried. 0.03 M carboxymethyl chitosan and 0.01 M sodium alendronate were added to the Tris solution and stirred. The solution was then placed in an environment of 2-8 °C for 10 h. The solution was then removed and activated with (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDC) and N-hydroxysuccinimide (NHS) (wt(EDC):(NHS)=0.5751g:0.1150g, 50mL) for 20 min. Subsequently, the regular array of metal ions loaded with metal ions was placed in the mixed solution and reacted fully for 8 h to obtain the final product.

[0039] Comparative Example 1: To compare the sustained-release effects of sodium alendronate and metal ions, equal amounts of sodium alendronate and Sr were loaded onto TNT via physical adsorption (TNT+ALN) and (TNT+Sr), respectively. The surface morphology, cell adhesion, and functional expression of the coating and control samples were also compared.

[0040] The coatings prepared in Example 2 and Comparative Example 1 were characterized and tested. The results are shown in the figure. Figures 1-7 As shown.

[0041] like Figure 1 As shown, (a) and (c) are TiO2 nanotube layers anodized on the surface of TA2 titanium alloy, with an inner diameter of about 70-90 nm and a length of about 3.8 μm; (b) and (d) are coatings that regulate the bone microenvironment on the implanted metal surface to promote osteogenic formation. After loading metal ions, depositing a polydopamine layer, and grafting carboxymethyl chitosan / alendronate, the inner diameter of the nanotubes becomes smaller than that of TiO2 nanotubes, about 40-50 nm, while the length remains almost unchanged.

[0042] like Figure 2As shown, (a) shows that the water contact angle of the TA2 alloy substrate surface is about 69.8°±3.1°, indicating poor hydrophilicity; while (b) shows that the water contact angle of the coating, which is a coating that regulates the bone microenvironment on the implanted metal surface to promote osteogenic formation, is 35.2°±1.7°, indicating further improvement in hydrophilicity.

[0043] like Figure 3 As shown, (a) is the release curve of Sr ions from the intermediate sample TNT+Sr after hydrothermal treatment of nanotubes, and (b) is the Sr ion release curve of the osteogenic coating that promotes the release of Sr ions by adjusting the bone microenvironment on the implanted metal surface. Clearly, the intermediate sample TNT+Sr exhibits an extremely rapid Sr ion release rate of 240 μg / ml within the first 48 hours, followed by a slow release over time, although the concentration remains high. The prepared coating, however, only shows a slow release within the first 48 hours, with a concentration not exceeding 50 μg / ml. Within 336 hours, the release concentration increases, but remains relatively low. Clearly, the prepared coating effectively controls the burst release of metal ions.

[0044] like Figure 4 As shown, (a) is the release curve of sodium alendronate (ALN) loaded onto the TNT+ALN intermediate sample, and (b) is the release curve of ALN on the prepared coating surface. It can be seen that the ALN released from the intermediate sample loaded directly onto the coating surface was suddenly released within the first 12 hours, with a concentration exceeding 9 mg / ml. As time went on, the concentration of released ALN also increased rapidly. However, the ALN released from the prepared coating surface had a much lower concentration than that of TNT+ALN within 168 hours.

[0045] like Figure 5 As shown, after osteoblasts were inoculated with TA2 alloy, the number of cells adhering to the surface (control) was relatively small after 1 day of culture, and the number of cells on the surface tended to increase with the extension of time; however, on the coating surface, the number of cells adhering to the surface increased significantly at different culture times, and the cells all extended filamentous pseudopodia, showing good adhesion and excellent cell compatibility.

[0046] like Figure 6 The figure shows the ALP expression of osteoblasts on the TA2 alloy surface (control) and coating surface (coating) prepared in Example 2 after 1, 3, and 7 days of cell culture. In comparison, the ALP expression concentration on the coating surface was higher than that on the titanium alloy surface. As a marker of early osteoblast differentiation, the ALP expression concentration indicates higher osteoblast activity and normal cell function on the coating surface.

[0047] like Figure 7The figure shows the release levels of OCN (a marker of osteoblast differentiation and maturation) after 1, 3, and 7 days of cell culture on the TA2 alloy surface (control) and coating surface obtained in Example 2. The OCN expression concentration on the coating surface was higher than that on the titanium alloy surface, indicating that osteoblasts differentiated and matured on the coating surface.

[0048] Example 3: The coating for implanting metal surfaces to regulate the bone microenvironment and promote osteoogenesis provided in this example uses a nickel-titanium alloy as a matrix and includes an oxide layer, an oxide nanotube layer carrying Zn ions, a polydopamine layer, and a carboxymethyl chitosan / alendronate sodium composite layer.

