Titanium implant material with functions of resisting infection and promoting synostosis and preparation method of titanium implant material

Abstract

The invention belongs to the technical field of medical materials, and discloses a titanium implant material with functions of resisting infection and promoting synostosis and a preparation method of the titanium implant material. The titanium implant material comprises a titanium material with the surface containing calcium and copper ions and a coating, and the coating is attached to the surface of the titanium material with the surface containing calcium and copper ions. The coating comprises hollow manganese dioxide nanoparticles modified by glucose oxidase and hyaluronic acid modified by alendronate. The titanium implant material disclosed by the invention has the functions of resisting infection and promoting synostosis, and also has a good anti-inflammatory effect.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials, and particularly relates to a titanium implant material having both anti-infection and bone-binding promoting functions and a preparation method thereof. Background Art

[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 the functions of anti-infection, anti-inflammatory and promoting bone integration, especially in the complex microenvironment of diabetic patients (such as high blood sugar, rich reactive oxygen species ROS, persistent inflammation and other complex microenvironments), implants are prone to infection or poor bone integration. In the prior art, although there is a coating technology to form a protective layer on the surface of titanium implants, most coatings have a single function and fail to simultaneously achieve multiple functions such as anti-infection, anti-oxidation and promoting bone healing.

[0003] Therefore, there is an urgent need to provide a titanium implant material that has both anti-infection and bone-binding promoting functions. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a titanium implant material having both anti-infection and bone-binding promoting functions and a preparation method thereof. The titanium implant material of the present invention has both anti-infection and bone-binding promoting functions and also has good anti-inflammatory effects.

[0005] The titanium implant material of the present invention is subjected to alkaline heat treatment of titanium material by alkali solution, calcium salt and copper salt to obtain titanium material with a surface rich in calcium and copper ions, and then a coating is further constructed on the surface of the titanium material with a surface rich in calcium and copper ions, and the coating comprises: hollow manganese dioxide nanoparticles (H-MnO 2 ) and alendronate-modified hyaluronic acid (AlnHA). The coating consumes glucose in the microenvironment in the body through enzymatic activity and generates H 2 O 2 , and further with Mn 2+ and Cu 2+ The mediated Fenton reaction produces ·OH, which has high bactericidal ability. 2 The superoxide dismutase (SOD) activity of the coating and the antioxidant properties of hyaluronic acid can remove excess ROS, reduce inflammation and promote bone healing. The material is suitable for bone implant applications with complex microenvironments such as diabetic patients. The coating can be Ca 2+ Tightly binds to the chelation effect of alendronate.

[0006] In addition, due to the glucose oxidase (GOx) modified hollow manganese dioxide nanoparticles (H-MnO 2), the coating has multi-enzyme activity, the coating has degradable function, and the nanoparticles can be degraded under acidic conditions.

[0007] A first aspect of the present invention provides a titanium implant material having both anti-infection and bone-binding promoting functions.

[0008] Specifically, a titanium implant material having both anti-infection and bone-binding promoting functions comprises a titanium material having calcium and copper ions on its surface, and a coating, wherein the coating is attached to the surface of the titanium material having calcium and copper ions on its surface;

[0009] The coating includes 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-MnO 2 ), as an "armored enzyme", provides multi-enzyme activity and has antibacterial, antioxidant and bone-binding promoting functions.

[0012] Alendronate-modified hyaluronic acid (AlnHA), as a matrix carrier, enhances the bonding 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 further contain a tannic acid (TA) structure.

[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) through a coupling reaction.

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

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

[0017] In some embodiments of the present invention, the titanium material is titanium material in various shapes such as titanium sheets and titanium rods.

[0018] The second aspect of the present invention provides a method for preparing a titanium implant material having both anti-infection and bone-binding promoting functions.

