Nanoporous titanium implants loaded with composite coatings, methods of construction and use thereof

Abstract

This invention discloses a nanoporous titanium implant loaded with a composite coating, its construction method, and its applications. First, by precisely controlling the anodizing time, a nanoporous structure with interconnected pore walls is constructed on the surface of a titanium substrate, replacing the traditional nanotube structure with poor mechanical properties. Then, a polydopamine / strontium ion composite coating is constructed on the nanoporous surface using a one-step impregnation method. This composite coating utilizes the strong coordination between polydopamine and strontium ions to achieve efficient loading, and achieves long-term, stable, and controllable release of strontium ions through coating degradation and coordination bond breaking. The implant of this invention generates a synergistic effect between its nanotopological structure and the released strontium ions, actively inducing macrophages to polarize towards the M2 anti-inflammatory phenotype, constructing a healing-promoting immune microenvironment, thereby systematically promoting osteogenic differentiation of bone marrow mesenchymal stem cells and angiogenic function of vascular endothelial cells, ultimately achieving rapid, stable, and high-quality bone integration.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a nanoporous titanium implant loaded with a composite coating, its construction method, and its application. Background Technology

[0002] Titanium and its alloys, due to their excellent mechanical properties, corrosion resistance, and biocompatibility, have long been the preferred materials for bone implants such as dental implants, artificial joints, and spinal fusion devices. However, titanium is inherently a bioinert material, making it difficult to form a rapid, strong, and durable biological bond, i.e., osseointegration, with the surrounding living bone tissue. Poor osseointegration leads to aseptic loosening of the implant in vivo, which is the leading cause of long-term implant failure, often requiring revision surgery, causing significant pain and financial burden on patients. Therefore, bio-activation modification of the titanium implant surface to actively guide the growth of new bone tissue onto the material surface and form a strong bond has become a core research direction in the field of biomedical materials.

[0003] To enhance the osseointegration capacity of titanium implants, existing technologies mainly focus on two strategies: surface topology design and loading of bioactive factors. Surface topology, especially nanoscale morphology, can mimic the microenvironment of the natural bone matrix and directly regulate cell adhesion, proliferation, and differentiation. Among these, highly ordered titanium dioxide nanotube arrays prepared on titanium surfaces via anodic oxidation have become one of the most widely studied nanostructures due to their relatively simple preparation process, controllable morphology, and large specific surface area. Studies have shown that titanium dioxide nanotube structures can significantly enhance the hydrophilicity of titanium surfaces and provide ideal physical support for osteoblast growth, thereby promoting osteoblast functional expression. However, this technology suffers from a major drawback that has not yet been effectively overcome—extremely poor mechanical stability. Specifically, anodic oxidation is a dynamic equilibrium process of oxide film formation and chemical dissolution. In the later stages of nanotube formation, the pore walls between adjacent nanopores are further dissolved, ultimately forming an array composed of numerous independent, thin-walled (typically only tens of nm) tubular units with significant physical gaps between them. While this structure provides an extremely large surface area, it also drastically degrades its mechanical properties. During implantation surgery (such as percussion implantation) or under long-term complex physiological loads in vivo (such as chewing forces and shear forces), these thin nanotube walls are highly susceptible to collapse, breakage, and even large-scale detachment from the substrate. The detached nanotube fragments are released as tiny abrasive particles into surrounding tissues, inducing persistent chronic inflammation and foreign body giant cell reactions, which in turn accelerates bone resorption, leading to peri-implant osteolysis and eventual aseptic loosening. Although some studies have attempted to improve the crystallinity of nanotubes through annealing to enhance their mechanical properties, the improvement in mechanical stability is extremely limited due to their inherent isolated tube wall structure, falling far short of the requirements for clinical application. Therefore, how to overcome the mechanical defects while preserving the excellent biological functions of the nanostructure is the primary technical challenge facing this field.

