A pH-responsive dual-controlled antibacterial-bone-promoting titanium-based implant and a preparation method and application thereof

Abstract

This invention discloses a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant, its preparation method, and its applications. The invention utilizes anodizing to construct a titanium dioxide nanotube coating structure on a titanium substrate. Dopamine is then grafted onto a portion of the nanotubes to attach amino groups, resulting in PMAA grafting. Simultaneously, His50 peptide is grafted onto another portion. Finally, HHC36 and kaempferol are loaded to obtain the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant. The titanium-based implant of this invention can respond to changes in the pH of the surrounding solution, regulating the release rate of the loaded drug. It can perform responsive burst release of antibacterial drugs under bacterial infection conditions and responsive burst release of drugs to promote bone growth under normal physiological conditions, while exhibiting excellent antibacterial effects. This titanium-based implant addresses the challenges of bacterial infection and implant loosening in clinical titanium-based implants, reducing the risk of bacterial infection and implant failure, and has excellent application prospects.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant, its preparation method, and its application. Background Technology

[0002] In current dental and orthopedic implant clinical practice, implant loosening and implant-related infections are the two main causes of related orthopedic complications and implant failure.

[0003] In clinical trials, poor osseointegration and low interfacial bonding can adversely affect implant lifespan, frequently leading to implant loosening. Simultaneously, the open environment of bone defects and a compromised immune system make titanium-based implants susceptible to bacterial infection during application, with clinically reported infection rates as high as 4%. Implant-related infections are typically caused by bacterial adhesion and biofilm formation on the implant surface. Bacterial cells in biofilms can withstand the host's immune response and are much less sensitive to antibiotics. Therefore, to prevent implant loosening and implant-related infections, achieve long-term antibacterial efficacy and osseointegration, reduce side effects, and improve patient convenience, it is essential to release antibacterial agents and osteogenic drugs through the surface of medical implants.

[0004] Titanium-based implants are widely used in the treatment of bone defects caused by accidents, avascular necrosis of the femoral head, and bone tumors due to their excellent mechanical and biocompatibility properties, making them one of the most commonly used orthopedic implant materials in clinical practice. Currently, the clinical method of preoperative and postoperative systemic administration of antibiotics is inefficient and difficult to effectively prevent and resolve these issues. Therefore, designing and constructing responsive drug delivery surfaces allows drug molecules to be implanted into the patient's body along with the implant. This enables the release of large amounts of antibacterial drugs when bacterial infection occurs at the implantation site, and the release of large amounts of bone-promoting drugs under normal physiological conditions. This achieves pH-responsive antibacterial and bone-promoting effects and may become an effective method for preventing and resolving implant loosening caused by bacterial infection. Summary of the Invention

[0005] The primary objective of this invention is to overcome the clinical problems of implant loosening and bacterial infection caused by existing titanium-based materials, and to provide a method for preparing a pH-responsive dual controlled-release antibacterial-promoting osteogenic titanium-based implant.

[0006] Another object of the present invention is to provide a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant obtained by the above preparation method. The prepared titanium-based implant has excellent antibacterial properties, which can eliminate adhering bacteria and biofilms; at the same time, the titanium-based implant has good biocompatibility and good osteogenic properties.

[0007] Another object of the present invention is to provide the application of the above-mentioned pH-responsive dual controlled-release antibacterial-promoting bone titanium-based implant.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a pH-responsive dual-controlled-release antibacterial-osteopromoting titanium-based implant involves constructing titanium dioxide nanotube structures on the surface of the titanium-based implant using anodizing technology. Then, dopamine is used to introduce amino reactive sites at the top of a portion of the titanium dioxide nanotubes, followed by grafting polymethyl methacrylate (PMAA) onto the top of the remaining titanium dioxide nanotubes. Finally, thiol-containing His groups are grafted onto the top of the remaining titanium dioxide nanotubes. 50 The peptides were then loaded with HHC36, an antibacterial peptide, and kaempferol, an osteogenic drug, to obtain a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant. The specific steps included are as follows:

[0010] (1) Using a titanium-based implant as the anode and a platinum sheet as the cathode, after anodizing in constant voltage mode, the implant is thoroughly rinsed and dried to obtain a titanium-based implant with a titanium dioxide nanotube structure on the surface.