[0019] Specifically, a method for preparing a titanium implant material having both anti-infection and bone-binding promoting functions comprises the following steps:

[0020] (1) Using silica nanoparticles (NPs) as templates, dropping potassium permanganate solution into a silica suspension by ultrasonic treatment, and then adding sodium carbonate solution for etching to prepare 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-MnO 2 -GOx);

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

[0023] (4) adding the titanium material to an alkaline solution and heating it to obtain a treated titanium material, and then immersing the titanium material in a calcium salt and a copper salt in sequence to obtain a titanium material containing calcium and copper ions on the surface;

[0024] (5) Mixing the glucose oxidase-modified hollow manganese dioxide nanoparticles with the alendronate-modified hyaluronic acid, coating the mixture on the surface of the titanium material containing calcium and copper ions, and drying the mixture 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: ultrasonically dispersing the hollow manganese dioxide nanoparticles in pure water, continuously stirring, adding tannic acid (TA) solution, reacting the obtained mixture at 35-40°C for 0.5-1 hour, and collecting the product by centrifugation, washing the product with pure water and anhydrous ethanol, and then drying overnight, dissolving glucose oxidase (GOx) in phosphate buffered saline to obtain a glucose oxidase solution, adding the product, reacting at 0-°C for 2-3 hours, and then centrifuging the product to obtain glucose oxidase-modified hollow manganese dioxide nanoparticles (H-MnO 2 -GOx).

[0027] In some embodiments of the present invention, a more specific process of step (2) is as follows: 1 g of hollow manganese dioxide nanoparticles is ultrasonically dispersed in 20 mL of pure water, stirred continuously at 40° C., 20 mL of tannic acid (TA) solution (25 mg / mL) is added, the resulting mixture is reacted at 40° C. for 1 hour, and the product is collected by centrifugation at 10,000 rpm, the product is washed 5 times with pure water, washed once with anhydrous ethanol, and then dried in a vacuum oven at 60° C. overnight, 5 mg of freeze-dried glucose oxidase (GOx) is dissolved in 2 mL of phosphate buffered saline (PBS) to obtain a glucose oxidase solution, 0.4 g of the product is added, the mixture is adjusted to a final volume of 10 mL with PBS, and the mixture is continuously shaken at 200 rpm and reacted at 4° C. for 3 hours, and then the product is separated by centrifugation at 10,000 rpm in a high-speed refrigerated centrifuge, and washed 3 times with PBS (0.1 M, pH 6.5) to remove free or weakly bound GOx to obtain glucose oxidase-modified hollow manganese dioxide nanoparticles.

[0028] In some embodiments of the present invention, the specific process of step (3) is: dissolving sodium hyaluronate, NHS (N-hydroxysuccinimide) and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) in PBS, stirring, then adding sodium alendronate, stirring, and purifying the obtained product with a dialysis bag, and then freeze-drying 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, a more specific process of step (3) is 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 obtained product is purified by a dialysis bag (3500 MW) for 3 days and then freeze-dried to obtain alendronate-modified hyaluronic acid (AlnHA).

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

[0032] In some embodiments of the present invention, in step (4), the concentration of the alkali 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 a halide salt of calcium, such as calcium chloride or 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 a copper halide salt, such as copper chloride or 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: cleaning the surface of the titanium material, then adding the titanium material to an alkali solution for heating, then taking out the titanium material and ultrasonically cleaning it with deionized water, then soaking the titanium material in calcium salt, taking out the titanium material, rinsing it with distilled water, and continuing to soak the titanium material in copper salt to exchange excess Na + and OH - , obtaining a titanium material with calcium and copper ions on the surface.

