A novel titanium-based implant material with oxygen-releasing, antibacterial and bone-promoting properties and a preparation method thereof

Abstract

The application belongs to the technical field of medical materials, and discloses a novel titanium-based implant material with oxygen release, antibacterial and bone formation promoting properties and a preparation method thereof. The titanium-based implant material comprises a titanium base material and calcium peroxide on the surface of the titanium base material. The application proposes that the dense or porous titanium-based implant material is modified by calcium peroxide. The characteristics of calcium peroxide itself, i.e., the reaction with water to release oxygen, hydrogen peroxide and calcium ions, and the biological activity of calcium ions in bone repair make the titanium-based implant material have application potential in preventing / relieving / treating bacterial infection of implants, oxygen deficiency of large-size bone defects and poor bone integration.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials, and particularly relates to a novel titanium-based implant material with oxygen release, antibacterial and bone formation promoting properties and a preparation method thereof. BACKGROUND

[0002] Titanium and titanium alloys are widely used as bone implant materials due to their excellent biocompatibility, mechanical properties and corrosion resistance. Limited by the biological inertness of titanium material itself, titanium-based bone implant materials, including dense and porous types, also face the risks of implant infection and poor bone integration, two "implant failure" risks. The former is caused by bacterial adhesion and biofilm formation on the surface of the implant, eventually leading to infectious loosening; the latter is mainly caused by the lack of biological activity of the device material, which is wrapped by fibrous tissue and cannot achieve "interface integration", eventually leading to aseptic loosening.

[0003] In addition, implant wear and particle release can also activate the mononuclear macrophage system, start a specific inflammatory cascade, excessively promote osteoclast differentiation and cause osteolysis, which will eventually also lead to aseptic loosening. Once the implant loosens, a second surgery is often required, causing additional pain and economic burden to the patient. Therefore, it is of great practical significance to develop a new type of titanium-based bone implant material with excellent biological activity and antibacterial properties.

[0004] Oxygen is essential for cell survival activities, as it is involved in respiratory metabolism, cell proliferation, and extracellular matrix synthesis. In bone defect repair, insufficient oxygen supply is often a key challenge to the growth of new tissue into a porous scaffold. Studies have shown that the effective penetration distance of oxygen through capillaries is only 100-200 microns, and the rupture of blood vessels at the defect site makes it impossible for oxygen carried by surrounding blood vessels to effectively penetrate the interior of a porous scaffold with a diameter of more than 1 millimeter, which causes cells in the center of the scaffold to tend to die due to hypoxia, severely hindering tissue growth. However, most clinical bone defects are usually larger than 1-2 centimeters in size, making it particularly urgent to address the oxygen supply problem.

[0005] In addition, bone regeneration is closely related to vascularization, and new blood vessels can provide oxygen and other nutrients required for bone tissue regeneration. However, the maturation period of capillaries in new bone is long, which can easily lead to early tissue hypoxic necrosis. Although moderate early hypoxia can activate anaerobic metabolism, stabilize hypoxia-inducible factor 1 alpha (HIF1a), and thereby upregulate the expression of vascular endothelial growth factor (VEGF), persistent hypoxia can hinder the formation of basement membrane, leading to unstable vascular sprouts, thereby inhibiting the persistence of angiogenesis.

[0006] Based on the above background, in view of the clinical situation, it is urgent to develop a kind of multifunctional titanium-based bone implant with antibacterial, promoting integration and promoting vascularization performance for preventing / relieving / treating bone implant-related surgical failure problems. SUMMARY

[0007] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a new titanium-based implant material with oxygen release, antibacterial and bone formation promoting properties and a preparation method thereof. The present application proposes to modify dense or porous titanium-based implant materials with calcium peroxide. The characteristics of calcium peroxide itself reacting with water to release oxygen, hydrogen peroxide and calcium ions and the biological activity of calcium ions in bone repair make the titanium-based implant material have application potential in preventing / relieving / treating implant bacterial infection, oxygen deficiency in large size bone defect, and poor bone integration.

[0008] The first aspect of the present application provides a titanium-based implant material.

[0009] A titanium-based implant material comprises a titanium substrate and calcium peroxide on the surface of the titanium substrate.

[0010] Preferably, the surface of the titanium substrate is in a planar structure, a porous structure or a multi-level structure; further preferably, the surface of the titanium substrate is in a porous structure.

[0011] Preferably, the surface of the titanium substrate further contains at least one of tannic acid, citric acid, phytic acid, malic acid, succinic acid, epicatechin, and dopamine. The modification of the titanium substrate surface by these substances further facilitates the loading of calcium peroxide on the titanium substrate surface.