[0011] (2) The polyvinyl alcohol modified ink pattern is partially covered by 3D printing technology to cover the surface of the titanium-based implant obtained in step (1), resulting in a titanium-based implant with part of the surface covered.

[0012] (3) Prepare a dopamine solution under alkaline conditions, immerse the titanium-based implant obtained in step (2) into the aforementioned dopamine solution, incubate in the dark, wash and dry to obtain a titanium-based implant with amino reactive groups introduced on the surface of the nanotube.

[0013] (4) Prepare a PMAA solution in an aqueous solution, and immerse the titanium-based implant obtained in step (3) into the aforementioned PMAA solution. Under the condition of introducing a catalyst, carry out an amide reaction, wash, and dry to obtain a titanium-based implant grafted with PMAA.

[0014] (5) Remove the ink pattern on the surface of the titanium-based implant obtained in step (4) with ethanol to expose the remaining surface; then remove the ink pattern from the surface of the implant. 50 The peptide is dissolved and mixed with the aforementioned titanium-based implant with its remaining surface exposed. A catalyst is added, and the reaction is carried out in the dark to obtain a product simultaneously grafted with PMAA and His. 50 Titanium-based implants containing peptides;

[0015] (6) HHC36 (amino acid sequence KRWWKWWRR) antibacterial peptide solution was dropped onto the surface of the titanium-based implant obtained in step (5), and the HHC36 peptide solution was absorbed into the titanium dioxide nanotube to obtain a titanium-based implant loaded with HHC36 peptide. The steps of dropping HHC36 peptide solution and absorbing it into the titanium dioxide nanotube were repeated. Then, kaempferol solution was dropped onto the surface, and the steps of dropping kaempferol solution and absorbing it into the titanium dioxide nanotube were repeated. The implant was washed and dried to finally obtain a pH-responsive dual controlled-release antibacterial-osteogenic titanium-based implant loaded with HHC36 antibacterial peptide and kaempferol osteogenic molecules.

[0016] Further, the titanium-based implant described in step (1) is first treated with a hydrofluoric acid solution with a concentration of 1-5% by volume, preferably 4%, for 1-5 minutes, preferably 1 minute, and then used as an anode for anodizing.

[0017] Further, the electrolyte for anodic oxidation in step (1) is a mixture of (NH4)2SO4 and NH4F, wherein the concentration of (NH4)2SO4 is 0.5-5 mol / L; the concentration of NH4F is 0.1-2 wt.%; and the pH is 5±1.

[0018] Further, the parameters for anodizing in step (1) are: voltage 10-40V, electrode spacing 2-5cm, and time 0.5-3h; preferably: voltage 15-25V, electrode spacing 2cm, and time 0.5-1h.

[0019] Furthermore, the ink described in step (2) is obtained by dissolving polyvinyl butyral in (anhydrous) DMSO at a concentration of 100-800 mg / mL.

[0020] Furthermore, the polyvinyl alcohol modified ink described in step (2) is dissolved in anhydrous DMSO at a concentration of 200–500 mg / mL, preferably 450 mg / mL.

[0021] Furthermore, the coverage mentioned in step (2) refers to coverage of 25% to 75%.

[0022] Further, the preparation of dopamine solution in step (3) is as follows: dopamine is dissolved in Tris / HCl buffer solution with pH = 8.5±1, 1±0.5mol / L, and the concentration of dopamine solution is 1~5mg / mL.

[0023] Furthermore, the incubation time in step (3) is 12 to 48 hours.

[0024] Further, the preparation of PMAA solution in step (4) is as follows: PMAA is dissolved in MES buffer solution with pH = 6.0 ± 0.5 and 0.1 ± 0.05 mol / L, and the amount of PMAA used is based on its concentration in the reaction system of 0.5 to 100 mmol / L.

[0025] Further, the catalysts mentioned in step (4) are EDC and NHS, the amount of NHS is based on its concentration in the reaction system of 1 to 25 mmol / L, and the molar ratio of EDC to NHS is 1 to 25:1.

[0026] Furthermore, the reaction time of the amide in step (4) is 4 to 36 hours.