[0004] Besides topological changes, introducing osteogenic biological factors is another important surface modification strategy. Strontium ions, due to their unique dual role—both promoting osteoblast bone formation activity and inhibiting osteoclast bone resorption activity—are widely recognized as an ideal osteogenic ion. Introducing strontium ions onto the surface of titanium implants has been proven to effectively enhance local osseointegration. Current methods for loading strontium ions onto titanium surfaces mainly include physical adsorption, ion exchange, chemical deposition, and the construction of strontium-containing coatings (such as strontium hydroxyapatite). However, these methods generally suffer from the following problems: uncontrollable ion release behavior: Strontium ions from physical adsorption or simple chemical deposition have weak binding to the substrate, often resulting in an initial burst release phenomenon after implantation, where most of the strontium ions are rapidly released within a very short time (hours to days). This not only leads to a huge waste of active ions, but more importantly, the high concentration of ions released during the burst may have toxic or adverse effects on local cells. Following the burst release phase, the lack of an effective ion reservoir often leads to insufficient subsequent release from the implant surface, failing to provide sustained and effective biological stimulation during the months-long bone healing process. Furthermore, conventional methods rely on limited surface adsorption sites, resulting in a low total strontium ion loading that is insufficient to maintain the effective dose required for long-term release.

[0005] In addition, existing technical solutions are often single-function. For example, simple nanotube structure research focuses on its direct effect on osteoblasts; simple strontium ion loading focuses on its direct osteogenic effect, lacking a systematic design for the synergistic regulation of multiple links in the bone integration process, including immunity, osteogenicity and angiogenesis.

[0006] Therefore, there is an urgent need in this field to develop a novel strategy for constructing titanium implant surfaces that can simultaneously achieve excellent mechanical stability, long-term stable and controllable release of bioactive ions, and actively regulate the local immune microenvironment, thereby systematically and synergistically promoting rapid, high-quality, and durable bone integration. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a nanoporous titanium implant loaded with a composite coating, its construction method, and its application. The implant forms a nanoporous structure with interconnected pore walls on its surface, exhibiting excellent mechanical properties and a strong bond with the substrate. This surface topology overcomes the tendency of traditional nanotube structures to peel off. Furthermore, a stable polydopamine / bioactive ion composite coating is constructed on the nanoporous surface in a one-step process, achieving efficient loading of bioactive ions and their long-term, continuous, and stable release in the physiological environment. Utilizing the synergistic effect of the surface nanoporous structure and the released ions, the local immune microenvironment is actively regulated, systematically promoting osteogenic-angiogenic coupling, thereby achieving excellent osteointegration.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a method for constructing a nanoporous titanium implant loaded with a composite coating, comprising the following steps: S1. A nanoporous structure with interconnected pore walls is constructed on the surface of a titanium implant by anodizing. S2. Immerse the titanium implant with a nanoporous structure on its surface into a mixed solution containing a polydopamine precursor and a bioactive ionic salt. A polydopamine / bioactive ionic composite coating is formed in situ on the surface and inside of the nanoporous structure in one step to obtain a nanoporous titanium implant loaded with the composite coating.

[0009] Furthermore, in step S1, the electrolyte used in the anodic oxidation method is an organic solvent system containing ammonium fluoride, the voltage is 30-50V, and the oxidation time is 0.5-3h. By precisely controlling the oxidation time, the complete transformation of nanopores into nanotubes can be prevented, thereby obtaining a nanoporous structure with interconnected pore walls and no inter-tube spacing.

[0010] Preferably, the electrolyte is a deionized aqueous solution containing 0.3-0.4 wt% ammonium fluoride and 4-6 vol% ethylene glycol; the voltage is 40 V and the oxidation time is 1-2 h.

[0011] Furthermore, in step S1, after the titanium plant body is anodized, it is then annealed at a temperature of 450-550℃ for 1-3 hours to improve the crystallinity of the oxide layer (mainly titanium dioxide).

