Titanate fiber with both antibacterial and osteogenic functions, preparation method and application thereof

Abstract

This invention relates to the field of biomaterials technology, specifically to a dual-functional titanate fiber with both antibacterial and osteogenic properties, its preparation method, and its application. A hydrothermal method is used to pre-grow a uniformly distributed nanoscale fiber layer on the surface of a β-titanium alloy. The excellent bioactivity of the fiber surface layer promotes osteoblast growth and increases the rate of bone formation. Furthermore, to impart antibacterial properties to the fiber surface layer, different concentrations of europium (Eu) ions are introduced into the fiber surface layer after hydrothermal treatment using a cation exchange method. The excellent antibacterial properties of Eu ions are utilized to achieve the goal of modifying the surface layer to possess both excellent bioactivity and antibacterial properties.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a bifunctional titanate fiber with both antibacterial and osteogenic properties, its preparation method, and its applications. Background Technology

[0002] Titanium has been widely used in orthopedics and dentistry due to its excellent mechanical, physicochemical properties and good biocompatibility. However, its bioinertness (it cannot form chemical bonds with bone tissue, which is detrimental to bone growth) and lack of antibacterial properties, which easily leads to surgical infections, greatly limit its widespread clinical application.

[0003] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0004] The purpose of this invention is to solve the problems that the existing metallic titanium has biological inertness (it cannot form chemical bonds with bone tissue, which is not conducive to bone growth) and lack of antibacterial properties, which easily causes surgical infections, greatly limiting its clinical application. The invention provides a bifunctional titanate fiber with both antibacterial and osteogenic properties, its preparation method, and its application.

[0005] To achieve the above objectives, this invention discloses a method for preparing bifunctional titanate fibers with both antibacterial and osteogenic properties, comprising the following steps:

[0006] S1, the titanium alloy block was polished and used as a sample;

[0007] S2, the sample from step S1 is ultrasonically cleaned with anhydrous ethanol and deionized water, then soaked in a mixture of HF and HNO3 acid, then etched in H2O2 solution, and finally placed in a reaction vessel containing NaOH solution to obtain the sample.

[0008] S3, react the sample obtained in step S2 in Eu(NO3)3 solution to obtain titanate fibers.

[0009] In step S1, the titanium alloy block has dimensions of 10mm×10mm×5mm and is sanded with 60#, 200#, 400#, 800#, and 3000# sandpaper respectively.

[0010] The ultrasonic cleaning time in step S2 is 15 minutes.

[0011] In step S2, the concentration of HF is 2% and the concentration of HNO3 is 30%.

[0012] In step S2, the concentration of the H2O2 solution is 15%, the corrosion temperature is 60℃, and the corrosion time is 1 hour.

[0013] In step S2, the concentration of the NaOH solution is 1 mol / L, the reaction temperature is 210℃, and the reaction time is 6 h.

[0014] In step S3, the concentration of the Eu(NO3)3 solution is 0.05–0.1 mol / L.

[0015] The present invention also discloses a bifunctional titanate fiber with both antibacterial and osteogenic properties prepared by the above preparation method, and the application of such bifunctional titanate fiber in the fields of orthopedics and dentistry.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. This invention employs a hydrothermal method to pre-grow a uniformly distributed layer of nanoscale fibers on the surface of a β-titanium alloy. The excellent bioactivity of the fiber surface promotes osteoblast growth and increases the rate of bone formation. Furthermore, to impart antibacterial properties to the fiber surface, different concentrations of europium (Eu) ions are introduced into the fiber surface using a cation exchange method after hydrothermal treatment. The good antibacterial properties of Eu ions are utilized to achieve the goal of modifying the surface to possess both excellent bioactivity and antibacterial properties.