[0039] In some embodiments of the present invention, a more specific process of step (4) is as follows: after polishing the titanium material with 400, 800, 1200 and 2000 sandpaper, ultrasonic cleaning is performed 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, ultrasonic cleaning is performed with 200 mL of deionized water for 20 minutes, twice, and then immersed in a 0.5M calcium chloride solution for 6 hours, rinsed with distilled water, and immersed in a 1mM copper chloride solution for 1 hour to exchange excess Na + and OH - , obtaining a titanium material 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, and then hollow manganese dioxide nanoparticles modified with glucose oxidase are added under stirring. The obtained mixture is coated on the surface of the titanium material containing calcium and copper ions on the surface, and dried to obtain the titanium implant material.

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

[0042] A device implanted in the body comprises the 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 the present invention is subjected to alkaline heat treatment of titanium material by alkali solution, calcium salt and copper salt to obtain titanium material with a surface rich in calcium and copper ions, and then a coating is further constructed on the surface of the titanium material with a surface rich in calcium and copper ions, and the coating comprises: hollow manganese dioxide nanoparticles (H-MnO 2 ) and alendronate-modified hyaluronic acid (AlnHA). The coating consumes glucose in the microenvironment in the body through enzymatic activity and generates H 2 O 2 , and further with Mn 2+ and Cu 2+ The mediated Fenton reaction produces ·OH, which has high bactericidal ability. 2 The superoxide dismutase (SOD) activity of the coating and the antioxidant properties of hyaluronic acid can remove excess ROS, reduce inflammation and promote bone healing. The material is suitable for bone implant applications with complex microenvironments such as diabetic patients. The coating can be Ca 2+ The titanium implant material of the present invention has the functions of anti-infection and promoting bone bonding, and also has good anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The scanning electron microscope images of the products prepared in Example 1 and Comparative Examples 1-3;

[0046] Figure 2 The results of photographing the colonies on the agar plates corresponding to the products prepared in Example 1 and Comparative Examples 1-3;

[0047] Figure 3 The statistical results of the number of bacteria on the agar plates corresponding to the products prepared in Example 1 and Comparative Examples 1-3;

[0048] Figure 4 The SOD activity test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3;

[0049] Figure 5 The CAT activity test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3;

[0050] Figure 6The GOx activity test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3;

[0051] Figure 7 The GPx activity test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3;

[0052] Figure 8 The POD activity test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3;

[0053] Fig. 9 The DPPH removal results corresponding to the products prepared in Example 1 and Comparative Examples 1-3;

[0054] Fig.10 The tissue section staining results corresponding to the products prepared in Example 1, Comparative Example 1 and Comparative Example 3;

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

[0056] Fig.12 The SBF mineralization test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3;

[0057] Fig.13 The 14-day alizarin red and Sirius red staining results of Ti, CaCu-Ti, and D-MAHTi are shown;

[0058] Fig.14 These are the quantitative analysis results of Alizarin red and Sirius red staining corresponding to Ti, CaCu-Ti, and D-MAHTi after 14 days. DETAILED DESCRIPTION

[0059] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

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

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

[0062] Example 1

[0063] A titanium implant material with both anti-infection and bone-binding promoting functions, comprising a titanium material containing calcium and copper ions on the surface, and a coating, wherein the coating is attached to the surface of the titanium material containing calcium and copper ions on the surface;

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

[0065] A method for preparing a titanium implant material having both anti-infection and bone-binding promoting functions comprises the following steps:

[0066] (1) The silica NPs template was prepared by the 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 mixed solution was stirred for 30 minutes. Finally, 500 μL of TEOS (tetraethyl orthosilicate) and 100 μL of APTES (3-aminopropyltrimethoxysilane) were mixed and added to the above solution, stirred at room temperature for 24 hours, and the silica nanoparticles were collected by centrifugation and repeated washing;

[0067] Then, hollow manganese dioxide nanoparticles (H-MnO 2 NPs), the specific process is: under ultrasonic treatment, 10 mg / mL potassium permanganate solution (150 mg) was dropped into a silica (20 mg) suspension (the suspension also contained water), and the ultrasonic treatment was continued for 6 hours to obtain silica nanoparticles coated with manganese dioxide shell (MnO 2 -SiO 2 NPs), adding the prepared silica nanoparticles coated with manganese dioxide shells into a 60°C sodium carbonate (2M) solution, reacting for 12 hours to dissolve and remove the silica template, and then centrifuging at 10000 rpm to obtain hollow manganese dioxide nanoparticles, which were washed with pure water for multiple times;

