A modified metal, a preparation method thereof and an application thereof

Abstract

The present invention relates to a modified metal, a preparation method thereof and an application. The modified metal comprises a metal and a film layer located on the surface of the metal. Among them, the metal comprises one or more of titanium, tantalum, and magnesium, and the film layer comprises a statin compound, and the statin compound comprises one or more of simvastatin, pravastatin, atorvastatin, and rosuvastatin. The modified metal of the present invention has good antibacterial properties and can effectively avoid the infection of the orthopedic implants produced.

Description

Technical Field

[0001] The present invention relates to a modified metal, and in particular to a modified metal that can be used for implants in medical devices. Background Art

[0002] Currently, almost all orthopedic surgeries use implants, and the absolute number of implant infections is extremely large. Taking joint replacement as an example, there are up to 800,000 joint replacement surgeries in the UK and the US every year, and it will exceed 4 million in 2030. The annual medical costs increased due to implant infections in the US are as high as $4 billion. Although there is a lack of statistical data in China, as a large populated country, the usage amount of implants is extremely large.

[0003] The infection risk after fracture internal fixation is as high as 0.4 - 16.1%, and even for elective surgeries, the prosthesis infection risk after joint replacement is as high as 0.3 - 9%. Therefore, the problem of postoperative concurrent infection of implants is not optimistic. Implant infection is a catastrophic complication in orthopedic surgeries. Once an infection occurs, the treatment is extremely difficult. In severe cases, it is necessary to remove the internal fixation and thoroughly debride to control the infection, causing great pain and heavy economic burden to the patients. And even after removing the internal fixation and thoroughly debriding, it may still develop into chronic osteomyelitis that does not heal for a long time or even affect life. After joint prosthesis infection, about half of the patients are ineffective in conservative treatment and often need secondary revision. In severe cases, joint fusion, amputation, or even death may occur. Summary of the Invention

[0004] In view of the above analysis, an embodiment of the present invention aims to provide a modified metal to solve the problem that existing orthopedic implants are prone to infection.

[0005] On the one hand, an embodiment of the present invention provides a modified metal, including a metal and a film layer on the surface of the metal. Among them, the metal includes one or more of titanium, tantalum, and magnesium, and the film layer includes statin compounds, and the statin compounds include one or more of simvastatin, pravastatin, atorvastatin, and rosuvastatin.

[0006] According to an embodiment of the present invention, the metal is titanium, a titanium alloy, tantalum, a tantalum alloy, magnesium, or a magnesium alloy.

[0007] According to an embodiment of the present invention, the film layer includes hydroxyapatite and the statin compounds.

[0008] On the other hand, an embodiment of the present invention provides a preparation method of a modified metal, including: forming a film layer on the surface of the metal through an electrochemical deposition process; wherein, the metal includes one or more of titanium, tantalum, and magnesium, and the film layer includes statin compounds, and the statin compounds include one or more of simvastatin, pravastatin, atorvastatin, and rosuvastatin.

[0009] According to an embodiment of the present invention, the method for preparing the modified metal includes:

[0010] Polishing the metal;

[0011] Using the metal as the cathode and an electrolyte solution containing the statin compound for electrochemical deposition to obtain the modified metal.

[0012] According to an embodiment of the present invention, the electrolyte solution contains calcium nitrate and ammonium dihydrogen phosphate.

[0013] According to an embodiment of the present invention, in the electrolyte solution, the concentration of calcium nitrate is 0.5 - 0.7 mmol / L, and the concentration of ammonium dihydrogen phosphate is 0.3 - 0.42 mmol / L.

[0014] According to an embodiment of the present invention, in the electrolyte solution, the concentration C of the statin compound is: 0 < C ≤ 10 -3 mol / L.

[0015] An embodiment of the present invention further provides an orthopedic implant, including any of the above-mentioned modified metals or the modified metal prepared by any of the above-mentioned methods.

[0016] An embodiment of the present invention further provides the application of statin compounds in the preparation of orthopedic implants for preventing infection, wherein the statin compounds include one or more of simvastatin, pravastatin, atorvastatin, and rosuvastatin.