[0018] 2. After alkaline heat treatment with NaOH, Ti-Nb β-titanium alloys yielded three-dimensional Na₂TiO₃ fibers with a diameter of approximately 20-40 nm and a well-crystallized state. Further experiments with different concentrations of Eu ions revealed that moderate ion exchange concentrations did not alter the morphology of the alkaline-thermally grown fiber layer. However, excessively high concentrations of the exchange ion solution led to localized fiber agglomeration and entanglement. This was because Eu ions entered the Na₂TiO₃ fibers after ion exchange, forming Na₂TiO₃ ions. 2-3X Eu X A solid solution of TiO3. The superior hydrophilicity of the surface-formed fibers promotes osteoblast adhesion and proliferation, while also significantly enhancing the biomimetic mineralization capacity of the surface. Although the Eu-doped fiber layer exhibits good bactericidal activity against both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), excessively high concentrations of doped Eu ions can cause toxicity to osteoblasts alongside bactericidal activity. These results confirm that a combined alkali-thermal treatment and Eu ion exchange method can prepare a layer of Eu-doped sodium titanate fibers with both antibacterial and osteogenic properties on the surface of β-titanium alloys. This fiber possesses excellent bioactivity and is expected to shorten bone healing time after orthopedic implantation of titanium-based metals. Furthermore, its excellent antibacterial properties effectively prevent infection problems during surgical implantation. This modified titanium alloy, possessing both good bioactivity and antibacterial function, has significant potential for future clinical applications. Attached Figure Description

[0019] Figure 1The surface morphology of β-titanium alloy samples after different processing techniques;

[0020] Figure 2 XRD patterns of different surface layers of β-titanium alloy samples;

[0021] Figure 3 XPS images of different surface layers of β-titanium alloy samples; where a is a broad XPS spectrum and b is a narrow Eu3d spectrum of the fiber surface of ST-Eu1 and ST-Eu2 Eu-doped samples.

[0022] Figure 4 Transmission electron microscopy of the ST-Eu1 fiber layer on the surface of a β-titanium alloy sample; where a is a bright-field image of the fiber, b is a diffraction spot, c is a high-resolution image of a typical fiber, and d is an elemental distribution map of each element in the fiber.

[0023] Figure 5 The hydrophilicity of different surfaces of the β-titanium alloy samples;

[0024] Figure 6 The growth morphology of Escherichia coli on the surface of different β-titanium alloy samples; where a is TLM sample, b is ST-Eu0 sample, c is ST-Eu1 sample, and d is ST-Eu2 sample.

[0025] Figure 7 The number of Escherichia coli on the surface of different β-titanium alloy samples was counted; where a is TLM sample, b is ST-Eu0 sample, c is ST-Eu1 sample, d is ST-Eu2 sample, and e is the number of bacteria on the surface of samples from different embodiments.

[0026] Figure 8 The growth morphology of Staphylococcus aureus on the surface of different β-titanium alloy samples; where a is TLM sample, b is ST-Eu0 sample, c is ST-Eu1 sample, and d is ST-Eu2 sample.

[0027] Figure 9 The number of Staphylococcus aureus on the surface of different β-titanium alloy samples was counted; where a is TLM sample, b is ST-Eu0 sample, c is ST-Eu1 sample, d is ST-Eu2 sample, and e is the number of bacteria on the surface of samples from different embodiments.

[0028] Figure 10 The biomimetic mineralization results of different β-titanium alloy samples after immersion in simulated body fluid for 28 days are shown; where a is TLM sample, b is ST-Eu0 sample, c is ST-Eu1 sample, and d is ST-Eu2 sample.

[0029] Figure 11 XRD results of different β-titanium alloy samples after immersion in simulated body fluid for 28 days;

[0030] Figure 12The MTT results of osteoblasts after culturing on different β-titanium alloy sample surfaces for different times;

[0031] Figure 13 The growth morphology of Escherichia coli and Staphylococcus aureus on the surface of Example 5 (the sample obtained by reacting ST-Eu0 sample in a low concentration (0.025mol / L) Eu(NO3)3 solution) for 12h; where a is Escherichia coli and b is Staphylococcus aureus. Detailed Implementation

[0032] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0033] Example 1

[0034] Ti-Nb titanium alloy (also known as TLM titanium alloy) sheets with a shape of 10cm×10cm and a thickness of 5mm were ground and polished with 60#, 200#, 400#, 800#, and 3000# sandpaper, respectively. The samples were then ultrasonically cleaned for 15 minutes with anhydrous ethanol and deionized water to obtain TLM samples. The results showed that the surface of this sample had no bactericidal effect against Escherichia coli and Staphylococcus aureus, and also did not promote the adhesion and proliferation of osteoblasts.