[0027] Furthermore, the ink pattern removal described in step (5) is achieved by ultrasonic treatment with anhydrous ethanol.

[0028] Furthermore, His described in step (5) 50 The polypeptides were dissolved in anhydrous ethanol at concentrations of 10–500 mmol / L.

[0029] Furthermore, the catalyst mentioned in step (5) is NaBH4, and the amount used is based on its concentration in the reaction system of 1 to 10 mM.

[0030] Furthermore, the light-protected reaction time described in step (5) is 1 to 12 hours.

[0031] Furthermore, the HHC36 antibacterial polypeptide solution and kaempferol solution mentioned in step (6) are both dissolved in ethanol, and the concentrations are both 50-1000 μmol / L.

[0032] The repeated steps described in step (6) are performed 1 to 50 times; preferably 10 to 30 times.

[0033] The process of drawing the titanium dioxide nanotubes into the air in step (6) is achieved by vacuuming, using a vacuum dryer.

[0034] The cleaning described in step (6) is performed using deionized water.

[0035] The drying process described in step (6) involves using nitrogen gas.

[0036] The PMAA mentioned in step (4) and the His mentioned in step (5) 50 The structural formulas of the HHC36 polypeptide and kaempferol mentioned in step (6) are shown in Formula I, Formula II, Formula III, and Formula V, respectively:

[0037] Formula I:

[0038] Formula II:

[0039] Formula III:

[0040] Formula V:

[0041] A pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant was prepared using the method described above.

[0042] The application of the above-mentioned pH-responsive dual controlled-release antibacterial-promoting titanium-based implant in the preparation of bone repair materials.

[0043] The technical principle of this invention is as follows: A titanium dioxide nanotube coating structure is constructed on the surface of a titanium substrate using anodizing. Dopamine is used to attach amino groups to a portion of the nanotubes, and PMAA is grafted by reacting the amino groups with the carboxyl groups on the PMAA via an amide reaction. Meanwhile, His50 peptide is grafted onto another portion by reacting titanium hydroxyl groups with thiol groups. Finally, the antibacterial peptide HHC36 and osteogenic molecule kaempferol are loaded into the nanotubes to obtain a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant.

[0044] The present invention has the following advantages and effects compared with the prior art:

[0045] (1) The pH-responsive dual controlled-release antibacterial-osteogenic titanium-based implant prepared in this invention can respond to pH changes in the surrounding solution environment and regulate the release rate of the loaded drug. Under normal physiological pH conditions, the antibacterial peptide is released slowly and the osteogenic drug is released rapidly; under pH < 7 conditions, the peptide is released rapidly and the osteogenic drug is released slowly.

[0046] (2) The pH-responsive dual controlled-release antibacterial-osteogenic titanium-based implant prepared in this invention has excellent antibacterial properties and can effectively remove bacteria and biofilms adhering to the surface.

[0047] (3) This invention uses medical titanium metal as the base material and prepares a responsive dual controlled-release system with "on-demand" release performance from the perspective of on-demand antibacterial administration and bone repair promotion. Under the condition of no bacterial infection, the osteogenic drug is released in a burst to promote bone integration under normal physiological conditions. At the same time, the antibacterial peptide is released slowly to effectively reduce the adverse effects of the peptide on the tissue. Under the condition of bacterial infection, the osteogenic drug is released slowly to reduce the waste of osteogenic drug. At the same time, the antibacterial peptide is released in a burst to quickly and efficiently kill bacteria and reduce the risk of bacterial infection. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the PMAA molecule used in the examples;

[0049] Figure 2 This is a schematic diagram of the His50 molecule used in the examples;

[0050] Figure 3 This is a schematic diagram of the structural formula of the HHC36 molecule used in the examples;

[0051] Figure 4 This is a schematic diagram of the structural formula of the kaempferol molecule used in the examples;

[0052] Figure 5 This is a scanning electron microscope image of the surface nanotubes of the pH-responsive dual controlled-release antibacterial-osteogenic titanium-based implant prepared in Example 1;

[0053] Figure 6 This is a low-magnification electron microscope image of the fluorescence morphology of the pH-responsive dual controlled-release antibacterial-osteopromoting titanium-based implant prepared in Example 1;

[0054] Figure 7 This is a comparison chart of drug loading of the pH-responsive dual controlled-release antibacterial-osteopromoting titanium-based implants prepared in Example 1;

[0055] Figure 8 This is an in vitro release curve of HHC36 peptide and kaempferol loaded into the pH-responsive dual controlled-release antibacterial-promoting osteoporotic titanium-based implant prepared in Example 1 under different pH conditions.

