Novel titanium-based implant material with oxygen-releasing, antibacterial and osteogenesis-promoting properties and preparation method of novel titanium-based implant material

Abstract

The invention belongs to the technical field of medical materials, and discloses a novel titanium-based implant material with oxygen-releasing, antibacterial and osteogenesis-promoting properties and a preparation method of the novel titanium-based implant material. The titanium-based implant material comprises a titanium substrate and calcium peroxide on the surface of the titanium substrate. The invention provides a method for modifying a compact or porous titanium-based implant material by using calcium peroxide. The characteristic that calcium peroxide reacts with water to release oxygen, hydrogen peroxide and calcium ions and the biological activity of calcium ions in the aspect of bone repair enable the titanium-based implant material to have application potential in the aspects of preventing / relieving / treating implant bacterial infection, large-size bone defect oxygen supply insufficiency and poor osseointegration.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and particularly relates to a novel titanium-based implant material with oxygen release, antibacterial, and osteogenic properties, and a preparation method thereof. Background Art

[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 itself, titanium-based bone implant materials, including dense and porous types, still face two major "implant failure" risks: implant infection and poor bone integration. The former stems from the adhesion of bacteria and the formation of biofilms on the implant surface, ultimately evolving into infectious loosening; the latter mainly results from the lack of bioactivity of the device material, which is encapsulated by fibrous tissue and unable to achieve "interface integration", ultimately evolving into aseptic loosening.

[0003] In addition, implant wear and particle release can also activate the mononuclear macrophage system, initiate specific inflammatory cascades, overly promote osteoclast differentiation of cells, and thus lead to osteolysis, which will ultimately also result in aseptic loosening. Once the implant becomes loose, a second operation is often required, bringing additional pain and economic burden to the patient. Therefore, developing a novel titanium-based bone implant material with excellent bioactivity and antibacterial properties has important practical significance.

[0004] Oxygen is crucial in cell survival activities and participates in multiple processes such as respiratory metabolism, cell proliferation, and extracellular matrix synthesis. In bone defect repair, insufficient oxygen supply is often a key challenge limiting the ingrowth of new tissue into porous scaffolds. Research shows that the effective distance of oxygen penetration through capillaries is only 100 - 200 micrometers. The rupture of blood vessels at the defect site prevents the oxygen carried by surrounding blood vessels from effectively penetrating into the porous scaffold with a diameter exceeding 1 millimeter, which causes the cells in the center of the scaffold to tend to die due to hypoxia, severely hindering tissue growth. However, the size of most clinical bone defects is usually larger than 1 - 2 centimeters, making it particularly urgent to solve the oxygen supply problem.

[0005] In addition, bone regeneration is closely related to vascularization, and new blood vessels can provide the oxygen and other nutrients required for bone tissue regeneration. However, the maturation cycle of capillaries in new bone is relatively long, easily leading to early tissue hypoxia and necrosis. Although moderate early hypoxia can activate anaerobic metabolism, stabilize hypoxia-inducible factor 1α (HIF1α), and then upregulate the expression of vascular endothelial growth factor (VEGF), continuous hypoxia may hinder the formation of the basement membrane, resulting in unstable vascular buds, thereby inhibiting the persistence of angiogenesis.

[0006] Based on the above background, in view of the clinical situation, there is an urgent need to develop a class of multifunctional titanium-based bone implants with antibacterial, integrative, and angiogenic properties for preventing / relieving / treating problems related to surgical failures of bone implant materials. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art described above. For this purpose, the present invention provides a novel titanium-based implant material with oxygen-releasing, antibacterial, and osteogenic properties and a preparation method thereof. The present invention proposes to modify a dense or porous titanium-based implant material with calcium peroxide. The characteristics of calcium peroxide itself reacting with water to release oxygen, hydrogen peroxide, and calcium ions, as well as the bioactivity of calcium ions in bone repair, endow the titanium-based implant material with application potential in preventing / relieving / treating bacterial infections of implants, insufficient oxygen supply for large bone defects, and poor bone integration.