[0068] (2) 1 g of hollow manganese dioxide nanoparticles was ultrasonically dispersed in 20 mL of pure water, stirred continuously at 40°C, and 20 mL of tannic acid (TA) solution (25 mg / mL) was added. The resulting mixture was reacted at 40°C for 1 hour, and the product was collected by centrifugation at 10,000 rpm. The product was washed 5 times with pure water, washed once with anhydrous ethanol, and then dried in a vacuum oven at 60°C overnight. 5 mg of freeze-dried glucose oxidase (GOx) was dissolved in 2 mL of phosphate buffered saline (PBS) to obtain a glucose oxidase solution, 0.4 g of the product was added, and the mixture was adjusted to a final volume of 10 mL with PBS, and the mixture was continuously shaken at 200 rpm and reacted at 4°C for 3 hours. The product was then separated by centrifugation at 10,000 rpm in a high-speed refrigerated centrifuge, and washed 3 times with PBS (0.1 M, pH 6.5) to remove free or weakly bound GOx, thereby obtaining 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 alendronate sodium was added and stirred for 3 days. The obtained product was purified with a dialysis bag (3500 MW) for 3 days and then freeze-dried to obtain alendronate-modified hyaluronic acid (AlnHA);

[0070] (4) After polishing the titanium material with 400, 800, 1200, and 2000 sandpaper, it was ultrasonically cleaned with 100 mL of acetone, ethanol, and deionized water for 15 minutes to remove surface contaminants, and a 5 M sodium hydroxide solution was prepared. The cleaned titanium material was added to the 5 M sodium hydroxide solution for alkaline heat treatment (80 ° C autoclave, 3 mL per piece, for 12 hours). After the reaction was completed, it was ultrasonically cleaned with 200 mL of deionized water for 20 minutes, twice, and then immersed in 0.5 M calcium chloride solution for 6 hours, rinsed with distilled water, and immersed in 1 mM copper chloride solution for 1 hour to exchange excess Na + and OH - , obtaining a titanium material with calcium and copper ions on the surface;

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

[0072] Comparative Example 1

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

[0074] Comparative Example 2

[0075] Comparative Example 2 is to polish the titanium material with 400, 800, 1200 and 2000 sandpaper, and then use 100mL acetone, ethanol and deionized water for ultrasonic cleaning for 15 minutes to remove surface contaminants, prepare 5M sodium hydroxide solution, add the cleaned titanium material to the 5M sodium hydroxide solution for alkaline heat treatment (80℃ autoclave, 3mL per piece, for 12 hours), after the reaction is completed, use 200mL deionized water for ultrasonic cleaning for 20 minutes, 2 times, and then soak in 0.5M calcium chloride solution for 6 hours, rinse with distilled water, and soak in 1mM copper chloride solution for 1 hour to exchange excess Na+ and OH - , a titanium material containing calcium and copper ions on the surface (denoted as CaCu-Ti) is obtained.

[0076] Comparative Example 3

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

[0078] Product effect testing

[0079] The products prepared in the above examples and comparative examples were tested for antibacterial, multi-enzyme activity, anti-inflammatory and osteogenic effects. The specific contents are as follows.