[0056] Figure 9 These are the antibacterial results of the pH-responsive dual controlled-release antibacterial-osteopromoting titanium-based implant prepared in Example 1, when immersed in solutions of different pH values.

[0057] Figure 10 This is a graph showing the surface circulation antibacterial results of the pH-responsive dual controlled-release antibacterial-promoting osteoporotic titanium-based implant prepared in Example 1;

[0058] Figure 11 This is a graph showing the absorbance values ​​of CCK-8 cells for the pH-responsive dual controlled-release antibacterial-osteopromoting titanium-based implant prepared in Example 1.

[0059] Figure 12 This is a scanning electron microscope image of the surface nanotubes of the pH-responsive dual controlled-release antibacterial-osteogenic titanium-based implant prepared in Example 2;

[0060] Figure 13 This is a scanning electron microscope image of the surface nanotubes of the pH-responsive dual controlled-release antibacterial-osteopromoting titanium-based implant prepared in Example 3;

[0061] Figure 14 This is a scanning electron microscope image of the surface nanotubes of the pH-responsive dual controlled-release antibacterial-osteogenic titanium-based implant prepared in Example 4;

[0062] Figure 15 This is a scanning electron microscope image of the surface nanotubes of the pH-responsive dual controlled-release antibacterial-osteogenic titanium-based implant prepared in Example 5.

[0063] Figure 16 This is a schematic diagram of a titanium-based implant with a titanium dioxide nanotube structure on its surface, prepared by anodizing according to the present invention; wherein, A is the titanium-based implant before anodizing; and B is the titanium-based implant with a titanium dioxide nanotube structure on its surface. Detailed Implementation

[0064] The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0065] Example 1

[0066] A method for preparing a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant includes the following steps:

[0067] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0068] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 0.5M ammonium sulfate and 0.2wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte;

[0069] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 10V, the anodizing time was 0.5h, and the electrode spacing was 2cm.

[0070] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0071] (5) Printing ink was prepared using polyvinyl butyral (Aladdin, catalog number: 63148-65-2): it was fully dissolved in anhydrous DMSO to 450 mg / mL, and then the ink was covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink could cover 50% of the titanium-based sample.

[0072] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 1mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 24h, and then wash it thoroughly with deionized water.

[0073] (7) PMAA (Sigma, part number: MKBS7506V, structural formula as follows) Figure 1 The product (shown) was dissolved in MES buffer solution (pH = 6.0, 0.1M). The product washed in step (6) was then immersed in MES buffer solution, and EDC / HNS was added to carry out the amide reaction. The concentration of PMAA was 0.5mM, the concentration of NHS was 1mM, the molar ratio of EDC to NHS was 2:1, and the amide reaction time was 4h.

[0074] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0075] (9) His 50 The polypeptide (synthesized by Shanghai Jier Biochemical Co., Ltd., catalog number: 1008447, structural formula as follows) Figure 2 (As shown) Dissolved in anhydrous ethanol, sodium borohydride catalyst was added, wherein the His50 concentration was 10 mM and the sodium borohydride concentration was 1 mM. The reaction was carried out in the dark for 6 hours, and then thoroughly washed with deionized water.

[0076] (10) The HHC36 polypeptide (Shanghai Qiangyao Biotechnology Co., Ltd., product number: 04010002609, structural formula as shown) Figure 3 The HHC36 polypeptide solution (shown) was dissolved in an ethanol solution (pH=5) to obtain a concentration of 100 μM. The grafted PMAA and His obtained in step (9) were then used in the reaction. 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0077] (11) Kaempferol (Sigma, product number: 60010, structural formula as follows) Figure 4The kaempferol solution (shown) was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 0.1 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of adding kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant.

