Titanium implant material with anti-infection and bone integration promoting functions and preparation method thereof

Abstract

This invention belongs to the field of medical materials technology and discloses a titanium implant material with both anti-infection and bone integration promoting functions, as well as its preparation method. The titanium implant material includes a titanium material with calcium-copper ion-containing surface, and a coating attached to the surface of the calcium-copper ion-containing titanium material. The coating includes glucose oxidase-modified hollow manganese dioxide nanoparticles and alendronate-modified hyaluronic acid. The titanium implant material of this invention possesses both anti-infection and bone integration promoting functions, and also exhibits good anti-inflammatory effects.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and specifically relates to a titanium implant material with both anti-infection and bone integration promotion functions and its preparation method. Background Technology

[0002] Titanium-based implants are widely used in orthopedic implants due to their good biocompatibility and mechanical properties. However, the surface of titanium implants generally lacks anti-infection, anti-inflammatory, and bone integration-promoting functions, especially in the complex microenvironment of diabetic patients (such as hyperglycemia, high levels of reactive oxygen species (ROS), and persistent inflammation), where implants are prone to infection or poor bone integration. While existing coating technologies have been developed to form a protective layer on the surface of titanium implants, most coatings have limited functionality and fail to simultaneously achieve multiple functions such as anti-infection, anti-oxidation, and bone healing promotion.

[0003] Therefore, there is an urgent need to provide a titanium implant material that combines anti-infection and bone integration promotion functions. Summary of the Invention

[0004] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a titanium implant material with both anti-infection and bone-integration promoting functions, and a method for preparing the same. The titanium implant material of this invention not only has anti-infection and bone-integration promoting functions, but also exhibits good anti-inflammatory effects.

[0005] The titanium implant material of this invention is obtained by alkaline heat treatment of titanium material with alkali solution, calcium salt, and copper salt to obtain titanium material with a surface rich in calcium and copper ions. Then, a coating is further constructed on the surface of the titanium material rich in calcium and copper ions. The coating includes: glucose oxidase (GOx) modified hollow manganese dioxide nanoparticles (H-MnO2) and alendronate-modified hyaluronic acid (AlnHA). This coating consumes glucose in the in vivo microenvironment through enzymatic activity and generates H2O2, which further reacts with Mn... 2+ and Cu 2+ The Fenton-mediated reaction generates ·OH, which possesses highly effective bactericidal capabilities. Simultaneously, the superoxide dismutase (SOD) activity of H-MnO2 and the antioxidant properties of hyaluronic acid scavenge excess ROS, reducing inflammation and promoting bone healing. This material is suitable for bone implant applications in patients with complex microenvironments, such as those with diabetes. The coating can be applied via Ca... 2+ It is tightly bound to alendronate through chelation.

[0006] In addition, due to the modification of hollow manganese dioxide nanoparticles (H-MnO2) with glucose oxidase (GOx), the coating has multi-enzyme activity and is degradable, and the nanoparticles are degradable under acidic conditions.

[0007] The first aspect of the present invention provides a titanium implant material that has both anti-infection and bone integration promotion functions.

[0008] Specifically, a titanium implant material with both anti-infection and bone integration promotion functions includes a titanium material with calcium and copper ions on its surface, and a coating, wherein the coating is attached to the surface of the titanium material with calcium and copper ions.

[0009] The coating comprises glucose oxidase-modified hollow manganese dioxide nanoparticles and alendronate-modified hyaluronic acid.

[0010] Titanium materials containing calcium and copper ions on their surface are represented by CaCu-Ti.

[0011] Hollow manganese dioxide nanoparticles (H-MnO2), acting as "armor enzymes," provide multi-enzyme activity and possess antibacterial, antioxidant, and bone integration-promoting functions.

[0012] Alenphosphonate-modified hyaluronic acid (AlnHA), as a matrix carrier, enhances the adhesion of the coating to titanium-based implants and promotes bone integration.

[0013] In some embodiments of the present invention, the glucose oxidase-modified hollow manganese dioxide nanoparticles also contain tannic acid (TA) structures.

[0014] In some embodiments of the present invention, the alendronate-modified hyaluronic acid is formed by grafting EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) via a coupling reaction.

[0015] In some embodiments of the present invention, the mass ratio of alendronate-modified hyaluronic acid to glucose oxidase-modified hollow manganese dioxide nanoparticles in the coating is (1-3) mg:(400-500) μg.

[0016] In some embodiments of the present invention, the thickness of the coating is 0.5-1 μm.

[0017] In some embodiments of the present invention, the titanium material is titanium sheet, titanium rod, or other titanium materials of various shapes.

[0018] A second aspect of the present invention provides a method for preparing a titanium implant material that has both anti-infection and bone integration promotion functions.