[0012] Further, in step S2, the polydopamine precursor is dopamine hydrochloride, and the bioactive ion is one or more of strontium ions, magnesium ions, zinc ions, and silver ions; the solvent of the mixed solution is a tris(hydroxymethyl)aminomethane buffer solution with a pH of 8-9.

[0013] Preferably, the bioactive ion is a strontium ion, or a combination of strontium ions and other ions.

[0014] Furthermore, the concentration of dopamine hydrochloride is 0.5-2 mg / mL, and the concentration of strontium chloride is 10-30 mg / mL.

[0015] Furthermore, the reaction conditions for the one-step method are as follows: at 20-25°C, the titanium implant with a nanoporous structure is immersed in a mixed solution for 30-60 hours.

[0016] During this process, dopamine undergoes self-polymerization under weakly alkaline conditions to form a highly adhesive polydopamine coating. Simultaneously, the abundant catechol and amino functional groups on the polydopamine molecular chain bind to bioactive ions in the solution through coordination, thereby efficiently and stably immobilizing the bioactive ions within the coating. After the reaction is complete, the sample is removed and ultrasonically cleaned with deionized water to remove unbound substances physically adsorbed on the surface. Finally, it is dried and stored in a vacuum-sealed container.

[0017] Furthermore, before anodizing the titanium implant, it undergoes pretreatment. Specifically, the surface of the titanium implant is polished step by step using silicon carbide sandpaper ranging from 320 to 1200 mesh. Then, it is ultrasonically cleaned in deionized water, anhydrous ethanol, and acetone for 15 minutes each to remove surface oil and impurities. Finally, it is dried with nitrogen gas for later use.

[0018] A second aspect of the present invention provides a nanoporous titanium implant loaded with a composite coating obtained by the above-described construction method, comprising a titanium substrate, a nanoporous titanium dioxide layer with interconnected pore walls formed on the surface of the titanium substrate, and a polydopamine / bioactive ion composite functional coating loaded on and inside the nanoporous titanium dioxide layer, wherein bioactive ions in the polydopamine / bioactive ion composite functional coating are bound to the polydopamine molecular chain through coordination bonds and are uniformly distributed therein.

[0019] The final product is a nanoporous / polydopamine / bioactive ion composite coating on a titanium surface, which has a "sandwich" or "multi-layer" structure. The bottom layer is a titanium implant substrate; the middle layer is a nanoporous titanium dioxide layer formed by anodizing, which has a uniform structure and is firmly bonded to the substrate; the outermost layer is a polydopamine / strontium ion composite functional coating formed by a one-step impregnation method. In this coating, strontium ions are uniformly distributed in the polydopamine network through coordination bonds, which can achieve long-term stable release.

[0020] Furthermore, the implant surface is hydrophilic and can synergistically induce macrophages to polarize towards the M2 anti-inflammatory phenotype, and promote osteogenic differentiation of bone marrow mesenchymal stem cells and tube-forming ability of vascular endothelial cells.

[0021] A third aspect of the present invention provides an application of the above-mentioned nanoporous titanium implant loaded with a composite coating, wherein the nanoporous titanium implant loaded with a composite coating is used to prepare orthopedic or dental implants, cardiovascular stents, neural electrodes or tissue engineering scaffolds that promote osseointegration.

[0022] Compared with the prior art, the present invention has the following beneficial effects: A surface structure with excellent and stable mechanical properties is provided. (1) This invention constructs an oxide layer with a uniform nanoporous structure on the surface of titanium implants by precisely controlling the anodic oxidation process parameters. The nanopore walls are interconnected, resulting in a more dense and stable structure. Compared with traditional nanotube structures, this structure has superior mechanical stability. Atomic force microscopy (DMT) mechanical testing results show that the modulus of the nanoporous structure is much higher than that of the nanotube structure, indicating that it has superior mechanical stability and resistance to damage, and can better withstand the mechanical stress during the implantation process, reducing the risk of coating peeling. Subsequently, by utilizing the strong coordination interaction between polydopamine and strontium ions, a polydopamine / strontium ion composite coating is constructed on the surface of the nanoporous structure in one step, achieving efficient loading of strontium ions. Under physiological conditions, this coating can achieve long-term, stable, and continuous release of strontium ions through the slow degradation of polydopamine and the breaking of coordination bonds. This sustained-release characteristic matches the bone healing cycle and can provide a more lasting biological effect.