[0078] The surface morphology of titanium nanotubes in a pH-responsive dual-release antibacterial-osteogenic titanium-based implant was observed using scanning electron microscopy. Nanotubes prepared by anodic oxidation exhibited stable morphology, as shown in the scanning electron microscopy results. Figure 5 As shown, the average diameter of the nanotubes was observed to be 67 nm, and the depth was 482 nm. Fluorescence images obtained after grafting with fluorescent phyllomolecules are shown below. Figure 6 As shown, the results indicate that the phyllomolecules are distributed in a banded pattern on the titanium-based surface. The loading amounts of HHC antimicrobial peptides and kaempferol loaded into the pH-responsive dual-controlled-release antibacterial-promoting bone-enhancing titanium-based implants are compared to... Figure 7 As shown, the results indicate that the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant contains 161.9 μg of HHC36 antimicrobial peptide and 100.5 μg of kaempferol.

[0079] The release rates of HHC36 peptide and kaempferol in pH-responsive dual-controlled-release antibacterial-osteopromoting titanium-based implants were determined using ultraviolet absorption spectroscopy. The pH-responsive dual-controlled-release antibacterial-osteopromoting titanium-based implants were immersed in PBS solutions at pH = 5 and pH = 7.4, and placed on a constant-temperature shaker (37℃, 87 rpm). 200 μL of the incubation solution was collected at specific time points of 0.5, 1, 2, 4, 8, 12, and 24 h, and the release amounts of HHC36 peptide and kaempferol in the pH-responsive dual-controlled-release antibacterial-osteopromoting titanium-based implants were measured using ultraviolet absorption spectroscopy. The results are as follows: Figure 8 As shown, the release rate of HHC36 antimicrobial peptides at pH 5 was significantly higher than that at pH 7.4, while the release rate of kaempferol at pH 5 was significantly lower than that at pH 7.4. This indicates that in a simulated normal microenvironment at pH 7.4, the antimicrobial drug is released slowly, reducing unnecessary release under normal physiological conditions, while a large amount of osteogenic drugs are released to improve osteogenic performance. Conversely, in a simulated bacterial infection environment at pH 5, the osteogenic drug is released slowly, reducing burst release under infected conditions, while a large amount of antimicrobial drugs are released, significantly improving the antimicrobial effect.

[0080] The pH-responsive dual-release antibacterial-promoting osteoporotic titanium-based implants were tested at different pH levels using an agar plate counting method. The bacterial suspension (Staphylococcus aureus ATCC 29213, purchased from VWR International, LLC, was diluted to 1×10⁻⁶ using PBS solutions at pH 5 and pH 7.4. 6 CFU / mL was added dropwise to 20 μL of a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant and a titanium sheet containing titanium dioxide nanotubes loaded with antimicrobial peptides and kaempferol. After incubation for 2 hours, the solution was diluted and plated. The antibacterial activity of each experimental group was determined by counting. Figure 9 As shown. The results indicate that the experimental group with titanium dioxide nanotubes loaded with antimicrobial peptides and kaempferol exhibited antibacterial activity of over 98% at both pH=5 and pH=7.4, without any responsive antibacterial effect. The pH-responsive dual-controlled-release antimicrobial-promoting titanium-based implant achieved an antibacterial performance of 98.98% in a microenvironment simulating bacterial infection at pH=5 due to the burst release of antimicrobial drugs, while in a normal microenvironment simulating pH=7.4, the antibacterial performance was only 64.24% due to slow release, demonstrating the pH-responsive antibacterial properties of the pH-responsive dual-controlled-release antimicrobial-promoting titanium-based implant. The preparation of the titanium nanotube-loaded antimicrobial peptide and kaempferol titanium sheet is as follows:

[0081] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0082] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 0.5M ammonium sulfate and 0.2wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte;

[0083] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the negative electrode and the platinum sheet as the positive electrode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 10V, the anodizing time was 0.5h, and the electrode spacing was 2cm.

[0084] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0085] (5)(10) Dissolve the HHC36 peptide in an ethanol solution (pH=5) to obtain a 100 μM HHC36 peptide solution. Then, combine the grafted PMAA and His obtained in step (9). 50The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0086] (6) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 0.1 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of adding kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The nanotubes were then washed with deionized water and dried with nitrogen gas to obtain titanium sheets loaded with antimicrobial peptides and kaempferol.