[0012] Preferably, the weight of the calcium peroxide accounts for 0.1-1% of the weight of the titanium-based implant material.

[0013] The loading of calcium peroxide on the surface of the titanium substrate can be achieved by directly loading calcium peroxide nanoparticles, or by in-situ growth. Preferably, it is achieved by in-situ growth.

[0014] Preferably, the titanium-based implant material comprises a titanium substrate and tannic acid / calcium peroxide nanoparticles on the surface of the titanium substrate. The tannic acid / calcium peroxide nanoparticles are denoted as TA / CaO2.

[0015] The second aspect of the present application provides a preparation method of a titanium-based implant material.

[0016] The preparation method of a titanium-based implant material comprises the following steps:

[0017] (1) dissolving ammonium fluoride, then adding alcohol, mixing, obtaining the mixture as electrolyte, using platinum sheet / graphite sheet as counter electrode, using titanium substrate as working electrode, anodizing under constant potential, then taking out the titanium substrate, obtaining the anodized titanium substrate;

[0018] (2) placing the anodized titanium substrate of step (1) in tannic acid solution, then taking out the titanium substrate, placing the titanium substrate in solution containing tannic acid and calcium salt, stirring, adding alkali, then adding oxidant, obtaining the titanium-based implant material.

[0019] Preferably, the ammonium fluoride is dissolved with deionized water.

[0020] Preferably, the alcohol includes methanol and ethylene glycol.

[0021] Preferably, the mass-volume ratio of ammonium fluoride to alcohol is 0.54 g:(80-95) mL.

[0022] Preferably, the anodizing is performed under a distance of 0.5-1 cm between the counter electrode and the working electrode, a constant potential of 50-60 V, and anodizing for 20-40 min.

[0023] Placing the anodized titanium substrate of step (1) in tannic acid solution is conducive to the adsorption of tannic acid into the TiO2 nanotubes on the surface of the titanium substrate, providing growth sites for chelate particles in the subsequent process.

[0024] Preferably, in the solution containing tannic acid and calcium salt, the mass ratio of tannic acid to calcium salt is 1:(10-30), and further preferably 1:20.

[0025] Preferably, the alkali is ammonia water with a concentration of 0.5-1 mol / L.

[0026] Preferably, the calcium salt is calcium chloride.

[0027] Preferably, the oxidant is H2O2 solution with a concentration of 30-40 wt%.

[0028] Preferably, the H2O2 solution is added at a rate of 0.05-0.08 mL / min using a syringe pump, and the amount added is 0.1-0.16 mL.

[0029] Preferably, the mass-volume ratio of tannic acid to ammonia water is (5-10) mg:0.8 mL.

[0030] After obtaining the titanium-based implant material, it is washed with anhydrous ethanol three times to remove unreacted substances and excess tannic acid, and the titanium-based implant material is stored in anhydrous ethanol.

[0031] The third aspect of the present application provides a titanium-based implant material.

[0032] A medical device comprising the titanium-based implant material described above.

[0033] The beneficial effects of the present application relative to the prior art are as follows:

[0034] (1) The present application proposes the use of calcium peroxide to modify dense or porous titanium-based implant materials. The characteristics of calcium peroxide itself, which releases oxygen, hydrogen peroxide and calcium ions when reacting with water, and the biological activity of calcium ions in bone repair make the titanium-based implant materials have application potential in preventing / relieving / treating implant bacterial infection, oxygen deficiency in large-size bone defects, and poor bone integration.

[0035] (2) The method of the present application is simple to operate, the raw materials are easy to obtain, and the process flow is less. Based on the specific modification method, the present application can grow calcium peroxide in situ on the surface of dense, porous and multi-level structure titanium materials while maintaining the mechanical properties of the materials unchanged.

[0036] (3) Based on the characteristics of calcium peroxide itself and the specific modification method, the calcium peroxide modified bone implant material of the present application has better wear resistance than the material prepared by the surface loading method and is not prone to particle shedding. Even if there is a small amount of shedding, calcium peroxide will react with water in the tissue to become ionic form and play a biological function, and there is no safety problem.

[0037] (4) The oxygen, hydrogen peroxide and calcium ions generated by the reaction of calcium peroxide existing in the micro-nano structure with water, as well as the interaction of calcium ions with phosphate, can endow the material with biological activity and have application potential in the repair of large-size bone defects and infected bone defects.