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

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

[0010] Preferably, the surface of the titanium substrate has a planar structure, a porous structure, or a hierarchical structure; more preferably, the surface of the titanium substrate has 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. Modifying the surface of the titanium substrate with these substances further facilitates the loading of calcium peroxide on the surface of the titanium substrate.

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

[0013] Loading 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 invention provides a preparation method of a titanium-based implant material.

[0016] A preparation method of a titanium-based implant material, comprising the following steps:

[0017] (1) Dissolve ammonium fluoride, then add alcohol and mix. The resulting mixture is used as the electrolyte. Using a platinum sheet / graphite sheet as the counter electrode and a titanium substrate as the working electrode, anodize at a constant potential, and then take out the titanium substrate to obtain an anodized titanium substrate.

[0018] (2) Place the anodized titanium substrate obtained 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 an alkali solution, and then add an oxidant to obtain the titanium-based implant material.

[0019] Preferably, the ammonium fluoride is dissolved in 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 anodization is carried out at a constant potential of 50 - 60 V for 20 - 40 min with the distance between the counter electrode and the working electrode being 0.5 - 1 cm.

[0023] Placing the anodized titanium substrate obtained in step (1) in a 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 chelating 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 more preferably 1:20.

[0025] Preferably, the alkali solution 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 an H2O2 solution with a concentration of 30 - 40 wt%.

[0028] Preferably, the H2O2 solution is added using a syringe pump at a rate of 0.05 - 0.08 mL / min, and the addition amount 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, wash it three times with absolute ethanol to remove unreacted substances and excess tannic acid, and store the titanium-based implant material in absolute ethanol.

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

[0032] A medical device includes the above-mentioned titanium-based implant material.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] (1) The present invention proposes to modify a dense or porous titanium-based implant material with calcium peroxide. The characteristics of calcium peroxide itself reacting with water to release oxygen, hydrogen peroxide, and calcium ions, as well as the bioactivity of calcium ions in bone repair, enable the titanium-based implant material to have application potential in preventing / relieving / treating implant bacterial infection, insufficient oxygen supply in large bone defects, and poor bone integration.

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

[0036] (3) Based on the characteristics of calcium peroxide itself and a specific modification method, the calcium peroxide-modified bone implant material of the present invention 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 an ionic form and play a biological function, without safety problems.

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

[0038] (5) The titanium-based implant material of the present invention utilizes the characteristics of calcium peroxide releasing oxygen, releasing hydrogen peroxide, releasing calcium ions in a specific environment, and the ability of calcium ions to be transformed into hydroxyapatite to induce bone mineralization, and has great potential in combating bacterial infection and promoting bone repair. Description of the Drawings

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

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

[0041] Figure 3 is the transmission electron micrograph and its energy spectrum diagram 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 prepared in Example 2 and the hydroxyapatite layer on its surface;

[0043] Figure 5 It is a diagram showing the antibacterial effect of titanium-based implant materials of the embodiment and comparative example;

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

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

[0046] Figure 8 These are 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 invention, but it should not be understood as a limitation of the present invention. Without violating the content and essence of the present invention, modifications to the method, conditions or steps of the present invention are all within the scope of protection claimed by the present invention.

[0048] Unless otherwise specified, the technical means used in the examples are conventional preparation and characterization means known to those skilled in the art. PB is phosphate buffered saline, and PBS is phosphate buffered saline.

[0049] The technical solution of the present invention is described in detail below with reference to examples.

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

[0051] Figure 1 "Wavelength" means wave number, "Transmittance" means transmittance. TA-CaO2 means TA / CaO2 nanoparticles.

[0052] Figure 2 The 2θ in the figure represents the diffraction angle, and “Intensity” represents the intensity.

[0053] Figure 3 The d here stands for diameter, “Overlap” stands for overlap, and “Electron” stands for electron.

[0054] Figure 4"TA-CaO2" in it represents TA / CaO2 nanoparticles, and "TNT@TA-CaO2" represents anodized titanium sheets loaded with TA / CaO2 nanoparticles. PB(-) represents the state before soaking in the PB solution, and PB(+) represents the state after soaking in the PB solution.