[0019] Specifically, a method for preparing a titanium implant material that combines anti-infection and bone integration promotion functions includes the following steps:

[0020] (1) Using silica nanoparticles (NPs) as templates, potassium permanganate solution was dropped into silica suspension by ultrasonic treatment, and sodium carbonate solution was added for etching to obtain hollow manganese dioxide nanoparticles.

[0021] (2) Tannic acid and glucose oxidase (GOx) were reacted with hollow manganese dioxide nanoparticles to prepare glucose oxidase modified hollow manganese dioxide nanoparticles (H-MnO2-GOx).

[0022] (3) Alendronate was grafted onto hyaluronic acid via a coupling reaction using EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to form alendronate-modified hyaluronic acid (AlnHA).

[0023] (4) Add the titanium material to the alkaline solution and heat it to obtain the treated titanium material. Then, immerse the titanium material in calcium salt and copper salt in sequence to obtain titanium material with calcium and copper ions on the surface.

[0024] (5) The glucose oxidase-modified hollow manganese dioxide nanoparticles are mixed with the alendronate-modified hyaluronic acid, coated on the surface of the titanium material containing calcium and copper ions, and dried to obtain the titanium implant material.

[0025] In some embodiments of the present invention, the mass ratio of tannic acid, glucose oxidase and hollow manganese dioxide nanoparticles is (20-40) mg: (2-10) mg: 1 g, for example, 25 mg: 5 mg: 1 g.

[0026] In some embodiments of the present invention, the specific process of step (2) is as follows: hollow manganese dioxide nanoparticles are ultrasonically dispersed in pure water, continuously stirred, and tannic acid (TA) solution is added. The resulting mixture is reacted at 35-40°C for 0.5-1 hours, and the product is collected by centrifugation. The product is washed with pure water and anhydrous ethanol, and then dried overnight. Glucose oxidase (GOx) is dissolved in phosphate buffered saline to obtain glucose oxidase solution. The product is added, and the mixture is reacted at 0°C for 2-3 hours. The product is then separated by centrifugation to obtain glucose oxidase-modified hollow manganese dioxide nanoparticles (H-MnO2-GOx).

[0027] In some embodiments of the present invention, step (2) is more specifically carried out as follows: 1g of hollow manganese dioxide nanoparticles are ultrasonically dispersed in 20mL of pure water, stirred continuously at 40°C, 20mL of tannic acid (TA) solution (25mg / mL) is added, the resulting mixture is reacted at 40°C for 1 hour, and the product is collected by centrifugation at 10000rpm. The product is washed 5 times with pure water and 1 time with anhydrous ethanol, and then dried overnight in a vacuum oven at 60°C. 5mg of lyophilized glucose oxidase (GOx) is dissolved in 2mL of phosphate buffered saline (PBS) to obtain glucose oxidase solution. 0.4g of product is added, and the mixture is adjusted to a final volume of 10mL with PBS. The mixture is continuously shaken at 200rpm and reacted at 4°C for 3 hours. The product is then separated by centrifugation at 10000rpm using a high-speed refrigerated centrifuge. The product is washed 3 times with PBS (0.1M, pH 6.5) to remove free or weakly bound GOx, and glucose oxidase-modified hollow manganese dioxide nanoparticles are obtained.

[0028] In some embodiments of the present invention, the specific process of step (3) is as follows: sodium hyaluronate, NHS (N-hydroxysuccinimide) and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) are dissolved in PBS and stirred. Then, sodium alendronate is added and stirred. The resulting product is purified by dialysis and then lyophilized to obtain alendronate-modified hyaluronic acid (AlnHA).

[0029] In some embodiments of the present invention, in step (3), the mass ratio of sodium hyaluronate, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is (450-550) mg:(100-200) mg:300 mg, for example, 500 mg:150 mg:300 mg.

[0030] In some embodiments of the present invention, step (3) is more specifically performed as follows: Sodium hyaluronate (500 mg), NHS (N-hydroxysuccinimide) and EDC (300 mg) are dissolved in 100 mL PBS (pH 5.0) and stirred for 3 hours. Then, 500 mg of alendronate sodium is added and stirred for 3 days. The resulting product is purified for 3 days using a dialysis bag (3500 MW) and then lyophilized to obtain alendronate-modified hyaluronic acid (AlnHA).

[0031] In some embodiments of the present invention, in step (4), the alkaline solution includes a sodium hydroxide solution or a potassium hydroxide solution.

[0032] In some embodiments of the present invention, in step (4), the concentration of the alkaline solution is 4-7 mol / L, for example, 5 mol / L.

[0033] In some embodiments of the present invention, in step (4), the heating temperature is 75-85°C and the heating time is 10-12 hours.