[0038] (5) The titanium-based implant material of the present application utilizes the characteristics of calcium peroxide in releasing oxygen, releasing hydrogen peroxide, and releasing calcium ions in a specific environment, as well as the ability of calcium ions to convert into hydroxyapatite to induce bone mineralization, and has great potential in combating bacterial infection and promoting bone repair. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is the Fourier transform infrared spectrum (FTIR) of TA / CaO2 nanoparticles prepared in Example 1 and commercial CaO2;

[0040] Figure 2 is the X-ray diffraction (XRD) analysis chart of TA / CaO2 nanoparticles prepared in Example 1 and commercial CaO2;

[0041] Figure 3 is the transmission electron micrograph and its energy spectrum chart of the titanium-based implant material prepared in Example 2;

[0042] Figure 4 is a scanning electron microscope micrograph of the titanium-based implant material and its surface hydroxyapatite layer prepared in Example 2;

[0043] Figure 5 is a graph of the antibacterial effect of the titanium-based implant material of the examples and comparative examples;

[0044] Figure 6 is the oxygen release of TA / CaO2 nanoparticles prepared in Example 1 in PBS with different pH concentrations;

[0045] Figure 7 is a graph of the oxygen release effect of the titanium-based implant material prepared in Example 2 and Comparative Example 1;

[0046] Figure 8 is the mechanical property results of the titanium-based implant materials of Example 2 and Example 4. DETAILED DESCRIPTION

[0047] The following is a further explanation of the content of the present application. However, it should not be understood as a limitation of the present application. Modifications to the method, conditions or steps of the present application, without departing from the content and substance of the present application, all belong to the scope of the present application claimed.

[0048] If not specifically pointed out, the technical means used in the examples are conventional preparation, characterization means known to those skilled in the art. PB is phosphate buffer, and PBS is phosphate buffered saline.

[0049] The technical solutions of the present application are described in detail below in conjunction with examples.

[0050] Figures 1 to 8 The English or symbols involved in the above are explained as follows:

[0051] Figure 1 In the above, "Wavelength" represents wave number, and "Transmittance" represents transmittance. TA-CaO2 represents TA / CaO2 nanoparticles.

[0052] Figure 2 In the above, 2θ represents diffraction angle, and "Intensity" represents intensity.

[0053] Figure 3 In the above, d represents diameter, "Overlap" represents overlap, and "Electron" represents electron.

[0054] Figure 4"TA-CaO2" means TA / CaO2 nanoparticles, "TNT@TA-CaO2" means an anodized titanium sheet loaded with TA / CaO2 nanoparticles, "PB(-)" means before PB solution immersion, and "PB(+)" means after PB solution immersion.

[0055] Figure 5 "TNT" means an anodized titanium sheet, "TNT@TA" means an anodized titanium sheet loaded with TA nanoparticles, "TNT@TA-CaO2" means an anodized titanium sheet loaded with TA / CaO2 nanoparticles, "TNT@CaO2" means an anodized titanium sheet loaded with CaO2 nanoparticles, "Ti@TA-CaO2" means a titanium sheet loaded with TA / CaO2 nanoparticles, "TA-Ca(NO3)2" means TA / Ca(NO3)2 nanoparticles, "TA-CaCl2" means TA / CaCl2 nanoparticles, and "IP6-CaO2" means phytic acid / CaO2 nanoparticles.

[0056] The preparation process of TA-CaCl2 is different from that of TA-CaO2 only in that an equal amount of calcium chloride is used instead of calcium peroxide.

[0057] The preparation process of TA-Ca(NO3)2 is different from that of TA-CaO2 only in that an equal amount of calcium nitrate is used instead of calcium peroxide.

[0058] The preparation process of IP6-CaO2 is different from that of TA-CaO2 only in that an equal amount of phytic acid is used instead of tannic acid.

[0059] "Viable bacterial count" means viable bacterial count.

[0060] "Control" means a blank control, i.e., no titanium sheet is added, and no other calcium-containing substance is added, which serves as a blank control.

[0061] Figure 6 ΔD.O in the table means the difference in dissolved oxygen, and "Time" means time.

[0062] Figure 8 "Before" in the table means the surface state of the titanium-based implant material before the grid knife is scored, and "After" means the surface state of the titanium-based implant material after the grid knife is scored.