[0017] The modified metal of an embodiment of the present invention has good antibacterial properties and can effectively avoid the infection of the orthopedic implants made.

[0018] In the present invention, the above technical solutions can also be combined with each other to achieve more preferred combination schemes. Other features and advantages of the present invention will be described in the subsequent specification, and some advantages can be made obvious from the specification or understood by implementing the present invention. The objectives and other advantages of the present invention can be realized and obtained from the content specifically pointed out in the specification and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings are only for the purpose of showing specific embodiments and are not considered to be a limitation of the present invention. Among them:

[0020] Figure 1A It is a laser confocal microscope photograph after live / dead cell staining of Staphylococcus aureus in Test 2 of Example 1;

[0021] Figure 1BLaser confocal microscopy photograph after live / dead cell staining of Staphylococcus epidermidis in Test 2 of Example 1;

[0022] Figure 2A SEM image of the titanium alloy in the control group of Example 2;

[0023] Figure 2B SEM image of the modified titanium alloy in the hydroxyapatite group of Example 2;

[0024] Figure 2C SEM image of the modified titanium alloy in the low-concentration drug group of Example 2;

[0025] Figure 2D SEM image of the modified titanium alloy in the high-concentration drug group of Example 2;

[0026] Figure 3A XRD pattern of the titanium alloy in the control group of Example 2;

[0027] Figure 3B XRD pattern of the modified alloy in the hydroxyapatite group of Example 2;

[0028] Figure 3C XRD pattern of the modified alloy in the low-concentration drug group of Example 2;

[0029] Figure 3D XRD pattern of the modified alloy in the high-concentration drug group of Example 2;

[0030] Figure 4 FTIR spectra of the modified alloys in the hydroxyapatite group, low-concentration drug group, and high-concentration drug group of Example 2;

[0031] Figure 5 Laser confocal microscopy photographs after live / dead cell staining of the control titanium alloy, hydroxyapatite-group modified alloy, low-concentration drug-group modified alloy, and high-concentration drug-group modified alloy in Example 2;

[0032] Figure 6A X-ray images of the femurs of rats at 3 weeks and 6 weeks after surgery in Example 2;

[0033] Figure 6B Cross-sectional views of different distal femurs of rats scanned by micro-CT at 6 weeks after surgery in Example 2;

[0034] Figure 7A SEM images of Kirschner wires taken from rats in different groups in Example 2;

[0035] Figure 7B Bacterial plating pictures on the surface of titanium alloy Kirschner wires in rats in different groups in Example 2;

[0036] Figure 7C Statistical chart of bacterial plate counts on the surface of titanium alloy Kirschner wires in rats of different groups in Example 2. Detailed implementation manners

[0037] The preferred implementation manners of the present invention will be specifically described below. Among them, the accompanying drawings form a part of the present invention and are used together with the implementation manners of the present invention to explain the principle of the present invention, rather than to limit the scope of the present invention.

[0038] Biofilm is a highly dense microbial population in which pathogenic microorganisms are wrapped by extracellular polymers produced by themselves to adapt to the living environment. It has extremely strong resistance to antibiotics and host immune defenses and is difficult to eradicate. The formation of biofilm by bacteria on the surface of internal implants is the main mechanism of internal implant infection. As a foreign body, the internal implant can reduce the bacterial concentration required for infection by 100,000 times; while the formation of biofilm increases the drug resistance of bacteria by 1,000 times. At present, prophylactic use of antibiotics commonly used in clinics is difficult to avoid the occurrence of internal implant infections. On the one hand, the "blood-bone barrier (BBB)" hinders the antibiotics from reaching the local bone; on the other hand, once the biofilm is formed, it makes the drug concentration reaching the local bone negligible.

[0039] An implementation manner of the present invention provides a modified metal, which can be used as a raw material for manufacturing orthopedic internal implants. The statin compounds contained in the modified metal can not only inhibit the growth of Staphylococcus epidermidis and Staphylococcus aureus, but also inhibit the formation of biofilm and can destroy mature biofilm. Therefore, it can carry out antibacterial from two aspects of inhibiting bacterial growth and inhibiting biofilm, effectively preventing the occurrence of internal implant-related infections; at the same time, the presence of statin compounds such as simvastatin also makes the internal implant have good osteogenic performance.