[0035] Example 2

[0036] Ti-Nb titanium alloy (also known as TLM titanium alloy) sheets with a shape of 10cm×10cm and a thickness of 5mm were ground and polished with 60#, 200#, 400#, 800# and 3000# sandpaper, respectively. After ultrasonic cleaning with anhydrous ethanol and deionized water for 15min, they were etched with a mixed acid of 2% HF and 30% HNO3. Then, they were placed in a reaction vessel containing 1mol / L NaOH and reacted at 210℃ for 6h to obtain ST-Eu0 samples. The results showed that a uniformly distributed sodium titanate fiber structure could be obtained on the surface of the sample, but Escherichia coli and Staphylococcus aureus grew well on the fiber surface, and the fiber surface did not have obvious antibacterial properties.

[0037] Example 3

[0038] Ti-Nb titanium alloy (also known as TLM titanium alloy) sheets with a shape of 10cm×10cm and a thickness of 5mm were polished with 60#, 200#, 400#, 800#, and 3000# sandpaper, respectively. After ultrasonic cleaning with anhydrous ethanol and deionized water for 15min, they were etched with a mixed acid of 2% HF and 30% HNO3. Then, they were placed in a reaction vessel containing 1mol / L NaOH and reacted at 210℃ for 6h. After that, they were placed in a reaction vessel containing 0.05mol / L Eu(NO3)3 and subjected to cation exchange at 100℃ for 24h to obtain ST-Eu1 samples. The results showed that the sample surface could obtain a uniformly distributed Eu-doped sodium titanate fiber structure, which had a good bactericidal effect on Escherichia coli and Staphylococcus aureus. While effectively promoting the deposition of apatite, it also greatly promoted the growth of osteoblasts on its surface.

[0039] Example 4

[0040] Ti-Nb titanium alloy (also known as TLM titanium alloy) sheets with a shape of 10cm×10cm and a thickness of 5mm were polished with 60#, 200#, 400#, 800# and 3000# sandpaper, respectively. After ultrasonic cleaning with anhydrous ethanol and deionized water for 15min, they were etched with a mixed acid of 2% HF and 30% HNO3. Then, they were placed in a reaction vessel containing 1mol / L NaOH and reacted at 210℃ for 6h. After that, they were placed in a reaction vessel containing 0.1mol / L Eu(NO3)3 and subjected to cation exchange at 100℃ for 24h to obtain ST-Eu2 samples. The results showed that local fiber agglomeration and knotting occurred on the surface of the samples, and that they had a certain degree of toxicity to osteoblasts cultured on them.

[0041] Example 5

[0042] Ti-Nb titanium alloy (also known as TLM titanium alloy) sheets with a shape of 10cm×10cm and a thickness of 5mm were ground and polished with 60#, 200#, 400#, 800#, and 3000# sandpaper, respectively. After ultrasonic cleaning with anhydrous ethanol and deionized water for 15 min, they were etched with a mixed acid of 2% HF and 30% HNO3. Subsequently, they were placed in a reaction vessel containing 1mol / L NaOH and reacted at 210℃ for 6 h. Then, they were placed in a reaction vessel containing 0.025mol / L Eu(NO3)3 and subjected to cation exchange at 100℃ for 24 h. Samples were obtained, and the results ( Figure 13 The results showed that Escherichia coli and Staphylococcus aureus grew well on the surface of the samples, and the samples did not have obvious antibacterial properties.

[0043] I. Evaluation of antibacterial test

[0044] 1. Antibacterial test

[0045] (1) Solid culture medium: Add 6.6g of nutrient agar to 200ml of deionized water, sonicate until completely dissolved, and refrigerate for later use.

[0046] (2) Liquid culture medium: Disperse 2.5g NaCl + 500mL deionized water by sonication. Dissolve 5g tryptone and 5g beef extract in a water bath and bring to volume in a volumetric flask. Add all the solution to the NaCl solution and adjust the pH to approximately 7.4. Store the liquid culture medium at 4℃ for later use.