[0034] In some embodiments of the present invention, in step (4), the calcium salt includes calcium halide salts, such as calcium chloride and calcium bromide.

[0035] In some embodiments of the present invention, in step (4), the concentration of the calcium salt is 0.1-1 mol / L.

[0036] In some embodiments of the present invention, in step (4), the copper salt includes copper halide salts, such as copper chloride and copper bromide.

[0037] In some embodiments of the present invention, in step (4), the concentration of the copper salt is 0.5-1.5 mmol / L.

[0038] In some embodiments of the present invention, the specific process of step (4) is as follows: the surface of the titanium material is cleaned, then the titanium material is added to an alkaline solution and heated, then the titanium material is taken out and ultrasonically cleaned with deionized water, then the titanium material is immersed in calcium salt, the titanium material is taken out and rinsed with distilled water, and then the titanium material is immersed in copper salt to exchange excess Na. + and OH - This yields titanium materials with calcium and copper ions on the surface.

[0039] In some embodiments of the present invention, step (4) is more specifically performed as follows: After polishing the titanium material with 400, 800, 1200 and 2000 grit sandpaper, it is ultrasonically cleaned with 100 mL of acetone, ethanol and deionized water for 15 minutes to remove surface contaminants. A 5M sodium hydroxide solution is prepared, and the cleaned titanium material is added to the 5M sodium hydroxide solution for alkaline heat treatment (80°C autoclave, 3 mL per piece, for 12 hours). After the reaction is completed, it is ultrasonically cleaned with 200 mL of deionized water for 20 minutes twice. Then it is soaked in 0.5M calcium chloride solution for 6 hours, rinsed with distilled water, and soaked in 1mM copper chloride solution for 1 hour to exchange excess Na. + and OH - This yields titanium materials with calcium and copper ions on the surface.

[0040] In some embodiments of the present invention, in step (5), PBS buffer and alendronate-modified hyaluronic acid are taken to obtain an alendronate-modified hyaluronic acid solution with a concentration of 1-3 mg / mL. Then, glucose oxidase-modified hollow manganese dioxide nanoparticles are added under stirring. The resulting mixture is coated on the surface of the titanium material containing calcium and copper ions, dried, and the titanium implant material is obtained.

[0041] In some embodiments of the present invention, the concentration of glucose oxidase-modified hollow manganese dioxide nanoparticles in the mixture is 400-500 μg / mL.

[0042] An implantable device comprising the aforementioned titanium implant material.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] The titanium implant material of this invention is obtained by alkaline heat treatment of titanium material with alkali solution, calcium salt, and copper salt to obtain titanium material with a surface rich in calcium and copper ions. Then, a coating is further constructed on the surface of the titanium material rich in calcium and copper ions. The coating includes: glucose oxidase (GOx) modified hollow manganese dioxide nanoparticles (H-MnO2) and alendronate-modified hyaluronic acid (AlnHA). This coating consumes glucose in the in vivo microenvironment through enzymatic activity and generates H2O2, which further reacts with Mn... 2+ and Cu 2+ The Fenton-mediated reaction generates ·OH, which possesses highly effective bactericidal capabilities. Simultaneously, the superoxide dismutase (SOD) activity of H-MnO2 and the antioxidant properties of hyaluronic acid scavenge excess ROS, reducing inflammation and promoting bone healing. This material is suitable for bone implant applications in patients with complex microenvironments, such as those with diabetes. The coating can be applied via Ca... 2+ It exhibits strong chelation with alendronate. The titanium implant material described in this invention combines anti-infection and bone integration promotion functions, and also has good anti-inflammatory effects. Attached Figure Description

[0045] Figure 1 Scanning electron microscope (SEM) images of the products prepared in Example 1 and Comparative Examples 1-3;

[0046] Figure 2 The images show the colony formation results on agar plates corresponding to the products prepared in Example 1 and Comparative Examples 1-3.

[0047] Figure 3 The results show the bacterial count on the agar plates corresponding to the products prepared in Example 1 and Comparative Examples 1-3.

[0048] Figure 4 The results of SOD activity tests are for the products prepared in Example 1 and Comparative Examples 1-3.

[0049] Figure 5 The results of CAT activity tests are for the products prepared in Example 1 and Comparative Examples 1-3.

[0050] Figure 6 The results of GOx activity tests are for the products prepared in Example 1 and Comparative Examples 1-3.

[0051] Figure 7 The results of GPx activity tests are for the products prepared in Example 1 and Comparative Examples 1-3.

[0052] Figure 8 The results of POD activity tests are for the products prepared in Example 1 and Comparative Examples 1-3.

[0053] Figure 9 The DPPH removal results are for the products prepared in Example 1 and Comparative Examples 1-3.