[0040] The modified metal of an implementation manner of the present invention includes a metal and a film layer on the surface of the metal. Among them, the metal contains titanium, and the film layer contains statin compounds. The statin compounds include one or more of simvastatin, pravastatin, atorvastatin, and rosuvastatin.

[0041] In one implementation manner, the metal is titanium, titanium alloy, tantalum, tantalum alloy, magnesium or magnesium alloy, and further is titanium or titanium alloy, such as TC4 titanium alloy (Ti-6Al-4V).

[0042] In one implementation manner, the film layer contains hydroxyapatite and simvastatin, and for example, it can be a hydroxyapatite layer containing simvastatin.

[0043] An embodiment of the present invention provides a method for preparing the above-mentioned modified metal, including: forming a film layer containing a statin compound on the metal surface through an electrochemical deposition process.

[0044] In one embodiment, the method for preparing the modified metal includes:

[0045] Performing surface treatment on the metal;

[0046] Using the surface-treated metal as the working electrode and an electrolyte solution containing a statin compound for electrochemical deposition to obtain the modified metal; wherein, the statin compound includes one or more of simvastatin, pravastatin, atorvastatin, and rosuvastatin.

[0047] In one embodiment, the solvent of the electrolyte solution includes water.

[0048] In one embodiment, the solvent of the electrolyte solution includes water and ethanol.

[0049] In one embodiment, the electrolyte solution contains calcium nitrate and ammonium dihydrogen phosphate. The concentration of calcium nitrate can be 0.5 - 0.7 mmol / L, such as 0.6 mmol / L; the concentration of ammonium dihydrogen phosphate can be 0.3 - 0.42 mmol / L, such as 0.36 mmol / L, 0.4 mmol / L.

[0050] In one embodiment, the molar ratio of calcium element to phosphorus element in the electrolyte solution is 1.67:1.

[0051] In one embodiment, in the electrolyte solution, the concentration C of the statin compound such as simvastatin can be: 0 < C ≤ 10 -3 mol / L, such as 10 -4 mol / L, 2×10 -4 mol / L, 5×10 -4 mol / L, 8×10 -4 mol / L, 10×10 -4 mol / L, etc.

[0052] In one embodiment, the electrolyte solution further contains sodium nitrate to increase conductivity, and the concentration of sodium nitrate can be 0.1 mol / L.

[0053] In one embodiment, the preparation method of the electrolyte solution includes:

[0054] Preparing a solution containing a statin compound;

[0055] Preparing an aqueous solution containing calcium nitrate and ammonium dihydrogen phosphate; and

[0056] Mixing the above two solutions to obtain the electrolyte solution.

[0057] In one embodiment, the steps of preparing a solution containing a statin compound include: dissolving the statin compound in ethanol or an ethanol solution (such as 95% ethanol), and adding an aqueous solution of sodium hydroxide thereto; subjecting the resulting system to a water bath at a temperature of 75-85 °C for 25-35 minutes, and then adjusting the pH value of the solution to 7.2 with dilute hydrochloric acid.

[0058] In one embodiment, in the step of preparing a solution containing a statin compound, the water bath temperature can be 76 °C, 78 °C, 79 °C, 80 °C, 81 °C, 82 °C, etc.; the water bath time can be 26 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, etc.

[0059] In one embodiment, the surface treatment of the metal includes the following steps:

[0060] a. Polishing and buffing the TC4 titanium alloy with sandpapers having roughnesses of 400 mesh, 600 mesh, 800 mesh, 1000 mesh, and 1200 mesh respectively, and successively performing ultrasonic oscillation cleaning in acetone, ethanol, and distilled water for 10-20 minutes, and then drying in an oven at a temperature of 45-55 °C; and

[0061] b. Placing the TC4 titanium alloy dried in step a into a mixed solution of nitric acid and hydrofluoric acid, standing at room temperature for 8-12 minutes, and then placing the TC4 titanium alloy in distilled water for ultrasonic vibration cleaning for 12-18 minutes, repeating twice; after cleaning the TC4 titanium alloy, drying it in an oven at a temperature of 45-55 °C, and then putting it into a mixed solution of concentrated sulfuric acid and concentrated hydrochloric acid with equal volume, performing a water bath at 80 °C for 30 minutes, taking it out and placing it in distilled water for ultrasonic oscillation cleaning for 12-18 minutes, repeating twice, and drying it in an oven at 45-55 °C for 24 hours.