[0023] (2) This invention constructs a multifunctional surface with both immunomodulatory and osteogenic activity based on a nanoporous structure and a polydopamine / strontium ion composite coating: through the synergistic effect of the nanoporous topology and the released strontium ions, macrophages are actively induced to polarize towards the anti-inflammatory M2 phenotype (upregulation of the M2 marker CD206 and downregulation of the M1 marker iNOS), regulating the local immune microenvironment, thereby synergistically promoting osteogenic differentiation of bone marrow mesenchymal stem cells and angiogenic function of vascular endothelial cells, ultimately achieving full-process promotion from immunomodulation to osteogenic-angiogenic coupling, significantly improving the osteointegration effect of the implant. The titanium implant surface can continuously release strontium ions and achieve the purpose of immunomodulation to improve the osteogenic effect.

[0024] (3) The main process of the present invention includes only two steps: anodizing and one-step impregnation. In particular, the construction of the polydopamine / strontium ion composite coating is a one-step process. The process is simple, easy to operate, requires no complicated equipment, and is inexpensive. It is suitable for titanium and titanium alloy implants of various complex shapes and has extremely high industrial transformation value. Attached Figure Description

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0026] Figure 1 This is a microscopic image of the surface morphology of the pretreated titanium implant; Figure 2 This is a test diagram of the surface mechanical properties of the pretreated titanium implant; Figure 3 The microstructure of the titanium surface after anodic oxidation for 0.5 h in Example 1 is shown in the figure; Figure 4 This is a microscopic morphology image of the titanium surface after 1 hour of anodizing in Example 2; Figure 5This is a graph showing the mechanical properties of titanium surface after 1 hour of anodizing in Example 2. Figure 6 This is a microscopic morphology image of the titanium surface after anodic oxidation for 2 hours in Example 3; Figure 7 This is a microscopic morphology image of the titanium surface after anodic oxidation for 3 hours in Example 4; Figure 8 This is a test diagram of the mechanical properties of titanium surface after 3 hours of anodizing in Example 4; Figure 9 The image shows the microstructure of the titanium surface with the nanoporous / polydopamine composite coating in Comparative Example 1. Figure 10 The image shows the microstructure of the titanium surface with the nanoporous / polydopamine / strontium ion composite coating prepared in Example 5. Figure 11 Fourier transform infrared (FTIR) spectra of the raw materials—titanium implants, polydopamine, the titanium implants with surface-constructed nanoporous structures prepared in Example 2, the titanium implants with nanoporous / polydopamine coatings in Comparative Example 1, and the titanium implants with nanoporous / polydopamine / strontium ion composite coatings prepared in Example 5. Figure 12 Static water contact angle test results were obtained for the raw material titanium implant, the titanium implant with a surface-constructed nanoporous structure prepared in Example 2, the titanium implant with a nanoporous / polydopamine coating in Comparative Example 1, and the titanium implant with a nanoporous / polydopamine / strontium ion composite coating prepared in Example 5. Figure 13 The in vitro release curve of strontium ions from the titanium implant with the nanoporous / polydopamine / strontium ion composite coating prepared in Example 5 is shown. Figure 14 Cell activity test diagrams for the raw material titanium implant, the titanium implant with a surface-constructed nanoporous structure prepared in Example 2, and the titanium implant with a nanoporous / polydopamine coating in Comparative Example 1; Figure 15 Alkaline phosphatase staining effect (A) of the raw material titanium implant, the titanium implant with surface-constructed nanoporous structure prepared in Example 2, the titanium implant with nanoporous / polydopamine coating in Comparative Example 1, and the titanium implant with nanoporous / polydopamine / strontium ion composite coating prepared in Example 5; Alizarin red staining effect on day 14 (B). Figure 16 Three-dimensional reconstructed image (A) and quantitative bone parameter analysis diagram (BE) of the titanium implant with nanoporous / polydopamine / strontium ion composite coating prepared in Example 5 after implantation into the distal femur. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Examples 1-4