[0047] (3) Bacterial culture: Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were cultured separately in liquid culture medium. After shaking on a 100W, 50Hz shaker for 30 minutes, they were cultured at 37℃ for 8-12 hours in a constant temperature shaking incubator. The bacteria diluted 10⁴ times were transferred to 24-well plates containing the samples and incubated for 4 hours. After incubation, the sample surface was rinsed three times with PBS solution to remove any unadhered bacteria. The samples were then fixed with 4% glutaraldehyde solution at 4℃ for 30 minutes. All samples were dehydrated for 10 minutes each with a gradient of ethanol solutions (30%, 50%, 70%, 90%, 100%), dried, and stored. When plating agar plates, Escherichia coli and Staphylococcus aureus were diluted 10 times each. 5 and 10 6 The bacterial suspension was then spread into an agar plate and cultured for 12 hours before observing the bacterial growth on the agar plate.

[0048] Experimental results showed that both *Escherichia coli* and *Staphylococcus aureus* grew well on the surfaces of TLM and ST-Eu0 samples, exhibiting a large number and relatively intact morphology. However, the Eu-doped fiber surface could disrupt the cell walls of the inoculated bacteria, thereby killing them. The bacterial count decreased sharply on the Eu-doped surface, and the bacterial morphology was damaged. Figure 6-9 ).

[0049] II. Simulated Body Fluid Immersion Experiment

[0050] Dissolve the reagents in the following order: NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, HCl, CaCl2, Na2SO4, Tris, and HCl in a solution at 36.5±1.5℃. Before dissolving Tris, insert a pH meter into the solution. The pH value of the solution should be 2.0±1.0. Heat the solution to 36.5±1.5℃. Maintain the solution temperature between 35℃ and 38℃, ideally at 36.5±0.5℃, and slowly add Tris to the solution, observing the pH change. When the pH value reaches 7.30±0.05, ensure the solution temperature remains at 36.5±0.5℃. At this temperature, add more Tris to bring the pH value closer to 7.45. After all Tris has dissolved, adjust the solution temperature to 36.5±0.2℃. Slowly add 1M HCl dropwise to bring the pH of the solution to 7.42±0.01 at 36.5±0.2℃. Finally, adjust the pH to 7.40 at 36.5℃, ensuring the temperature rise / fall rate is less than 0.1℃ / min during the adjustment process. Remove the pH meter electrode from the solution and rinse it with distilled water. Pour the pH-adjusted solution from the beaker into a 1000ml volumetric flask. Rinse the beaker surface repeatedly with distilled water and pour the rinsing solution into the volumetric flask, using a stirring rod to guide the flow of the solution into the flask. Add distilled water to the solution until it reaches the mark. After mixing the solution in the flask, cool it in water to 20℃. After the solution cools to 20℃, add distilled water to bring the solution to the mark and store it in a 5°C refrigerator. Clean the samples thoroughly with ethanol and deionized water using ultrasonication, then dry them. Place them in plastic bottles, adding 50mL of simulated body fluid to each sample and immersing them for 28 days, changing the simulated body fluid every three days. After cleaning and drying, remove the samples for characterization and testing.

[0051] Experimental results showed that after soaking in SBF solution for 28 days, no new material appeared on the surface of the TLM sample, while a large number of white spherical deposits were induced on the surfaces of ST-Eu0, ST-Eu1, and ST-Eu2 samples. Figure 10 XRD results Figure 11 The results confirmed that the phase of these sediments was hydroxyapatite, indicating that the modified fiber surface has good biomimetic mineralization ability.