[0062] In one embodiment, the ultrasonic oscillation cleaning time in step a of the metal surface treatment can be 12 minutes, 14 minutes, 15 minutes, 16 minutes, 18 minutes; the drying temperature can be 47 °C, 49 °C, 50 °C, 51 °C, 53 °C, etc.

[0063] In one embodiment, the room temperature standing time in step b of the metal surface treatment can be 9 minutes, 10 minutes, 11 minutes; the ultrasonic oscillation cleaning time can be 14 minutes, 15 minutes, 16 minutes, etc.; the drying temperature in the oven can be 47 °C, 49 °C, 50 °C, 51 °C, 53 °C, etc.

[0064] In one embodiment, the voltage of the electrochemical deposition process can be 1.5V to 3.0V, such as 2.0V, 2.5V; the temperature can be 75°C to 85°C, such as 78°C, 80°C, 82°C, 83°C; the deposition time can be 15 to 30 minutes, such as 20 minutes, 25 minutes.

[0065] One embodiment of the present invention provides an orthopedic implant made of any of the above-mentioned modified metals.

[0066] In one embodiment, the orthopedic implant can be a Kirschner wire, a screw, a titanium plate, an artificial joint prosthesis, etc.

[0067] The orthopedic implant of one embodiment of the present invention can be used in animals, such as the human body.

[0068] One embodiment of the present invention further provides the use of statin compounds in the preparation of orthopedic implants capable of preventing infection, wherein the statin compounds are active components for preventing infection; further, the statin compounds include one or more of simvastatin, pravastatin, atorvastatin, and rosuvastatin.

[0069] Hereinafter, the preparation method and application of the modified metal of one embodiment of the present invention will be further described in conjunction with the accompanying drawings and specific examples.

[0070] Example 1 Antibacterial effects of statin compounds against Staphylococcus epidermidis and Staphylococcus aureus in vitro

[0071] Experiment 1: Different concentrations of simvastatin, pravastatin, atorvastatin, and rosuvastatin solutions were added to sterile 96-well plates by the microbroth dilution method. Then, the overnight-cultured and diluted Staphylococcus epidermidis ATCC35984 and Staphylococcus aureus ATCC25923 were added to the wells containing different concentrations of the drugs, and they were placed in a 37°C bacterial incubator for cultivation. After incubation for 16 h, the results were visually observed and judged. The lowest drug concentration that completely inhibited bacterial growth in the small wells was the MIC (minimum inhibitory concentration) of the statin compounds. Table 1 lists the MICs of simvastatin and other statin compounds against the two bacteria (Staphylococcus epidermidis ATCC35984 and Staphylococcus aureus ATCC25923). Among them, the MIC of simvastatin was the lowest at 64 μg / ml, showing the strongest antibacterial effect among the four statin compounds. Therefore, simvastatin was selected as the representative of the statin compounds for subsequent experiments.

[0072] Table 1: MICs (μg / ml) of simvastatin and other statin compounds against Staphylococcus aureus and Staphylococcus epidermidis

[0073]

[0074] Experiment 2: After culturing bacteria in the control group without drugs and the simvastatin group with drugs for 24 hours, the bacteria were stained with STYO9 dye and PI dye for live / dead cell staining, and then observed through a laser confocal microscope. For details, see Figure 1A and 1B . From Figure 1A and 1B , it can be seen that compared with the control group, 64 μg / ml simvastatin has an obvious inhibitory effect on the formation of bacterial biofilms of Staphylococcus epidermidis ATCC35984 and Staphylococcus aureus ATCC25923.