[0028] Examples 1-4 all provide a method for constructing nanoporous structures on the surface of titanium implants, including the following steps: Step 1: Titanium Implant Pretreatment: The titanium implant undergoes surface pretreatment. Pretreatment includes sequentially polishing the titanium implant with silicon carbide sandpaper ranging from 320 to 1200 grit, followed by ultrasonic cleaning in deionized water, anhydrous ethanol, and acetone for 15 minutes each to remove surface oil and impurities. Finally, it is dried with nitrogen gas for later use. The resulting titanium implant's surface microstructure is shown below. Figure 1 As shown, the surface mechanical properties of titanium obtained from the test are as follows: Figure 2 As shown.

[0029] Step 2: Construct a nanoporous structure on the surface of the titanium implant. The titanium implant pretreated in Step 1 is used as the anode and placed in an electrolyte for anodizing treatment to form a uniform nanoporous structure on its surface.

[0030] The electrolyte was an ethylene glycol solution containing 0.35 wt% ammonium fluoride and 5 vol% deionized water. The anodizing process parameters were: voltage 40 V, and oxidation time controlled between 0.5 and 3 h. By precisely controlling the oxidation time, the complete transformation of nanopores into nanotubes could be prevented, thus obtaining a nanoporous structure with interconnected pore walls and no inter-tube spacing. After anodizing, the sample was annealed at 500 °C for 2 h to improve the crystallinity of the oxide layer (mainly titanium dioxide).

[0031] In the specific experiments, the anodizing time was set to 0.5 h in Example 1, 1 h in Example 2, 2 h in Example 3, and 3 h in Example 4.

[0032] Example 1: Microstructure of titanium surface after anodic oxidation for 0.5 h is as follows Figure 3 As shown, a sporadic, dispersed initial nanoporous structure is formed, with the nanopores spaced far apart.

[0033] Example 2: Microstructure of titanium surface after 1 hour of anodizing. Figure 4 As shown, a uniformly distributed, regular nanoporous structure is formed, with interconnected pore walls and no independent tubular structures. The mechanical properties of the titanium surface after 1 hour of anodizing are as follows... Figure 5As shown, it exhibits excellent mechanical properties.

[0034] Example 3: Microstructure of titanium surface after 2 hours of anodizing. Figure 6 As shown, a uniformly distributed nanoporous structure is formed, with most of the pore walls still connected to each other, but the pore wall thickness is thinner than that of 1 hour.

[0035] Example 4: Microstructure of titanium surface after 3 hours of anodizing. Figure 7 As shown, some areas of the surface begin to transition into nanotube structures, with obvious gaps appearing between the tube walls. The mechanical properties of the titanium surface after 3 hours of anodizing are as follows... Figure 8 As shown, its DMT modulus is significantly lower than that of the sample oxidized for 1 hour, indicating a decrease in mechanical properties. Example 5

[0036] The titanium implant with a surface-constructed nanoporous structure was prepared by anodizing for 1 hour as described in Example 2.

[0037] The sample was annealed at 500℃ for 2 hours at a heating rate of 5℃ / min to improve the crystallinity of the oxide layer (mainly titanium dioxide).

[0038] Then, a polydopamine / strontium ion composite coating that continuously releases strontium is constructed, as follows: The prepared titanium implant with titanium nanoporous structure was immersed in a dopamine-strontium mixed solution, and a polydopamine / strontium ion composite coating was constructed on the surface of the titanium nanoporous structure by a one-step impregnation method to obtain the final product, nanoporous / polydopamine / strontium ion composite coated titanium.