[0054] Figure 10 The staining results of tissue sections corresponding to the products prepared in Example 1, Comparative Example 1, and Comparative Example 3;

[0055] Figure 11 The results of quantitative analysis of stained inflammatory cells corresponding to the products prepared in Example 1, Comparative Example 1, and Comparative Example 3;

[0056] Figure 12 The results of SBF mineralization experiments are for the products prepared in Example 1 and Comparative Examples 1-3.

[0057] Figure 13 The results of 14-day Alizarin Red and Sirius Red staining for Ti, CaCu-Ti, and D-MAHTi are shown.

[0058] Figure 14 The results of quantitative analysis of Alizarin Red and Sirius Red staining for Ti, CaCu-Ti, and D-MAHTi at 14 days are presented. Detailed Implementation

[0059] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0060] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0061] The titanium material used below is metallic titanium sheet.

[0062] Example 1

[0063] A titanium implant material with both anti-infection and bone integration promotion functions includes a titanium material containing calcium and copper ions on its surface, and a coating that is attached to the surface of the titanium material containing calcium and copper ions.

[0064] The coating comprises glucose oxidase-modified hollow manganese dioxide nanoparticles and alendronate-modified hyaluronic acid.

[0065] A method for preparing a titanium implant material with both anti-infection and bone integration promotion functions includes the following steps:

[0066] (1) Preparation of silica NPs template by reverse microemulsion method: Specifically, Triton X-100 (5.3 mL), cyclohexane (22.5 mL) and n-hexanol (5.4 mL) were mixed and stirred for 5 minutes. Then, ammonia (0.75 mL) and water (1 mL) were quickly added and the mixture was stirred for 30 minutes. Finally, 500 μL of TEOS (tetraethyl orthosilicate) and 100 μL of LAPTES (3-aminopropyltrimethoxysilane) were mixed and added to the above solution. The mixture was stirred at room temperature for 24 hours. Silica nanoparticles were collected by centrifugation and repeated washing.

[0067] Then, using silica nanoparticles as templates, hollow manganese dioxide nanoparticles (H-MnO2 NPs) were prepared. The specific process was as follows: under ultrasonic treatment, 10 mg / mL potassium permanganate solution (150 mg) was added dropwise to a silica (20 mg) suspension (the suspension also contained water), and ultrasonic treatment was carried out continuously for 6 hours to obtain silica nanoparticles coated with a manganese dioxide shell (MnO2-SiO2 NPs). The prepared silica nanoparticles coated with a manganese dioxide shell were added to a sodium carbonate (2 M) solution at 60 °C and reacted for 12 hours to dissolve and remove the silica template. Then, the hollow manganese dioxide nanoparticles were obtained by centrifugation at 10,000 rpm and washed multiple times with pure water.

[0068] (2) 1g of hollow manganese dioxide nanoparticles were ultrasonically dispersed in 20mL of pure water and stirred continuously at 40℃. 20mL of tannic acid (TA) solution (25mg / mL) was added. The resulting mixture was reacted at 40℃ for 1 hour and the product was collected by centrifugation at 10000rpm. The product was washed 5 times with pure water and 1 time with anhydrous ethanol. Then it was dried overnight in a vacuum oven at 60℃. 5mg of lyophilized glucose oxidase (GOx) was dissolved in 2mL of phosphate buffered saline (PBS) to obtain glucose oxidase solution. 0.4g of product was added and the mixture was adjusted to a final volume of 10mL with PBS. The mixture was continuously shaken at 200rpm and reacted at 4℃ for 3 hours. Then the product was separated by centrifugation at 10000rpm using a high-speed refrigerated centrifuge. The product was washed 3 times with PBS (0.1M, pH 6.5) to remove free or weakly bound GOx and obtain glucose oxidase modified hollow manganese dioxide nanoparticles.

[0069] (3) Sodium hyaluronate (500 mg), NHS (N-hydroxysuccinimide) and EDC (300 mg) were dissolved in 100 mL PBS (pH 5.0) and stirred for 3 hours. Then, 500 mg of sodium alendronate was added and stirred for 3 days. The product was purified by dialysis bag (3500 MW) for 3 days and then lyophilized to obtain alendronate-modified hyaluronic acid (AlnHA).