[0080] 1. Antibacterial effect verification

[0081] Antibacterial experiment: Staphylococcus aureus was used as a model bacterium to evaluate its antibacterial properties. Staphylococcus aureus was cultured in LB broth at 37°C with continuous shaking (200 rpm) overnight, and the bacterial concentration was 10 9 CFU / mL, stored at 4°C before use. Take 30 μL of Staphylococcus aureus suspension (10 9 CFU / mL), and 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 samples in the illumination group (referred to as NIR+MAHTi) were illuminated with NIR light (near infrared light, 1 W / cm 2 ) for 5 min, and the temperature was maintained at 50°C. After incubation for 1 h in all groups, 1 mL of LB broth was introduced and cultured for another 4 h. Ultrasonic separation of bacteria adhering to the surface of each group of products was performed, and the stock solution was diluted 10 5 After that, 50 μL of bacterial suspension was placed on LB agar for 12 h, and digital photos of colony formation were taken (the results are shown in Figure 2 On the other hand, the bacterial count was determined by the plate count method (the results are shown in Figure 3 shown).

[0082] Figure 2 The results of photographing the colonies on the agar plates corresponding to the products prepared in Example 1 and Comparative Examples 1-3; Figure 3 The results of bacterial count 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 illumination group sample of the product of example 1 under NIR.

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

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

[0086] SOD enzyme activity: According to the total superoxide dismutase detection kit, the WST-8 riboflavin colorimetric method was used to study the SOD-like enzyme activity. In a typical analysis, the WST-8 / enzyme working solution (320 μL) was mixed with the products of the embodiments 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 minutes, and the absorbance at 560nm was measured and recorded as Ab. At the same time, 40 μL SOD detection buffer was used to replace the product to prepare a blank control group 1 (A1), and 80 μL SOD detection buffer was used to replace the product and the reaction initiation working solution to prepare another control group 2 (A2). The absorbance was converted to the inhibition rate of the sample by the formula: inhibition rate (%) = (A1-A2-Ab) / (A1-A2) × 100%. The higher the SOD activity, the lower the absorbance and the higher the inhibition rate.

[0087] CAT enzyme (catalase) activity: CAT can catalyze the decomposition of hydrogen peroxide into water and oxygen. The change in oxygen content in the closed solution system detected by the dissolved oxygen meter reflects the simulated CAT enzyme activity of the product of Example 1. The measurement is carried out at room temperature. In a typical experiment, the products of the examples and comparative examples are added to 10mL PBS (pH=5.5 or pH=7.4), an appropriate amount of vegetable oil is dripped into the sealing surface, and then 10μL of 30% mass fraction hydrogen peroxide solution is added with a syringe. First, the solution content is recorded, and after 15 minutes, the oxygen content in the solution is recorded (unit: mg / L). The higher the CAT activity, the more oxygen is released.

[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. The change in oxygen content in the closed solution system is detected by a dissolved oxygen meter to reflect the GOx enzyme activity of MAHTi. The measurement was carried out at room temperature. The products of the embodiments and comparative examples were added to pure water to a volume of 9.75 mL, and an appropriate amount of vegetable oil was dropped to seal the surface. Then, 250 μL of glucose solution (2.5 mg / mL) was added with a syringe. The oxygen content in the solution was recorded every 5 seconds (unit: mg / L). The higher the oxygen consumption, the stronger the GOx activity.

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

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

[0091] Figure 4 These are the SOD activity test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3; "Negative" represents the negative control, i.e., the pure water control, which has no SOD activity at all; and "Inh" represents the inhibition ratio, i.e., the ability to inhibit superoxide anion from oxidizing WST-8.

[0092] Figure 5 The CAT activity test results of the products prepared in Example 1 and Comparative Examples 1-3 are as follows; 2 "Release" indicates the amount of oxygen released.

[0093] Figure 6 The GOx activity test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3; "add the glucose" means adding glucose, "2 "Concentration" means oxygen content, and "Time" means time.

[0094] Figure 7 These are the GPx activity test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3; "Positive" represents the positive control, "Negative" represents the negative control, and "Intensity" represents the intensity.