[0039] The dopamine-strontium mixed solution is prepared by dissolving dopamine hydrochloride (1 mg / mL) and strontium chloride (20 mg / mL) in 10 mM Tris buffer solution at pH 8.5 and stirring until homogeneous.

[0040] The impregnation process parameters are as follows: at room temperature (20-25℃), the titanium nanoporous sample is immersed in the above mixed solution for 48 hours.

[0041] During this process, dopamine undergoes self-polymerization under weakly alkaline conditions, forming a highly adhesive polydopamine coating. Simultaneously, the abundant catechol and amino functional groups on the polydopamine molecular chain bind to strontium ions in the solution through coordination, thereby efficiently and stably immobilizing the strontium ions within the coating. After the reaction, the sample is removed and ultrasonically cleaned with deionized water to remove unbound substances physically adsorbed on the surface. Finally, it is dried and stored in a vacuum-sealed container. The microstructure of the nanoporous / polydopamine / strontium ion composite coating on titanium surface is shown below. Figure 10As shown, it has a "sandwich" or "multi-layer" structure. The bottom layer is a titanium implant substrate; the middle layer is a nanoporous titanium dioxide layer formed by anodizing. This layer has a uniform structure and is firmly bonded to the substrate; the outermost layer is a polydopamine / strontium ion composite functional coating formed by a one-step impregnation method. In this coating, strontium ions are uniformly distributed in the polydopamine network through coordination bonds, which can achieve long-term stable release. Comparative Example 1

[0042] Comparative Example 1 is a comparative test example of Example 1, and its difference from Example 1 is as follows: A polydopamine coating was constructed on the surface of a nanoporous titanium implant using a dopamine solution. The resulting nanoporous / polydopamine composite coating on the titanium surface exhibits the following microstructure: Figure 9 As shown.

[0043] Fourier transform infrared spectroscopy (FTIR) was performed on the following materials: titanium implant, polydopamine, the titanium implant with a nanoporous surface structure prepared in Example 2, the titanium implant with a nanoporous / polydopamine coating in Comparative Example 1, and the titanium implant with a nanoporous / polydopamine / strontium ion composite coating prepared in Example 5. The test results are shown in [Figure 1]. Figure 11 This demonstrates that polydopamine and strontium ions were successfully immobilized on the nanoporous titanium surface in Example 5.

[0044] Static water contact angle test ( Figure 12 This indicates that the nanoporous / polydopamine / strontium ion composite coated titanium surface prepared in Example 5 is hydrophilic.

[0045] In vitro release behavior test of strontium ions: The sample prepared in Example 5 (a titanium implant with a nanoporous / polydopamine / strontium ion composite coating) was immersed in 10 mL of phosphate buffer solution at pH 7.4 and placed in a constant temperature shaker at 37°C. The release solution was collected at predetermined time points, and an equal volume of fresh phosphate buffer solution was added. The concentration of strontium ions in the release solution was detected using inductively coupled plasma atomic emission spectrometry (ICP-AES).

[0046] Test results are as follows Figure 13 As shown, the titanium implant with the nanoporous / polydopamine / strontium ion composite coating exhibits a small release (approximately 15% of the total release) within the initial 24 hours, after which it enters a continuous and stable release plateau, with a release time exceeding 384 hours (16 days), and the release curve approaches zero-order kinetics. These results demonstrate that the present invention successfully achieves long-term, stable, and controllable release of strontium ions.

[0047] In vitro biological performance evaluation: 1. Cell compatibility The cytotoxicity of the materials (raw material - titanium implant, titanium implant with surface-constructed nanoporous structure prepared in Example 2, titanium implant with nanoporous / polydopamine coating in Comparative Example 1, and titanium implant with nanoporous / polydopamine / strontium ion composite coating prepared in Example 5) to mouse embryonic osteoblast precursor cells (MC3T3-E1) was evaluated using a cell viability assay kit.