[0070] (4) After polishing the titanium material with 400, 800, 1200, and 2000 grit sandpaper, ultrasonically clean it for 15 minutes with 100 mL of acetone, ethanol, and deionized water to remove surface contaminants. Prepare a 5M sodium hydroxide solution and add the cleaned titanium material to the 5M sodium hydroxide solution for alkaline heat treatment (80℃ autoclave, 3 mL per piece, for 12 hours). After the reaction is complete, ultrasonically clean it twice with 200 mL of deionized water for 20 minutes each time. Then, soak it in a 0.5M calcium chloride solution for 6 hours, rinse it with distilled water, and soak it in a 1mM copper chloride solution for 1 hour to exchange excess Na. + and OH - This yields titanium material with calcium and copper ions on its surface;

[0071] (5) Mix PBS buffer and alendronate-modified hyaluronic acid to obtain an alendronate-modified hyaluronic acid solution with a concentration of 2 mg / mL. Then, add glucose oxidase-modified hollow manganese dioxide nanoparticles under stirring. The resulting mixture (the concentration of glucose oxidase-modified hollow manganese dioxide nanoparticles in the mixture is 500 μg / mL) is coated on the surface of a titanium material containing calcium and copper ions. The mixture is then vacuum dried overnight at room temperature to obtain a titanium implant material (denoted as MAHTi).

[0072] Comparative Example 1

[0073] Comparative Example 1 is a titanium material (denoted as Ti) that was polished with 400, 800, 1200 and 2000 grit sandpaper and then ultrasonically cleaned for 15 minutes with 100 mL of acetone, ethanol and deionized water to remove surface contaminants.

[0074] Comparative Example 2

[0075] Comparative Example 2 involved polishing titanium materials with 400, 800, 1200, and 2000 grit sandpaper, followed by ultrasonic cleaning with 100 mL of acetone, ethanol, and deionized water for 15 minutes to remove surface contaminants. A 5M sodium hydroxide solution was then prepared, and the cleaned titanium materials were immersed in the solution for alkaline heat treatment (80°C autoclave, 3 mL per piece, for 12 hours). After the reaction, the materials were ultrasonically cleaned twice with 200 mL of deionized water for 20 minutes each. They were then soaked in a 0.5M calcium chloride solution for 6 hours, rinsed with distilled water, and finally soaked in a 1 mM copper chloride solution for 1 hour to exchange excess sodium.+ and OH - This yields a titanium material with calcium-copper ions on its surface (denoted as CaCu-Ti).

[0076] Comparative Example 3

[0077] Comparative Example 3 is a titanium material (denoted as AHTi) with a surface coating of alendronate-modified hyaluronic acid containing calcium and copper ions. The preparation methods of alendronate-modified hyaluronic acid and the titanium material with surface calcium and copper ions are the same as in Example 1.

[0078] Product effectiveness test

[0079] The products prepared in the above examples and comparative examples were subjected to experiments on antibacterial, multi-enzyme activity, anti-inflammatory, and osteogenic effects. Specific details are as follows.

[0080] 1. Verification of antibacterial effect

[0081] Antibacterial test: The antibacterial properties were evaluated using Staphylococcus aureus as a model bacterium. Staphylococcus aureus was cultured overnight in LB broth at 37°C with continuous shaking (200 rpm), resulting in a bacterial concentration of 102. 9 CFU / mL, store at 4°C before use. Take 30 μL of Staphylococcus aureus suspension (10 9 CFU / mL), then 10 μL of glucose solution (20 mg / mL) and 10 μL of hyaluronidase solution (2 mg / mL) were added to the surface of the products prepared in Example 1 and Comparative Examples 1-3. The light-illuminated samples (denoted as NIR+MAHTi) were subjected to NIR light (near-infrared light, 1 W / cm²). 2 Irradiate for 5 minutes, maintaining the temperature at 50℃. After incubating all groups for 1 hour, introduce 1 mL of LB broth and incubate for another 4 hours. Use ultrasound to separate bacteria adhering to the surface of each product group, and dilute the stock solution 10... 5 Then, 50 μL of bacterial suspension was placed on LB agar and incubated for 12 h. The colony formation was then digitally photographed (results are shown in Figure 1). Figure 2 (As shown). On the other hand, bacterial counts were determined using the plate count method (results shown). Figure 3 (As shown).

[0082] Figure 2 The images show the colony formation results on agar plates corresponding to the products prepared in Example 1 and Comparative Examples 1-3. Figure 3 The bacterial count results on the agar plates corresponding to the products prepared in Example 1 and Comparative Examples 1-3 are shown.

[0083] “Ti” corresponds to Comparative Example 1, “CaCu-Ti” corresponds to Comparative Example 2, “AHTi” corresponds to Comparative Example 3, “MAHTi” corresponds to Example 1, and “NIR+MAHTi” corresponds to the sample of Example 1 under NIR illumination.

[0084] Antibacterial conclusion: Compared with untreated pure Ti, CaCu-Ti, and AHTi, MAHTi can effectively inhibit the growth of Staphylococcus aureus (results are shown in Figure 1). Figure 2 (As shown). Quantitatively, compared with the three comparative examples of hollow manganese dioxide nanoparticles without glucose oxidase modification, the MAHTi-treated Staphylococcus aureus CFU count decreased by two orders of magnitude, corresponding to an antibacterial rate of 99.5% (results are shown in Figure 1). Figure 3 (As shown).