[0049] The preparation method of the above coating is as follows: A nickel-titanium alloy sample of 70mm×50mm×2.5mm was prepared. The surface was polished by sanding with 320, 400, 600, 800, and 1000# sandpaper, then polished, and electropolished for 5s with a mixed solution of HF+HNO3+H2O2. After washing with water and drying, anodizing was performed in an electrolyte of ethylene glycol + 0.5wt% ammonium fluoride + 20ml / L phosphoric acid + 2vol.% deionized water. The voltage was maintained at 60V, the distance between the anode and cathode was controlled at 3cm, and the oxidation was performed for 50min. After that, the sample was ultrasonically cleaned with water, ethylene glycol, and ethanol for 5min each, and then dried. Subsequently, the sample was placed in a reaction vessel, 0.1M ZnCl2 was added, and the reaction was carried out at 180℃ for 2h. After that, the sample was taken out, washed with water 3 times, and air-dried. Then, the sample was annealed at 400℃ for 5h. A dopamine solution of 2mg / ml was prepared using Tris buffer, and the pH of the solution was adjusted to 8.0. The prepared metal ion-loaded oxide nanotubes were placed in the above-mentioned dopamine solution and reacted at room temperature for 12 h. They were then washed with water and dried. 0.03 M carboxymethyl chitosan and 0.01 M sodium alendronate were added to the Tris solution and stirred. The solution was then placed in an environment of 2-8 °C for 10 h. The solution was then removed and activated with N,N-dimethylaminopropyltrimethoxysilane (DMTMM) (wt(DMTMM)=0.9330 g, 50 mL) for 20 min. Subsequently, the regular array of metal ion-loaded nanotubes was placed in the mixed solution and reacted fully for 8 h to obtain the final product.

[0050] Example 4: The coating for implanting metal surfaces to regulate the bone microenvironment and promote osteoogenesis provided in this example uses pure zirconium as a matrix and includes zirconium oxide and oxide nanotube layers carrying Ca ions, a polydopamine layer and a carboxymethyl chitosan / alendronate sodium composite layer.

[0051] The preparation method of the above coating is as follows: Pure zirconium is made into a sample of 70mm×50mm×2.5mm. The surface is polished by sanding with 320, 400, 600, 800 and 1000# sandpaper, then polished, electropolished for 5s with a mixed solution of HF+HNO3+H2O, washed with water and dried; anodic oxidation is performed in an electrolyte of glycerol + 0.5wt% ammonium fluoride + 2vol.% deionized water, the voltage is maintained at 60V, the distance between the anode and cathode is controlled at 3cm, and the oxidation is performed for 40min. After that, the sample is ultrasonically cleaned with water, ethylene glycol and ethanol for 5min each, and dried; then, the sample is placed in a reaction vessel, saturated Ca(OH)2 solution is added, and the reaction is carried out at 200℃ for 2h. After that, the sample is taken out, washed with water 3 times and air-dried; then, it is annealed at 400℃ for 5h; a dopamine solution of 1mg / ml is prepared with Tris buffer and the pH of the solution is adjusted to 8.0. The prepared Fe oxide nanotubes loaded with metal ions were placed in the above-mentioned dopamine solution and reacted at room temperature for 12 h. They were then washed with water and dried. 0.02 M carboxymethyl chitosan and 0.01 M sodium alendronate were added to the Tris solution and stirred. The solution was then placed in an environment of 2-8 °C for 10 h. N,N'-diisopropylcarbodiimide (DCC) (wt(DCC)=0.3786 g, 50 mL) was added to activate the solution for 30 min. Subsequently, the regular nanotube array loaded with metal ions was placed in the mixed solution and reacted fully for 9 h to obtain the final product.

[0052] Example 5: The coating for implanting metal surfaces to regulate the bone microenvironment and promote osteoogenesis provided in this example has a tantalum alloy matrix, including oxide nanotubes and oxide nanotube layers carrying Cu ions, a polydopamine layer and a carboxymethyl chitosan / alendronate sodium composite layer.

[0053] The preparation method of the above coating is as follows: A tantalum alloy sample of 70mm×50mm×2.5mm was prepared. The surface was polished with 320, 400, 600, 800, and 1000# sandpaper, then electropolished with a mixed solution of HF and HNO3 for 5 seconds, followed by washing with water and drying. Anodizing was performed in an electrolyte of ethylene glycol + 0.5wt% ammonium fluoride + 2vol.% deionized water, with a voltage maintained at 45V, a distance of 3cm between the anode and cathode, and oxidation for 60 minutes. The sample was then ultrasonically cleaned with water, ethylene glycol, and ethanol for 5 minutes each, and dried. Subsequently, the sample was placed in a reaction vessel, saturated CuCl2 solution was added, and the reaction was carried out at 150℃ for 4 hours. The sample was then washed with water three times and air-dried. Afterward, it was annealed at 500℃ for 5 hours. A 1mg / ml dopamine solution was prepared using Tris buffer, and the pH of the solution was adjusted to 8.0. The prepared metal ion-loaded oxide nanotubes were placed in the above-mentioned dopamine solution and reacted at room temperature for 12 h. They were then washed with water and dried. 0.04 M carboxymethyl chitosan and 0.02 M sodium alendronate were added to the Tris solution and stirred. The solution was then placed in an environment of 2-8 °C for 10 h. The solution was then removed and activated with N-acetyl-N'-(dimethylamino)pyrrolidine (DEAP) (wt(DEAP)=0.8532 g, 50 mL) for 15 min. Subsequently, the regular array of metal ion-loaded nanotubes was placed in the mixed solution and reacted fully for 8 h to obtain the final product.