[0048] The biocompatibility of the material with cells was assessed using a cell counting kit (CCK-8). MC3T3-E1 cells in the logarithmic growth phase were seeded at a density of 2*10-1. 4 Cells were seeded at a density of [number] cells / mL on the surface of different groups of materials and cultured in a cell culture incubator for 1, 3, and 5 days, respectively. After discarding the supernatant, CCK-8 assay reagent prepared according to the instructions was added, and the cells were incubated again for 2 hours. Finally, the optical density (OD) value at a wavelength of 450 nm was measured using a microplate reader to reflect the cell viability and proliferation status.

[0049] Test results are as follows Figure 14 As shown, compared with smooth titanium (Ti), the titanium implant with a surface-constructed nanoporous structure prepared in Example 2, and the titanium implant with a nanoporous / polydopamine coating in Comparative Example 1, the titanium implant with a nanoporous / polydopamine / strontium ion composite coating prepared in Example 5 showed the highest cell activity (OD value) on days 1, 2, and 3, indicating that the material is non-cytotoxic and can even promote cell proliferation and metabolism.

[0050] 2. Osteogenic differentiation capacity MC3T3-E1 cells were cultured on different materials (raw material - titanium implants, titanium implants with surface-constructed nanoporous structures prepared in Example 2, titanium implants with nanoporous / polydopamine coatings in Comparative Example 1, and titanium implants with nanoporous / polydopamine / strontium ion composite coatings prepared in Example 5). Alkaline phosphatase (ALP) staining was performed on day 7, and Alizarin Red (ARS) staining was performed on day 14.

[0051] Osteogenic induction factors were added to 10% FBS and 1% penicillin / streptomycin α-MEM medium to make the final medium contain 100 nmol / L dexamethasone, 10 mmol / L β-glycerophosphate sodium, and 50 μg / mL ascorbic acid. MC3T3-E1 cells in the logarithmic growth phase were seeded at a density of 2*102. 4 Different groups of materials were seeded at a density of cells / mL, and the solution was replaced with bone mineralization solution every 2 days.

[0052] After 7 days of mineralization induction, discard the supernatant and wash 2-3 times with PBS. Add 4% paraformaldehyde and fix at room temperature for 15-30 min. Then wash 3-5 times with PBS, 3-5 min each time. Prepare BCIP / NBT staining working solution according to the alkaline phosphatase chromogenic kit instructions. Add the staining working solution to the wells of the plate, fully covering the cells. Incubate at room temperature in the dark for 5-30 min until the desired staining depth is achieved. Remove the staining solution and add ddH2O to terminate the chromogenic reaction. Finally, observe the staining results and take photos.

[0053] After 14 days of induction for cell mineralization, cells were fixed with 4% paraformaldehyde and washed with PBS. Alizarin Red staining solution was added, and staining was performed at room temperature for 5-10 minutes. After removing the dye, the cells were washed three times with ddH2O, and the staining results were observed and photographed.

[0054] Test results are as follows Figure 15 As shown, the titanium implant with the nanoporous / polydopamine / strontium ion composite coating has the deepest ALP staining color. Figure 15 A), and the number and area of ​​the mineralized nodules formed are the largest (red). Figure 15 B) indicates that it has the strongest ability to promote osteogenic differentiation.

[0055] 3. Assessment of in vivo osseointegration capacity Using a rat femoral condyle defect model, a titanium implant with a nanoporous / polydopamine / strontium ion composite coating prepared in Example 5 was implanted into the distal femur. New bone formation was evaluated by Micro-CT scan at 4 and 8 weeks postoperatively.

[0056] Sixteen male SD rats (approximately 200 grams each) were randomly divided into two groups (n=8 each: control group and experimental group) after one week of acclimatization to their new environment. In short, after anesthetizing, shaving, and disinfecting the rats, a 3mm incision was made on the medial side of the knee joint. The soft tissue was pushed aside through periosteal dissection to expose the surgical area. A round hole was drilled at the proximal plateau of the tibia using a drill bit slightly smaller in diameter than the implant. Simultaneously, the surgical area was flushed with saline to lower the local temperature and minimize tissue damage. Experimental group titanium implants were randomly implanted into the tibia of the rats, while blank group titanium implants were implanted on the other side. The soft tissue was then sutured in layers. Postoperatively, the rats received intramuscular injections of penicillin for three consecutive days to reduce infection.