[0052] III. Osteoblast Activity Detection Experiment

[0053] 1. Cell Culture

[0054] Human osteoblasts (hfOB1.19) were cultured in a 1:1 mixture of 10% fetal bovine serum, 0.5 mM pyruvate, and 0.3 mg·mL⁻¹ DMEM. The cell line grew to approximately 1 × 10⁻⁶ cells / year. 6When the cell count is 1 / mL, wash with PBS buffer, add 0.25% trypsin for about 2-3 minutes, then neutralize with culture medium to stop digestion. Pipette to prepare a single-cell suspension, wash, centrifuge, and re-prepare a single-cell suspension. Subculture and inoculate into two 25cm² cells. 2 The culture medium was placed in disposable culture flasks. The flasks were then placed in an incubator at 37°C and 5% CO2 saturated humidity for incubation. The culture medium was changed every 2-3 days, and cells passaged 2-4 times were used as experimental cells.

[0055] 2. Cell viability detection

[0056] osteoblasts were charged at 4 × 10 4 The samples were seeded at a density of 1 / cm² in wells containing the samples. After incubation for 3, 7, 24, 72, 150, and 360 hours, the culture medium was aspirated. The samples were then transferred to new empty wells after PBS buffering, and 50 μL of 5 mg·mL⁻¹ MTT reagent and 450 μL of culture medium were added. The plates were incubated for another 4 hours. The original culture medium was carefully aspirated, and 500 μL of DMSO reagent was added to each well. The plates were incubated at room temperature with shaking for 15 min. 200 μL of the dissolved solution was measured at 490 nm using an ELISA reader. Four replicates were used for each sample group at each time point, and the tests were performed four times to examine the statistical significance (n=4).

[0057] Experimental results showed that cell activity on the sample surface increased with time. Compared with the TLM sample, the other three groups of samples exhibited higher cell activity, indicating that both surface-modified and ion-doped samples significantly promoted osteoblast growth. Notably, at each culture time, the cell activity on the surface of the undoped ST-Eu0 sample was slightly higher than that of the ST-Eu1 and ST-Eu2 groups, suggesting that doped Eu ions exhibit slight toxicity to osteoblasts. Nevertheless, the cell activity measured on the surface of the ST-Eu1 and ST-Eu2 groups was still much higher than that of the untreated TLM titanium alloy sample, indicating that doping the nanofiber surface with Eu can prepare a dual-functional medical coating with both good bioactivity and antibacterial properties on the titanium alloy surface. Figure 12 ).

[0058] IV. Microstructure Characterization Experiment of Samples

[0059] 1. X-ray diffraction (XRD) spectroscopy test

[0060] The crystal structure of the sample was analyzed and tested using an X-ray diffraction instrument (X-ray diffraction, Shimadzu, XRD7000, copper source) at a speed of 5° / min and a test range of 20-80°.

[0061] Experimental results show that the TLM titanium alloy has three diffraction peaks of β-Ti (110), (200), and (211) at 2θ positions of approximately 39°, 56°, and 70°. Excluding the β-Ti diffraction peaks, the ST-Eu0 sample spectrum shows three diffraction peaks belonging to Na2TiO3. After cation exchange, the Na2TiO3 peak shows a significant leftward shift, indicating that Eu... 3+ It entered the crystal lattice of Na2TiO3 fibers and formed Na 2-3x Eu x TiO3 solid solution ( Figure 2 ).

[0062] 2. Scanning Electron Microscopy (SEM) Testing

[0063] The surface morphology of the samples was observed using a scanning electron microscope (FESEM, Gemini 300).

[0064] Experimental results show that the surface of the TLM titanium alloy sample is smooth and flat. The surface of the undoped ST-Eu0 sample forms a uniformly distributed three-dimensional nanofiber layer. The surface structure of the ST-Eu1 sample doped with 0.05 mol / L Eu ions is similar to that of ST-Eu0, while the surface of the ST-Eu2 sample doped with 0.1 mol / L Eu ions shows slight fiber agglomeration. Figure 1 ).

[0065] 3. Transmission electron microscopy (TEM) testing

[0066] The surface of the sample was investigated with a transmission electron microscope (JEOL JEM-2100F) to explore more detailed microscopic morphology information. The surface fibers were first dispersed in ethanol and then retrieved with a carbon film during the test. The fiber morphology was observed under the TEM microscope, and the crystallization was judged by high resolution and diffraction.