[0063] Example 1

[0064] TA / CaO2 nanoparticles are prepared, including the following steps:

[0065] Take 5 mg of tannic acid dissolved in 15 mL of anhydrous ethanol, then 100 mg of anhydrous calcium chloride is fully dissolved in 15 mL of anhydrous ethanol with different TA (tannic acid) content, and is uniformly dispersed under magnetic stirring, then 0.8 mL of ammonia water (concentration 1M) is added and reacted for 2 min, then 0.16 mL of H2O2 solution (35wt%) is added at a rate of 0.05 mL / min using a syringe pump, the mixture is reacted overnight, centrifuged at 10000 rpm for 10 min, and the TA / CaO2 nanoparticles are collected and washed with anhydrous ethanol three times, and the TA / CaO2 nanoparticles are stored in anhydrous ethanol.

[0066] Example 2

[0067] A preparation method of a titanium-based implant material, comprising the following steps:

[0068] (1) Dissolve 0.54 g of ammonium fluoride in 5 mL of deionized water, then add 5 ml of methanol and 90 mL of ethylene glycol, and mix uniformly under ultrasonic stirring, use platinum / graphite sheet as the counter electrode and titanium sheet as the working electrode, the distance between the counter electrode and the working electrode is 1 cm, anodize at a constant potential of 60 V for 40 min, and fully wash the titanium sheet with anhydrous ethanol and deionized water to obtain an anodized titanium sheet (the surface of the anodized titanium sheet has a layer of TiO2 nanotubes);

[0069] (2) Place the anodized titanium sheet obtained in step (1) in a 10 mg TA anhydrous ethanol solution and vacuum load for 30 min to allow the TA to fully adsorb into the nanotubes on the surface of the anodized titanium sheet to provide chelation particle growth sites for subsequent steps, then place the anodized titanium sheet in an anhydrous ethanol solution containing 5 mg of TA and 100 mg of anhydrous calcium chloride, add 0.8 mL of ammonia water (concentration 1M) under magnetic stirring and react for 2 min, at which time the mixture solution becomes turbid, then add 0.16 mL of concentrated H2O2 solution (35wt%) at a rate of 0.05 mL / min using a syringe pump, and fully react to obtain an anodized titanium sheet with in-situ grown TA / CaO2 nanoparticles, finally wash with anhydrous ethanol three times to remove unreacted substances and excess tannic acid, and store the obtained titanium-based implant material in anhydrous ethanol.

[0070] Example 3

[0071] Compared with Example 2, the difference between Example 3 is only that Example 3 directly uses a titanium sheet instead of the anodized titanium sheet in Example 2, i.e., Example 3 does not perform an anodization treatment on the surface of the titanium sheet.

[0072] Example 4

[0073] Compared with Example 2, the difference between Example 4 is only that Example 4 does not add tannic acid.

[0074] Comparative Example 1

[0075] Compared with Example 2, Comparative Example 1 only performed step (1) to obtain anodized titanium sheet.

[0076] Product effectiveness test

[0077] 1. Product structure characterization

[0078] (1) The TA / CaO2 nanoparticles prepared in Example 1 and commercial CaO2 were subjected to infrared spectroscopy detection, and the test results are as follows: Figure 1 As shown, the absorption peaks of commercially available CaO2 and synthetic TA / CaO2 were characterized by FTIR, with the peak at 875 cm⁻¹ being the highest. -1 The peak at 1488cm is attributed to the OO stretching. -1 The peak at that point belongs to the O-Ca-O vibration. This proves that the synthesized TA / CaO2 contains CaO2.

[0079] (2) X-ray diffraction analysis was performed on the TA / CaO2 nanoparticles prepared in Example 1 and commercial CaO2. The test results are as follows: Figure 2 As shown, the TA / CaO2 nanoparticles exhibit obvious CaO2 diffraction peaks.

[0080] (3) The titanium-based implant material prepared in Example 2 was subjected to transmission electron scanning analysis and energy dispersive spectroscopy analysis. The test results are as follows: Figure 3 As shown, the TA / CaO2 nanoparticles in the prepared titanium-based implant material are uniformly distributed in the TiO2 nanotubes on the surface of the titanium sheet.

[0081] 2. Mineralization performance test of titanium-based implant materials in PB solution (phosphate buffer)

[0082] The titanium-based implant material prepared in Example 2 was subjected to mineralization treatment by immersion in PB solution and then analyzed by scanning electron microscopy. The test results are as follows: Figure 4 As shown, the titanium-based implant material changes from a spherical shape to an irregular porous shape, indicating that the titanium-based implant material and the porous titanium-based scaffold form hydroxyapatite in the PB solution.