[0057] Test results are as follows Figure 16 As shown, the three-dimensional reconstructed image ( Figure 16 A) shows that the amount of new bone formation (bone volume fraction), trabecular bone thickness, and bone mineral density around titanium implants with nanoporous / polydopamine / strontium ion composite coatings are significantly higher than those of raw titanium implants, indicating a tighter bone-implant interface. Quantitative bone parameter analysis ( Figure 16The results (B-16E) showed that the titanium implant with nanoporous / polydopamine / strontium ion composite coating had significantly higher bone volume fraction (BV / TV), trabecular bone thickness (Tb.Th), and bone mineral density (BMD) at all time points than the control group (raw material-titanium implant), while the trabecular bone separation (Tb.Sp) was significantly reduced, demonstrating that the nanoporous / polydopamine / strontium ion composite coating promotes denser and more mature new bone formation.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a nanoporous titanium implant loaded with a composite coating, characterized in that, Includes the following steps: S1. A nanoporous structure with interconnected pore walls is constructed on the surface of a titanium implant by anodizing. S2. Immerse the titanium implant with a nanoporous structure on its surface into a mixed solution containing a polydopamine precursor and a bioactive ionic salt. A polydopamine / bioactive ionic composite coating is formed in situ on the surface and inside of the nanoporous structure in one step to obtain a nanoporous titanium implant loaded with the composite coating.

2. The construction method according to claim 1, characterized in that, In step S1, the electrolyte used in the anodic oxidation method is an organic solvent system containing ammonium fluoride, the voltage is 30-50V, and the oxidation time is 0.5-3h.

3. The construction method according to claim 2, characterized in that, The voltage is 40V and the oxidation time is 1-2 hours.

4. The construction method according to claim 1, characterized in that, In step S1, after the titanium plant body is anodized, it is then annealed at a temperature of 450-550℃ for 1-3 hours.

5. The construction method according to claim 1, characterized in that, In step S2, the polydopamine precursor is dopamine hydrochloride, and the bioactive ion is one or more of strontium ions, magnesium ions, zinc ions, and silver ions; the solvent of the mixed solution is a tris(hydroxymethyl)aminomethane buffer solution with a pH of 8-9.

6. The construction method according to claim 5, characterized in that, The concentration of dopamine hydrochloride is 0.5-2 mg / mL, and the concentration of strontium chloride is 10-30 mg / mL.

7. The construction method according to claim 6, characterized in that, The reaction conditions for the one-step method are as follows: at 20-25°C, the titanium implant with a nanoporous structure is immersed in a mixed solution for 30-60 hours.

8. A nanoporous titanium implant loaded with a composite coating, obtained by the construction method as described in any one of claims 1-7, characterized in that, The invention includes a titanium substrate, a nanoporous titanium dioxide layer with interconnected pore walls formed on the surface of the titanium substrate, and a polydopamine / bioactive ion composite functional coating loaded on and inside the nanoporous titanium dioxide layer, wherein bioactive ions in the polydopamine / bioactive ion composite functional coating are bound to the polydopamine molecular chain through coordination bonds and are uniformly distributed therein.

9. The nanoporous titanium implant loaded with a composite coating according to claim 8, characterized in that, The implant has a hydrophilic surface and can synergistically induce macrophages to polarize towards the M2 anti-inflammatory phenotype, and promote osteogenic differentiation of bone marrow mesenchymal stem cells and tube-forming ability of vascular endothelial cells.

10. An application of a nanoporous titanium implant loaded with a composite coating as described in claim 8 or 9, characterized in that, Nanoporous titanium implants loaded with composite coatings are used to prepare orthopedic or dental implants that promote osseointegration, cardiovascular stents, neural electrodes or tissue engineering scaffolds.

Images