[0085] 2. Verification of multi-enzyme activity:

[0086] SOD-like enzyme activity: SOD-like enzyme activity was studied using the WST-8 riboflavin colorimetric method according to the total superoxide dismutase assay kit. In a typical analysis, WST-8 / enzyme working solution (320 μL) was mixed with the product from the examples and comparative examples. Then, the reaction initiator solution (xanthine solution, 40 μL) was added. The reaction mixture was incubated on a shaker at 37°C for 30 min, and the absorbance at 560 nm was measured and recorded as Ab. Simultaneously, a blank control group 1 (A1) was prepared using 40 μL of SOD detection buffer instead of the product, and another control group 2 (A2) was prepared using 80 μL of SOD detection buffer instead of the product and reaction initiator working solution. The absorbance was converted to the inhibition rate of the sample using the formula: Inhibition rate (%) = (A1 - A2 - Ab) / (A1 - A2) × 100%. Higher SOD activity corresponds to lower absorbance and a higher inhibition rate.

[0087] CAT enzyme (catalase) activity: CAT catalyzes the decomposition of hydrogen peroxide into water and oxygen. Changes in oxygen content in a closed solution system, detected by a dissolved oxygen meter, reflect the simulated CAT enzyme activity of the product from Example 1. Measurements were performed at room temperature. In a typical experiment, the product from the examples and comparative examples was added to 10 mL of PBS (pH 5.5 or pH 7.4), a suitable amount of vegetable oil was added to seal the surface, and then 10 μL of 30% hydrogen peroxide solution was added using a syringe. The solution concentration was recorded first, and the oxygen content in the solution (in mg / L) was recorded after 15 minutes. Higher CAT activity indicates greater oxygen release.

[0088] GOx activity: Catalyzes the decomposition of glucose to produce hydrogen peroxide and gluconic acid, a process that consumes an equal molar amount of oxygen. Changes in oxygen content in the sealed solution system were measured using a dissolved oxygen analyzer to reflect the GOx enzyme activity of MAHTi. Measurements were performed at room temperature. The products from the examples and comparative examples were added to pure water to a volume of 9.75 mL, and a suitable amount of vegetable oil was added to seal the surface. Then, 250 μL of glucose solution (2.5 mg / mL) was added using a syringe. The oxygen content in the solution (in mg / L) was recorded every 5 seconds. Higher oxygen consumption indicates stronger GOx activity.

[0089] GPx enzyme (glutathione peroxidase) activity: GPx enzyme activity was assessed using DTNB (5,5'-dithiobis(2-nitrobenzoic acid)). The products from examples and comparative studies were placed in 900 μL of Tris-HCl buffer (50 mM, pH 8) and GSH (8 mM glutamyl-cysteine) and allowed to react at room temperature. After 30 minutes of reaction, 100 μL of 100 mM DTNB solution was added. The absorbance of the mixture at 412 nm was measured to assess GSH consumption. Addition or omission of hydrogen peroxide served as positive and negative controls, respectively. Lower absorbance indicated higher GPx activity.

[0090] POD (peroxidase) activity verification: ·OH production was measured using an ESR (electron spin resonance) spectrometer. DMPO (5,5-dimethyl-1-pyrrolline-N-oxide) was used as a trap. During the experiment, MAHTi was added to a mixture of hydrogen peroxide and DMPO (100 mM), and the reaction was allowed to proceed for 5 minutes. At pH 5.5, a distinct four-peak signal with an intensity ratio of 1:2:2:1 appeared in the ESR spectrum.

[0091] Figure 4 The results are the SOD activity test results for the products prepared in Example 1 and Comparative Examples 1-3; "Negative" indicates the negative control, which is the pure water control, with no SOD activity at all; "Inh" indicates the inhibition ratio, which is the ability to inhibit the oxidation of WST-8 by superoxide anions.

[0092] Figure 5 The results are the CAT activity test results for the products prepared in Example 1 and Comparative Examples 1-3; "O2 release" indicates the amount of oxygen released.

[0093] Figure 6The results of GOx activity tests are for the products prepared in Example 1 and Comparative Examples 1-3; "add theglucose" means adding glucose, "O2 concentration" means oxygen content, and "Time" means time.

[0094] Figure 7 The results of GPx activity tests are for the products prepared in Example 1 and Comparative Examples 1-3; "Positive" indicates a positive control, "Negative" indicates a negative control, and "Intensity" indicates the strength.

[0095] Figure 8 The results of POD activity tests are for the products prepared in Example 1 and Comparative Examples 1-3. "Intensity" indicates the intensity, "Control" indicates the control group, i.e., the ESR test result of MAHTi at pH=7.4, i.e., the POD activity is weakened at pH=7.4, and "Magnetic Field" indicates the magnetic field.