[0054] like Figure 8 As shown, the amount of ALN released by the coatings prepared in Examples 1-5 was relatively low, and the release rate was slow. This indicates that the coating surface effectively controlled the release of ALN. Examples 1 and 5 were loaded with 0.02M ALN, while Examples 2, 3, and 4 were loaded with 0.01M ALN. Comparatively, Examples 1 and 5 released relatively more ALN than Examples 2, 3, and 4, but the overall concentration of ALN released by the coatings in Examples 1-5 was very low.

Claims

1. A coating implanted on a metal surface to regulate the bone microenvironment and promote osteoogenesis, characterized in that, Using a metal-based biomaterial as a matrix, the coating comprises, from the inside out, an oxide nanotube layer, a polydopamine layer, and a carboxymethyl chitosan / alendronate composite layer. The oxide nanotube layer is formed by metal ions mounted within the oxide nanotube layer. The method for preparing the coating for regulating the bone microenvironment and promoting osteoogenesis on the implanted metal surface includes the following steps: (1) Using metal-based biomaterials as anodes, regular oxide nanotube layers are prepared on their surface by electrochemical oxidation. (2) The material obtained in step (1) is subjected to hydrothermal treatment in a solution containing metal ions; (3) Anneal the material obtained in step (2) to obtain an oxide nanotube layer with a crystal structure; (4) The material obtained in step (3) is placed in a dopamine solution to carry out a self-polymerization deposition reaction; (5) Place the material obtained in step (4) in a mixed solution of carboxymethyl chitosan / alendronate, shake to react, and then wash and dry to obtain a coating that can be implanted on the metal surface to regulate the bone microenvironment and promote osteoogenesis. In step (5), the preparation method of the carboxymethyl chitosan / alendronate mixed solution is as follows: after dissolving carboxymethyl chitosan in a solvent, add alendronate, stir thoroughly, then add an activator for activation, and obtain the solution after sufficient reaction; the solvent is a weakly acidic aqueous solution or alcohol solution with pH=4-7; the activator is a substance that has an activating effect on carboxyl groups, the molar ratio of the activator to the carboxyl groups in carboxymethyl chitosan is ≥1, and the activation time is 10-30 min; the shaking reaction time is 2-24 h.

2. The coating according to claim 1, characterized in that, In the polydopamine layer and the carboxymethyl chitosan / alendronate composite layer, the molar ratio of the carboxyl groups in the carboxymethyl chitosan to the amino groups in the alendronate is >1, the relative molecular mass of the carboxymethyl chitosan is 4000-500000, and the alendronate is sodium alendronate.

3. The coating according to claim 1, characterized in that, The metal ion is one or more of strontium ions, calcium ions, zinc ions, copper ions, and magnesium ions; the oxide is an oxide of titanium, zirconium, tantalum, cobalt, nickel metal or their alloys; and the inner diameter of the oxide nanotube layer is 30-120 nm and the tube length is 0.1-5 µm.

4. The coating according to claim 1, characterized in that, The implanted metal is titanium, zirconium, tantalum, cobalt, nickel, or an alloy thereof.

5. The coating according to claim 1, characterized in that, In step (1), the conditions for electrochemical oxidation are: oxidation voltage of 10-200V, oxidation time of 1-120min, reaction temperature controlled at 0℃-50℃, and distance between anode and cathode controlled at 1-5cm.

6. The coating according to claim 1, characterized in that, In step (2), the concentration of the metal ion-containing solution is 0.002-2.5 mol / L, the hydrothermal treatment temperature is 150-200℃, and the hydrothermal treatment time is 1-5h.

7. The coating according to claim 1, characterized in that, In step (3), the annealing temperature is 200-900℃, the annealing time is 1-5h, and the temperature increases from room temperature to the maximum temperature by 5-15℃ per minute.

8. The coating according to claim 1, characterized in that, In step (4), the solvent of the dopamine solution is Tris buffer or physiological saline, the final solution pH is controlled between 7 and 9, and the dopamine concentration is 0.5 mg to 10 mg / ml; the conditions for the self-polymerization deposition reaction are: deposition at room temperature for 12 to 24 hours.

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