[0083] 3. Antibacterial performance test

[0084] The titanium-based implant material prepared in Example 2 and the titanium sheet prepared in Example 4 were subjected to antibacterial experiments. The antibacterial efficiency of the samples was determined by plate counting method using Gram-positive Staphylococcus aureus co-cultured for 4 hours. The results are as follows: Figure 5As shown, the number of bacterial colonies is lower in the presence of CaO2, indicating that CaO2 has antibacterial properties. Furthermore, a comparison between Ti@TA-CaO2 and TNT@TA-CaO2 shows that the anodic oxidation step promotes the performance of the coating material. Compared with IP6-CaO2, TA-Ca(NO3)2, and TNT@TA-CaCl2, TA-CaO2 demonstrates that TA and CaO2 synergistically promote the antibacterial effect.

[0085] 4. Test on the oxygen release performance of titanium-based implant materials in reaction with water

[0086] Oxygen release from TA / CaO2 nanoparticles prepared in Example 1 in PBS at different pH concentrations. Figure 6 As shown, the long-term cumulative release of oxygen is better under acidic conditions.

[0087] The titanium-based implant material prepared in Example 2 and the untreated porous titanium-based scaffold (i.e., the anodic titanium oxide sheet of Comparative Example 1) were immersed in PBS solution at pH 2 and allowed to stand. The generation of bubbles was then observed. Figure 7 As shown, the EP tube wall of the titanium-based implant material prepared in Example 2 has many air bubbles, indicating that the titanium-based implant material prepared in Example 2 has significant oxygen production performance.

[0088] 5. Mechanical property testing of titanium-based implant materials

[0089] Using a crisscross tool, uniform squares were drawn on the surface of the titanium-based implant material. The depth of the scratches needed to penetrate the coating but not damage the substrate. Then, adhesive tape was evenly applied to the crisscrossed areas, and the tape was quickly peeled off. A panoramic depth microscope was used to photograph the crisscrossed areas before and after the tape was applied. The results are as follows: Figure 8 As shown, the TNT@Ti-CaO2 coating (TNT@Ti-CaO2 represents the titanium-based implant material prepared in Example 2) using TA as the intermediate layer hardly peels off, indicating that TNT@Ti-CaO2 has better adhesion than the TNT@CaO2 coating (TNT@CaO2 represents the titanium-based implant material prepared in Example 4).

Claims

1. A titanium-based implant material, characterized in that, The titanium-based implant material comprises a titanium substrate and tannic acid / calcium peroxide nanoparticles on the surface of the titanium substrate. The surface of the titanium substrate is in a planar structure, a porous structure or a multi-stage structure. The preparation method of the titanium-based implant material comprises the following steps: (1) Dissolve ammonium fluoride, then add alcohol, mix, and obtain the mixture as an electrolyte, use platinum sheet / graphite sheet as a counter electrode, and use a titanium substrate as a working electrode to perform anodic oxidation under a constant potential, then take out the titanium substrate to obtain an anodically oxidized titanium substrate; (2) Place the anodically oxidized titanium substrate in step (1) in a tannic acid solution, then take out the titanium substrate, place the titanium substrate in a solution containing tannic acid and calcium salt, stir, add lye, and then add an oxidizing agent to obtain the titanium-based implant material.

2. The method of producing a titanium-based implant material according to claim 1, characterized in that, comprises the following steps: (1) Dissolve ammonium fluoride, then add alcohol, mix, and obtain the mixture as an electrolyte, use platinum sheet / graphite sheet as a counter electrode, and use a titanium substrate as a working electrode to perform anodic oxidation under a constant potential, then take out the titanium substrate to obtain an anodically oxidized titanium substrate; (2) Place the anodically oxidized titanium substrate in step (1) in a tannic acid solution, then take out the titanium substrate, place the titanium substrate in a solution containing tannic acid and calcium salt, stir, add lye, and then add an oxidizing agent to obtain the titanium-based implant material.

3. The preparation method according to claim 2, characterized in that, The alcohol comprises methanol and ethylene glycol; and / or, the mass-volume ratio of the ammonium fluoride to alcohol is 0.54 g:(80-95) mL.

4. The production method according to claim 2, characterized by, The anodic oxidation is performed under a constant potential of 50-60 V for 20-40 min with a distance of 0.5-1 cm between the counter electrode and the working electrode; and / or, in the solution containing tannic acid and calcium salt, the mass ratio of tannic acid to calcium salt is 1:(10-30); and / or, the lye is ammonia water with a concentration of 0.5-1 mol / L.

5. The preparation method according to claim 2, characterized in that, The calcium salt is calcium chloride; and / or, the oxidizing agent is an H2O2 solution with a concentration of 30-40 wt%.

6. A medical device, comprising: The titanium-based implant material of claim 1 is included.

Images