[0096] Conclusions on multi-enzyme activity: Compared with other treatment groups, such as Ti, MAHTi showed significantly higher multi-enzyme activity (GPx and GOx), exhibiting POD activity under acidic conditions and SOD and CAT activity under neutral conditions. The activity of SOD and CAT also provides evidence for its anti-inflammatory ability.

[0097] 3. Verification of anti-inflammatory effects

[0098] Free radical scavenging validation: The antioxidant performance of MAHTi was evaluated using the 1,1-diphenyl-2-pyridinium hydrazine (DPPH) free radical scavenging method. A DPPH working solution in anhydrous ethanol (500 μg / mL) was prepared and thoroughly mixed with the products (Ti, CaCu-Ti, AHTi, MAHTi) of the comparative and example studies, respectively. The reaction was incubated in the dark at 37°C for a period of time. The absorbance of each reaction solution was measured at 520 nm using a multi-mode microplate reader. The absorbance values ​​of the blank solution and the test solution were recorded as Ac and Ab, respectively. The DPPH free radical scavenging rate was calculated as follows: D% = (Ab / Ac) × 100%. All experiments were repeated. Lower absorbance indicated better anti-inflammatory effect.

[0099] In vivo anti-inflammatory performance verification: BALB / c male mice (albino lab mice, 20-25g) were anesthetized and their skin prepared. Subcutaneous pockets were formed on both sides of the dorsal side, and the bacterial-contaminated samples were introduced into both sides. For bacterial inoculation, the comparative and example samples were immersed in a Staphylococcus aureus suspension (10... 7The tissue was treated with NIR (CFU / mL) for 1 hour. After implantation, the wound site was irradiated with 808 nm NIR for 5 minutes. Histological analysis was performed. Tissue around the implant was collected on postoperative days 3 and 7, stained with hematoxylin and eosin (HE), and the degree of tissue inflammation was observed.

[0100] Figure 9 The DPPH removal results are for the products prepared in Example 1 and Comparative Examples 1-3; "Absorbance" represents absorbance, and "control" represents blank solution.

[0101] Figure 10 The staining results of tissue sections corresponding to the products prepared in Example 1, Comparative Example 1, and Comparative Example 3 are shown; "3d" means 3 days and "7d" means 7 days.

[0102] Figure 11 The results of quantitative analysis of stained inflammatory cells corresponding to the products prepared in Example 1, Comparative Example 1, and Comparative Example 3 are shown; "Neutrophi percentage" represents the percentage of neutrophils.

[0103] Anti-inflammatory effect conclusion: In vitro, compared with Ti, MAHTi can effectively scavenge various reactive oxygen species that induce inflammation, such as superoxide anion (SOD activity), hydrogen peroxide (CAT activity), and DPPH free radicals. In vivo, the MAHTi group had the fewest inflammatory cells at both 3 and 7 days after implantation, and could significantly reduce the inflammatory response in mice.

[0104] 4. Verification of bone-promoting effects

[0105] SBF mineralization experiment: The bone bioactivity of the products (implant materials) prepared in Example 1 and Comparative Examples 1-3 was evaluated using a well-established simulated body fluid (SBF) model. In short, the bone interface material was immersed in an SBF solution at 37°C to simulate the in vivo mineralization process, promoting ion supersaturation and precipitation of minerals such as calcium and phosphorus on the material surface, forming minerals like hydroxyapatite, thereby evaluating the material's bioactivity and bone integration capacity.

[0106] Cell-mediated osteogenic experiment: 2x10 5 MC3T3-E1 cells were seeded on the surface of MAHTi from Example 1 after soaking in PBS solution containing 50 μL and 2 mg / mL hyaluronidase for 6 h, and on the surface of products (implant materials) prepared in Comparative Examples 1-3. The cells were cultured until they were completely covered, then replaced with osteogenic induction medium, with the medium changed every 3 days. After 14 days of culture, Alizarin Red and Sirius Red staining was performed to reflect calcium nodules and collagen deposition.

[0107] Figure 12The results of SBF mineralization experiments are for the products prepared in Example 1 and Comparative Examples 1-3; "DAY" means day, and "Atomic ratio" means atomic ratio.

[0108] Figure 13 The results of 14-day staining with Alizarin Red and Sirius Red for Ti, CaCu-Ti, and D-MAHTi are shown; "PicroSirius Red" represents Sirius Red, and "Alizarin Red S" represents Alizarin Red.

[0109] Figure 14 The results of 14-day alizarin red and Sirius red staining for Ti, CaCu-Ti, and D-MAHTi are presented. "Intensity" indicates the intensity.