[0067] Experimental results show that the nanofibers on the surface of the ST-Eu1 sample are approximately 150 nm long and 30 nm in diameter. Diffraction patterns and high-resolution imaging indicate good crystallinity of the nanofibers. Mapping patterns of individual fibers show that the distribution of Eu ions is similar to the distribution of Ti, O, and Na elements within the fibers, confirming that Eu ions tend to combine with Ti, O, and Na to form compounds. Figure 4 ).

[0068] 4. Photoelectron spectroscopy (XPS) test

[0069] X-ray photoelectron spectroscopy (Thermo SCIENTIFIC ESCALAB 250Xi) was used to investigate the surface chemical state and elemental valence states of the samples. Charge correction was performed using C1s 285 eV, and the samples were placed at 10... -5 The test was conducted in a vacuum chamber.

[0070] Experimental results show that the surface of the TLM sample is mainly composed of Ti, O, Nb, and C elements, while the ST-Eu0 sample surface, in addition to the above elements, also showed a Na1s peak. The ST-Eu1 and ST-Eu2 sample surfaces exhibited three characteristic peaks at binding energies of 128.2 eV, 1134.1 eV, and 1164.1 eV, which, upon comparison, correspond to the 4d and 3d energies of Eu, respectively. 5 / 2 and 3D 3 / 2 The peak indicates that Eu ions were successfully doped onto the sample surface. Figure 3 a). Analysis of the narrow spectrum of Eu ( Figure 3 b) The Eu peak positions on the surfaces of ST-Eu1 and ST-Eu2 samples are both at 1134.1 eV and 1164.1 eV, respectively, which are similar to the Eu peak positions on the surface of ST-Eu1 and ST-Eu2 samples. 3+ 3D 5 / 2 and Eu 3+ 3D 3 / 2 The peak positions are highly consistent, indicating that the Eu incorporated into the fiber exists in the form of trivalent Eu.

[0071] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A method for preparing bifunctional titanate fibers with both antibacterial and osteogenic properties, characterized in that, Includes the following steps: S1, the titanium alloy block was polished and used as a sample; S2, the sample from step S1 is ultrasonically cleaned with anhydrous ethanol and deionized water, then soaked in a mixture of HF and HNO3 acid, then etched in H2O2 solution, and finally placed in a reaction vessel containing NaOH solution to obtain the sample. S3, react the sample obtained in step S2 in Eu(NO3)3 solution to obtain titanate fiber; The concentration of the Eu(NO3)3 solution in step S3 is 0.05~0.1 mol / L; The titanium alloy is a Ti-Nb based β-titanium alloy; Titanate fibers have a diameter of 20-40 nm, are in a crystalline state, and are composed of Na+. 2-3X Eu X Solid solution of TiO3.

2. The method for preparing a bifunctional titanate fiber with both antibacterial and osteogenic properties as described in claim 1, characterized in that, In step S1, the titanium alloy block has dimensions of 10 mm × 10 mm × 5 mm and is sanded with 60#, 200#, 400#, 800#, and 3000# sandpaper, respectively.

3. The method for preparing a bifunctional titanate fiber with both antibacterial and osteogenic properties as described in claim 1, characterized in that, The ultrasonic cleaning time in step S2 is 15 minutes.

4. The method for preparing a bifunctional titanate fiber with both antibacterial and osteogenic properties as described in claim 1, characterized in that, In step S2, the concentration of HF is 2% and the concentration of HNO3 is 30%.

5. The method for preparing a bifunctional titanate fiber with both antibacterial and osteogenic properties as described in claim 1, characterized in that, In step S2, the concentration of the H2O2 solution is 15%, the corrosion temperature is 60°C, and the corrosion time is 1 hour.

6. The method for preparing a bifunctional titanate fiber with both antibacterial and osteogenic properties as described in claim 1, characterized in that, In step S2, the concentration of the NaOH solution is 1 mol / L, the reaction temperature is 210°C, and the reaction time is 6 h.

7. A bifunctional titanate fiber with both antibacterial and osteopromoting properties, prepared by the method described in any one of claims 1 to 6.

8. The application of a dual-function titanate fiber with both antibacterial and osteogenic properties as described in claim 7 in the fields of orthopedics and dentistry.

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