[0055] Figure 5 "TNT" in it represents anodized titanium sheets, "TNT@TA" represents anodized titanium sheets loaded with TA nanoparticles, "TNT@TA-CaO2" represents anodized titanium sheets loaded with TA / CaO2 nanoparticles, "TNT@CaO2" represents anodized titanium sheets loaded with CaO2 nanoparticles, "Ti@TA-CaO2" represents titanium sheets loaded with TA / CaO2 nanoparticles, "TA-Ca(NO3)2" represents TA / Ca(NO3)2 nanoparticles, "TA-CaCl2" represents TA / CaCl2 nanoparticles, and "IP6-CaO2" represents phytic acid / CaO2 nanoparticles.

[0056] The difference between the preparation process of TA-CaCl2 and that of TA-CaO2 is only that calcium chloride is used to replace calcium peroxide in equal amounts.

[0057] The difference between the preparation process of TA-Ca(NO3)2 and that of TA-CaO2 is only that calcium nitrate is used to replace calcium peroxide in equal amounts.

[0058] The difference between the preparation process of IP6-CaO2 and that of TA-CaO2 is only that phytic acid is used to replace tannic acid in equal amounts.

[0059] "Viable bacterial count" represents viable cell count.

[0060] "Control" represents a blank control, that is, no titanium sheets are added, nor are other calcium-containing substances added, which serves as a blank control.

[0061] Figure 6 △D.O in it represents the difference in dissolved oxygen, and "Time" represents time.

[0062] Figure 8 "Before" in it represents the surface state of the titanium-based implant material before scribing with a scribe knife, and "After" represents the surface state of the titanium-based implant material after scribing with a scribe knife.

[0063] Example 1

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

[0065] Weigh 5 mg of tannic acid and dissolve it in 15 mL of absolute ethanol. Then, dissolve 100 mg of anhydrous calcium chloride in 15 mL of absolute ethanol with different TA (tannic acid) contents by ultrasonic treatment, disperse it evenly under magnetic stirring, add 0.8 mL of ammonia water (concentration 1 M) and react for 2 min. Subsequently, use a syringe pump to add 0.16 mL of H2O2 solution (35 wt%) at a rate of 0.05 mL / min, react the mixture overnight, centrifuge at 10000 rpm for 10 min, collect the TA / CaO2 nanoparticles, and wash them three times with absolute ethanol. The TA / CaO2 nanoparticles are stored in absolute 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, stir and mix evenly by ultrasonic treatment. Use a platinum sheet / graphite sheet as the counter electrode and a 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 wash the titanium sheet thoroughly with absolute ethanol and deionized water to obtain an anodized titanium sheet (there is a layer of TiO2 nanotubes on the surface of the anodized titanium sheet);

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

[0070] Example 3

[0071] Compared with Example 2, the difference in Example 3 is only that in Example 3, a titanium sheet is directly used instead of the anodized titanium sheet in Example 2, that is, the anodization treatment on the surface of the titanium sheet is not carried out in Example 3.

[0072] Example 4

[0073] Compared with Example 2, the difference in Example 4 is only that tannic acid is not added in Example 4.

[0074] Comparative Example 1

[0075] Compared with Example 2, in Comparative Example 1, only step (1) was carried out to obtain an anodized titanium sheet.

[0076] Product Effect 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 Figure 1 shown. The absorption peaks of commercially purchased CaO2 and synthesized TA / CaO2 were characterized by FTIR. Among them, the peak at 875 cm -1 is attributed to O-O stretching, and the peak at 1488 cm -1 is attributed to O-Ca-O vibration. It is proved that the synthesized TA / CaO2 contains CaO2.

[0079] (2) The TA / CaO2 nanoparticles prepared in Example 1 and commercial CaO2 were subjected to X-ray diffraction analysis, and the test results are as Figure 2 shown, among which the TA / CaO2 nanoparticles have obvious CaO2 diffraction peaks.