[0110] Conclusion on promoting bone formation: SBF experiments showed that after 3 days of immersion, the MAHTi group had formed hemispherical apatite aggregates (results as shown in the figure). Figure 12 (As shown). After 7 days of immersion induction, the surfaces of AHTi and MAHTi were completely covered with apatite-like aggregates. The hemispherical aggregates on the MAHTi surface gradually transformed into more stable aggregates, while no mineral particles were observed on the unmodified Ti implants. Elemental analysis showed that Ca and P were mainly distributed on the MAHTi surface, with Ca and P contents of 13.03% and 13.2%, respectively. In contrast, only trace amounts of calcium (0.33%) were detected on the Ti surface. In the cell-mediated osteogenic assay, D-MAHTi (D-MAHTi refers to MAHTi material soaked in PBS solution containing 50 μL, 2 mg / mL hyaluronidase for 6 h) showed significant calcium deposition and collagen formation at 14 days (results are shown in Figure 1). Figure 13 , 14 (as shown), while the other two groups have relatively fewer.

[0111] It should be noted that the above embodiments of the present invention are merely demonstrations of a specific technical solution and do not constitute a limitation on the scope of protection of the present invention. Based on Embodiment 1 and within the scope of protection claimed by the present invention, by changing the process parameters in the preparation process of titanium implant materials, such as changing the amount, concentration, temperature, etc. of raw material components, products with similar effects to Embodiment 1 can also be obtained.

Claims

1. A titanium implant material, characterized in that, The invention includes a titanium material containing calcium and copper ions on its surface, and a coating thereof, the coating being adhered to the surface of the titanium material containing calcium and copper ions. The coating comprises glucose oxidase-modified hollow manganese dioxide nanoparticles and alendronate-modified hyaluronic acid.

2. The titanium implant material according to claim 1, characterized in that, The glucose oxidase-modified hollow manganese dioxide nanoparticles also contain tannic acid structures.

3. The titanium implant material according to claim 1, characterized in that, The alendronate-modified hyaluronic acid is formed by grafting 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide via a coupling reaction.

4. The titanium implant material according to claim 1, characterized in that, In the coating, the mass ratio of alendronate-modified hyaluronic acid to glucose oxidase-modified hollow manganese dioxide nanoparticles is (1-3) mg: (400-500) μg.

5. The method for preparing the titanium implant material according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Using silica nanoparticles as templates, potassium permanganate solution was dropped into silica suspension by ultrasonic treatment, and sodium carbonate solution was added for etching to obtain hollow manganese dioxide nanoparticles. (2) Tannic acid and glucose oxidase were reacted with hollow manganese dioxide nanoparticles to prepare glucose oxidase-modified hollow manganese dioxide nanoparticles. (3) Alendronate was grafted onto hyaluronic acid via a coupling reaction using 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to form alendronate-modified hyaluronic acid. (4) Add the titanium material to the alkaline solution and heat it to obtain the treated titanium material. Then, immerse the titanium material in calcium salt and copper salt in sequence to obtain titanium material with calcium and copper ions on the surface. (5) The glucose oxidase-modified hollow manganese dioxide nanoparticles are mixed with the alendronate-modified hyaluronic acid, coated on the surface of the titanium material containing calcium and copper ions, and dried to obtain the titanium implant material.

6. The preparation method according to claim 5, characterized in that, The mass ratio of tannic acid, glucose oxidase and hollow manganese dioxide nanoparticles is (20-40) mg: (2-10) mg: 1 g.

7. The preparation method according to claim 5, characterized in that, The specific process of step (3) is as follows: Sodium hyaluronate, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are dissolved in PBS and stirred. Then, sodium alendronate is added and stirred. The resulting product is purified by dialysis and then lyophilized to obtain alendronate-modified hyaluronic acid.

8. The preparation method according to claim 5, characterized in that, In step (4), the alkaline solution includes a sodium hydroxide solution or a potassium hydroxide solution; and / or, the concentration of the alkaline solution is 4-7 mol / L; and / or, the heating temperature is 75-85℃ and the heating time is 10-12 hours; and / or, the calcium salt includes a calcium halide; and / or, the concentration of the calcium salt is 0.1-1 mol / L; and / or, the copper salt includes a copper halide; and / or, the concentration of the copper salt is 0.5-1.5 mmol / L.

9. The preparation method according to claim 5, characterized in that, The specific process of step (4) is as follows: clean the surface of the titanium material, then add the titanium material to the alkaline solution for heating, then take out the titanium material and clean it with deionized water using ultrasound, then soak the titanium material in calcium salt, take out the titanium material, rinse it with distilled water, and continue to soak the titanium material in copper salt to obtain titanium material with calcium and copper ions on the surface.

10. An implantable device, characterized in that, Includes the titanium implant material as described in any one of claims 1-4.

Images