[0075] Example 2

[0076] Preparation of Titanium Alloy with Simvastatin-Hydroxyapatite Coating

[0077] (1) Prepare the stock solution of simvastatin: Dissolve 210 mg of simvastatin in 5 ml of 95% ethanol, then add 7.5 ml of 0.1 mol / L sodium hydroxide solution. Heat the resulting solution in a water bath at 80 °C for 30 minutes, adjust the pH value of the solution to 7.2 with dilute hydrochloric acid, and make up the volume to 50 ml with deionized water. The concentration of the simvastatin stock solution is 10 mmol / L, and it is stored frozen at -20 °C for later use.

[0078] (2) Polish the TC4 (Ti-6Al-4V) titanium alloy with sandpapers of 400 mesh, 600 mesh, and 800 mesh roughness respectively, then ultrasonically clean it in acetone, ethanol, and distilled water for 15 minutes in sequence, and finally dry it in an oven at 50 °C.

[0079] (3) Immerse the TC4 titanium alloy dried in step (2) into a mixed solution of 0.09 mol / L nitric acid and 0.11 mol / L hydrofluoric acid, let it stand at room temperature for 10 minutes, then place the TC4 titanium alloy in distilled water for ultrasonic vibration cleaning for 15 minutes, and repeat twice. After cleaning, dry the TC4 titanium alloy in an oven at 50 °C, then put it into a sealed container containing a mixed solution of concentrated sulfuric acid and concentrated hydrochloric acid with equal volume, place the container in a water bath at 80 °C and let it stand for 30 minutes, take it out and place it in distilled water for ultrasonic oscillation cleaning for 15 minutes, repeat twice, and dry it in the oven at 50 °C for 24 hours for later use. Prepare 3 pieces of surface-treated TC4 titanium alloy in the same way as in steps (2) and (3).

[0080] (4) Prepare the electrolyte solution in the reaction cell: Prepare a deionized aqueous solution containing 0.6 mmol / L calcium nitrate and 0.36 mol / L ammonium dihydrogen phosphate of analytical grade, so that the calcium-phosphorus ratio in the solution is 1.67. Then add analytical grade sodium nitrate (final concentration 0.1 mol / L) to the above mixed solution of calcium nitrate and ammonium dihydrogen phosphate to increase the conductivity, and obtain the electrolyte preparation solution.

[0081] In the same way, three electrolyte preparation solutions with exactly the same components, concentrations, and volumes were prepared respectively. Different concentrations of simvastatin solutions were added to two of them, and finally three electrolyte solutions with different simvastatin contents were prepared, namely the hydroxyapatite group (simvastatin concentration: 0 mol / L), the low-concentration drug group (simvastatin concentration: 10 - 4 mol / L), and the high-concentration drug group (simvastatin concentration: 10 -3 mol / L).

[0082] (5) Electrochemical deposition to construct the TC4 titanium alloy surface coating: Respectively take the three processed TC4 titanium alloys above as the working electrodes of the cathode, and use platinum sheet electrodes as the anodes; respectively add the solutions prepared in step (4) into the reaction cell for electrochemical deposition. The deposition process is controlled by an electrochemical workstation, and electrochemical deposition is carried out for 30 minutes under the conditions of 1.5 V voltage and 85 °C, and the modified alloys of the hydroxyapatite group, the low-concentration drug group, and the high-concentration drug group are obtained in sequence.

[0083] (6) Instrument characterization: Using TC4 (Ti-6Al-4V) titanium alloy as the control group, scanning electron microscopy (SEM) and X-ray diffraction analysis (XRD) were carried out on the TC4 titanium alloy of the control group and the modified alloys of the hydroxyapatite group, the low-concentration drug group, and the high-concentration drug group respectively, and FTIR infrared spectroscopy detection was carried out on the three groups of titanium alloy coatings except the control group, specifically as Figures 2A to 4 shown.

[0084] Figures 2A to 4 The results shown indicate that the surface of the titanium alloy in the control group is relatively flat, and columnar hydroxyapatite crystals are formed on the surfaces of the modified titanium alloys in the hydroxyapatite group, the low-concentration drug group, and the high-concentration drug group. The XRD results show that there is hydroxyapatite on the surface coatings of the modified titanium alloys in the hydroxyapatite group, the low-concentration drug group, and the high-concentration drug group. The FTIR infrared spectroscopy results show that the coating of the hydroxyapatite group is a hydroxyapatite coating without drugs, and the titanium alloy coatings of the low-concentration and high-concentration drug groups are hydroxyapatite coatings containing simvastatin.