[0080] (3) The titanium-based implant material prepared in Example 2 was subjected to transmission electron scanning analysis and energy spectrum analysis, and the test results are as Figure 3 shown. It can be seen that 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 Material in PB Solution (Phosphate Buffer Solution)

[0082] The titanium-based implant material prepared in Example 2 was immersed in PB solution for mineralization treatment and subjected to scanning electron microscopy analysis. The test results are as Figure 4 shown. It can be seen that the titanium-based implant material changes from spherical to irregular porous, 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 an antibacterial experiment. The antibacterial efficiency of the samples was determined by the plate counting method of Gram-positive Staphylococcus aureus after 4 h of co-culture. The results are as Figure 5As shown, it can be seen that the number of colonies is less in the presence of CaO2, indicating that CaO2 has antibacterial properties. Comparing Ti@TA-CaO2 with TNT@TA-CaO2, it can be seen that the anodization step promotes the performance of the coating material. Comparing TA-CaO2 with the compared IP6-CaO2, TA-Ca(NO3)2, and TNT@TA-CaCl2, it can be seen that TA and CaO2 synergistically promote the antibacterial effect.

[0085] 4. Performance test of oxygen release from titanium-based implant materials reacting with water

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

[0087] The titanium-based implant material prepared in Example 2 and the untreated porous titanium-based scaffold (i.e., the anodized titanium sheet of Comparative Example 1) were immersed in a PBS solution with pH = 2 and left standing, and the generation of bubbles was observed. As Figure 7 shown, it can be seen that there are many bubbles on the EP tube wall of the titanium-based implant material prepared in Example 2, indicating that the titanium-based implant material prepared in Example 2 has significant oxygen-producing performance.

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

[0089] A scribing knife was used to scribe uniform squares on the surface of the titanium-based implant material, and the scratch depth needed to penetrate the coating but not damage the substrate. Then, tape was evenly applied to the scribed area and quickly torn off, and a panoramic depth microscope was used to photograph the scratch area before and after the tape adhesion. The results are as Figure 8 shown, it can be seen that the coating of TNT@Ti-CaO2 (TNT@Ti-CaO2 represents the titanium-based implant material prepared in Example 2) using TA as the intermediate layer hardly falls off, indicating that the TNT@Ti-CaO2 coating has better adhesion compared to 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 invention comprises a titanium substrate and calcium peroxide on the surface of the titanium substrate.

2. The titanium-based implant material according to claim 1, characterized in that: The surface of the titanium substrate has a planar structure, a porous structure or a multi-level structure.

3. The titanium-based implant material according to claim 1, characterized in that: The surface of the titanium substrate also contains at least one of tannic acid, citric acid, phytic acid, malic acid, succinic acid, epicatechin, and dopamine.

4. The titanium-based implant material according to claim 3, 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.

5. The method for preparing the titanium-based implant material according to any one of claims 3 to 4, characterized in that: The following steps are involved: (1) dissolving ammonium fluoride, then adding alcohol, mixing, using the obtained mixture as an electrolyte, using a platinum sheet / graphite sheet as a counter electrode, and a titanium substrate as a working electrode, performing anodization at a constant potential, and then removing the titanium substrate to obtain an anodized titanium substrate; (2) placing the anodized titanium substrate of step (1) in a tannic acid solution, then taking out the titanium substrate, placing the titanium substrate in a solution containing tannic acid and calcium salt, stirring, adding alkali solution, and then adding an oxidant to obtain the titanium-based implant material.

6. The preparation method according to claim 5, characterized in that: The alcohol includes methanol and ethylene glycol; and / or, the mass volume ratio of ammonium fluoride to alcohol is 0.54 g: (80-95) mL.

7. The preparation method according to claim 5, characterized in that: The anodic oxidation is carried out at a constant potential of 50-60V for 20-40 minutes at a distance of 0.5-1cm 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 alkali solution is ammonia water with a concentration of 0.5-1mol / L.

8. The preparation method according to claim 5, characterized in that: The calcium salt is calcium chloride; and / or the oxidant is H2O2 solution, and the concentration of the H2O2 solution is 30-40wt%.

9. The preparation method according to claim 5, characterized in that: The mass volume ratio of the tannic acid to the ammonia water is (5-10) mg:0.8 mL.

10. A medical device, characterized in that: The invention comprises the titanium-based implant material as described in any one of claims 1 to 4.

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