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

[0096] Conclusion of multi-enzyme activity: Compared with other treatment groups, such as Ti, MAHTi showed obvious multi-enzyme activity (GPx and GOx), including POD activity under acidic conditions and SOD, CAT, etc. under neutral conditions. Among them, SOD and CAT activities also provide evidence for its anti-inflammatory ability.

[0097] 3. Verification of anti-inflammatory effect

[0098] Free radical scavenging verification: The antioxidant property of MAHTi was evaluated by 1,1-diphenyl-2-pyridine hydrazine (DPPH) free radical scavenging method. A DPPH anhydrous ethanol (500 μg / mL) working solution was prepared and thoroughly mixed with the products of the comparative example and the embodiment (Ti, CaCu-Ti, AHTi, MAHTi), 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 a wavelength of 520 nm using a multifunctional microplate reader. The absorbance values ​​of the blank solution and the test solution were recorded as Ac and Ab, respectively. The calculation formula for the DPPH free radical scavenging rate is as follows: D% = (Ab / Ac) × 100%. All experiments were repeated. The lower the absorbance, the better the anti-inflammatory effect.

[0099] In vivo anti-inflammatory performance verification: BALB / C male mice (albino laboratory mice, 20-25 g) were anesthetized and skinned, and subcutaneous pockets were formed on both sides of the back, and the samples contaminated with bacteria were introduced into both sides. When inoculating bacteria, the comparative example and the example were soaked in a Staphylococcus aureus suspension (10 7 CFU / mL) for 1h. After implantation, the wound site was irradiated with 808nm NIR for 5min. For histological analysis, the tissue around the implant was collected on the 3rd and 7th day after surgery and stained with hematoxylin and eosin (HE) to observe tissue inflammation.

[0100] Fig. 9 These are the DPPH removal results corresponding to the products prepared in Example 1 and Comparative Examples 1-3; “Absorbance” indicates absorbance, and “control” indicates blank solution.

[0101] Fig.10 These are the tissue section staining results corresponding to the products prepared in Example 1, Comparative Example 1, and Comparative Example 3; "3d" means 3 days, and "7d" means 7 days.

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

[0103] Conclusion of anti-inflammatory effect: In vitro, compared with Ti, MAHTi can efficiently remove superoxide anions (SOD activity), hydrogen peroxide (CAT activity), DPPH free radicals and other reactive oxygen species that cause inflammation. In vivo, the number of inflammatory cells in the MAHTi group was the least at 3 or 7 days of implantation, which can significantly slow down the inflammatory response of mice.

[0104] 4. Verification of osteogenic effect

[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 mature simulated body fluid (SBF) model. Briefly, the bone interface material was immersed in SBF solution at 37°C to simulate the in vivo mineralization process, promoting the supersaturation of ions and precipitation of calcium and phosphorus to form minerals such as hydroxyapatite on the surface of the material, thereby evaluating the bioactivity and bone integration ability of the material.

[0106] Cell-induced osteogenic assay: 2x10 5 MC3T3-E1 cells were inoculated on the surface of the MAHTi material of Example 1 after being immersed in 50 μL, 2 mg / mL hyaluronidase PBS solution for 6 h, and on the surface of the product (implant material) prepared in Comparative Examples 1-3, and cultured until completely confluent. The medium was then replaced with osteogenic induction medium, and the medium was changed every 3 days for 14 days. Alizarin red and Sirius red staining were performed to reflect calcium nodules and collagen deposition.

[0107] Fig.12 These are the SBF mineralization test results corresponding to the products prepared in Example 1 and Comparative Examples 1-3; "DAY" means day, and "Atomic ratio" means atomic ratio.

[0108] Fig.13These are the 14-day Alizarin Red and Sirius Red staining results corresponding to Ti, CaCu-Ti, and D-MAHTi; “PicroSirius Red” means Sirius Red, and “Alizarin Red S” means Alizarin Red.

[0109] Fig.14 The quantitative analysis results of Alizarin Red and Sirius Red staining for Ti, CaCu-Ti, and D-MAHTi after 14 days. “Intensity” indicates intensity.