[0085] In vitro antibacterial effect of the coating-modified titanium alloy biomaterial

[0086] After culturing bacteria on the control group titanium alloy and the modified alloys of the hydroxyapatite group, the low-concentration drug group, and the high-concentration drug group for 24 h respectively, the bacteria were stained with STYO9 dye and PI dye for live / dead cell staining, and then observed through a laser confocal microscope. The results are as Figure 5 shown. Through Figure 5It can be seen that compared with the uncoated titanium alloy in the control group and the modified alloy in the hydroxyapatite group, the coated modified alloys in the low concentration drug group and the high concentration drug group were able to inhibit the formation of Staphylococcus aureus bacterial biofilm in vitro.

[0087] In vivo antibacterial and osteogenic effects of coating-modified titanium alloy implants

[0088] Kirschner wire implants were prepared using TC4 titanium alloy in the control group, modified alloy in the hydroxyapatite group, modified alloy in the low-concentration drug group, and modified alloy in the high-concentration drug group as materials.

[0089] In vivo experiment: Adult SD rats were randomly divided into four groups, and the same amount of Staphylococcus aureus suspension was injected into the bone marrow cavity of the four groups of rats. The Kirschner wire implants of TC4 titanium alloy in the control group, modified alloy in the hydroxyapatite group, modified alloy in the low-concentration drug group, and modified alloy in the high-concentration drug group were implanted into the distal femoral bone marrow cavity of the four groups of rats to construct a femoral implant-related infection model. After surgery, X-rays, micro-CT, and bacteriological tests were used to evaluate the antibacterial and osteogenic effects of different titanium alloy implants. For details, see Figures 6A to 7C .

[0090] according to Figure 6A It can be seen that at 3 and 6 weeks after surgery, the distal femoral osteolysis of rats in the low-concentration drug group and the high-concentration drug group was milder and accompanied by milder periosteal reaction compared with the control group and the hydroxyapatite group. Figure 6B It can be seen that 6 weeks after surgery, compared with the control group and hydroxyapatite group, the proportion of distal femoral bone tissue in the low-concentration drug group and the high-concentration drug group was significantly increased, among which the proportion of bone tissue in the high-concentration drug group increased more than that in the low-concentration drug group.

[0091] Figure 7B , 7C The results of bacterial plate counts on the surface of Kirschner wires showed that 6 weeks after surgery, the number of bacteria on the surface of the implants in the low-concentration drug group and the high-concentration drug group was significantly reduced compared with the control group and the hydroxyapatite group, and the number of bacteria in the high-concentration drug group was lower than that in the low-concentration group. Figure 7A The scanning electron microscopy results of the Kirschner wires after sampling showed that compared with the control group and the hydroxyapatite group, the number of Staphylococcus aureus on the surface of the Kirschner wires in the low-concentration drug group and the high-concentration drug group was significantly reduced, and the coverage of cells and other tissues was higher.

[0092] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. Use of statin compounds in orthopedic implants against Staphylococcus epidermidis and Staphylococcus aureus, wherein, The statin compound is simvastatin; and The orthopedic implant is made of antibacterial modified metal; The antibacterial modified metal includes a metal and a film layer on the surface of the metal. Among them, the metal includes one or more of titanium alloy, tantalum, and magnesium, and the film layer includes a statin compound, and the statin compound is simvastatin; The antibacterial modified metal is prepared by an electrochemical deposition method, wherein the concentration of the statin compound in the electrolyte solution is 10 -3 mol / L.

2. Use of the statin compound according to claim 1 in orthopedic implants against Staphylococcus epidermidis and Staphylococcus aureus, wherein, The metal is titanium alloy, tantalum, tantalum alloy, magnesium or magnesium alloy.

3. Use of the statin compound according to claim 1 in orthopedic implants against Staphylococcus epidermidis and Staphylococcus aureus, wherein, The film layer includes hydroxyapatite and the statin compound.

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