[0110] Conclusion of osteogenic effect: In the SBF experiment, after 3 days of immersion, hemispherical apatite aggregates have been formed in the MAHTi group (results are shown in Fig.12 As shown). After 7 days of immersion induction, the surfaces of AHTi and MAHTi were completely covered with apatite-like aggregates, and the hemispherical aggregates on the surface of MAHTi 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 surface of MAHTi, with the contents of Ca and P being 13.03% and 13.2%, respectively. In contrast, only trace amounts of calcium (0.33%) were detected on the Ti surface. In the cell-induced osteogenic experiment, D-MAHTi (D-MAHTi refers to the material after MAHTi was immersed in a PBS solution containing 50 μL, 2 mg / mL hyaluronidase for 6 hours) showed obvious calcium deposition and collagen formation at 14 days (the results are shown in Fig.13 , 14 The remaining two groups were relatively small.

[0111] It should be pointed out that the above-mentioned embodiment of the present invention is only a demonstration of a specific technical solution and does not limit the scope of protection of the present invention. On the basis of Example 1 and within the scope of protection requested by the present invention, by changing the process parameters in the preparation process of the titanium implant material, such as changing the amount, concentration, temperature, etc. of the raw material components, a product with similar effects to Example 1 can also be produced.

Claims

1. A titanium implant material, characterized in that: It includes a titanium material with calcium copper ions on the surface, and a coating, wherein the coating is attached to the surface of the titanium material with calcium copper ions on the surface; The coating includes 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 through a coupling reaction.

4. The titanium implant material according to claim 1, characterized in that: In the coating, the mass ratio of the alendronate-modified hyaluronic acid to the 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 to 4, characterized in that: The following steps are involved: (1) Using silica nanoparticles as templates, dropping potassium permanganate solution into a silica suspension by ultrasonic treatment, and adding sodium carbonate solution for etching to prepare hollow manganese dioxide nanoparticles; (2) reacting tannic acid and glucose oxidase with hollow manganese dioxide nanoparticles to prepare hollow manganese dioxide nanoparticles modified with glucose oxidase; (3) using 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to graft alendronate onto hyaluronic acid through a coupling reaction to form alendronate-modified hyaluronic acid; (4) adding the titanium material to an alkaline solution and heating it to obtain a treated titanium material, and then immersing the titanium material in a calcium salt and a copper salt in sequence to obtain a titanium material containing calcium and copper ions on the surface; (5) Mixing the glucose oxidase-modified hollow manganese dioxide nanoparticles with the alendronate-modified hyaluronic acid, coating the mixture on the surface of the titanium material containing calcium and copper ions, and drying the mixture to obtain the titanium implant material.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the 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: dissolving sodium hyaluronate, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in PBS, stirring, then adding sodium alendronate, stirring, purifying the obtained product with a dialysis bag, and then freeze-drying to obtain alendronate-modified hyaluronic acid.

8. The preparation method according to claim 5, characterized in that: In step (4), the alkali solution includes sodium hydroxide solution or potassium hydroxide solution; and / or the concentration of the alkali solution is 4-7 mol / L; and / or the heating temperature is 75-85° C., and the heating time is 10-12 hours; and / or the calcium salt includes a halide of calcium; and / or the concentration of the calcium salt is 0.1-1 mol / L; and / or the copper salt includes a halide of copper; 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: cleaning the surface of the titanium material, then adding the titanium material to an alkaline solution for heating, then taking out the titanium material and ultrasonically cleaning it with deionized water, then immersing the titanium material in calcium salt, taking out the titanium material, rinsing it with distilled water, and continuing to immerse the titanium material in copper salt to obtain a titanium material containing calcium and copper ions on the surface.

10. A device implanted in the body, characterized in that: The titanium implant material comprises the titanium implant material according to any one of claims 1 to 4.

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