[0087] The antibacterial experiment was repeated three times, and the results are shown in the figure below. Figure 10 As shown, both the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant and the titanium dioxide nanotube loaded with antimicrobial peptides and kaempferol group exhibited antibacterial activity exceeding 99.99% in the first antibacterial experiment. In subsequent cyclic antibacterial experiments, the antibacterial activity of the titanium dioxide nanotube loaded with antimicrobial peptides and kaempferol group decreased with each cycle, reaching 86.74% in the second cycle and 29.69% in the third cycle. In contrast, the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant demonstrated stable release, exhibiting excellent antibacterial activity of 99.31% and 87.59% in the second and third cycles, respectively.

[0088] Seeds with a density of 1×10⁻⁶ were applied to the surfaces of unmodified pure titanium-based implants and pH-responsive dual-release antibacterial-osteogenic titanium-based implants. 4 Cells were subjected to CCK-8 assays on days 1 and 3, and the absorbance values ​​of each sample were measured. The results are as follows: Figure 11 As shown, the absorbance value of the pH-responsive dual-controlled-release antibacterial-promoting titanium-based implant group was higher than that of the unmodified pure titanium-based implant group, indicating that the pH-responsive dual-controlled-release antibacterial-promoting titanium-based implant group has good biocompatibility and no cytotoxicity.

[0089] Example 2

[0090] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0091] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 1M ammonium sulfate and 0.3wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte.

[0092] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 15V, the anodizing time was 0.5h, and the electrode spacing was 2cm.

[0093] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0094] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 450 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 33% of the titanium-based sample.

[0095] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 1.5 mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 24 h, and then wash it thoroughly with deionized water.

[0096] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 1mM, the concentration of NHS is 1mM, the molar ratio of EDC to NHS is 1:1, and the amide reaction time is 4h.

[0097] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0098] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 15 mM and the sodium borohydride concentration was 2 mM. The reaction was carried out in the dark for 6 hours, and then thoroughly washed with deionized water.

[0099] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain a 100 μM HHC36 peptide solution. Then, combine the grafted PMAA and His obtained in step (9). 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0100] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 0.1 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping the kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0101] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy. Figure 12 The release rates of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting bone titanium-based implant were tested using ultraviolet absorption spectroscopy; the antibacterial properties of the pH-responsive dual controlled-release antibacterial-promoting bone titanium-based implant were tested using the agar plate counting method.

[0102] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0103] Example 3

[0104] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0105] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 2M ammonium sulfate and 0.5wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte.

[0106] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 20V, the anodizing time was 0.5h, and the electrode spacing was 2cm.

[0107] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0108] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 450 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 25% of the titanium-based sample.

[0109] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 2mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 24h, and then wash it thoroughly with deionized water.

[0110] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 5mM, the concentration of NHS is 1mM, the molar ratio of EDC to NHS is 5:1, and the amide reaction time is 8h.

[0111] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0112] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 20 mM and the sodium borohydride concentration was 5 mM. The reaction was carried out in the dark for 6 hours, and then thoroughly washed with deionized water.

[0113] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain a 200 μM HHC36 peptide solution. Then, combine the grafted PMAA and His obtained in step (9). 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0114] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 0.2 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping the kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0115] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy. Figure 13The release rates of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting bone titanium-based implant were tested using ultraviolet absorption spectroscopy; the antibacterial properties of the pH-responsive dual controlled-release antibacterial-promoting bone titanium-based implant were tested using the agar plate counting method.

[0116] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0117] Example 4

[0118] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0119] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 1M ammonium sulfate and 0.8wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte.

[0120] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 20V, the anodizing time was 0.5h, and the electrode spacing was 2cm.

[0121] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0122] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 450 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 66% of the titanium-based sample.

[0123] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 5mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 24h, and then wash it thoroughly with deionized water.

[0124] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 5mM, the concentration of NHS is 2mM, the molar ratio of EDC to NHS is 2.5:1, and the amide reaction time is 12h.

[0125] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0126] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 50 mM and the sodium borohydride concentration was 5 mM. The reaction was carried out in the dark for 6 hours, and then thoroughly washed with deionized water.

[0127] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain a 500 μM HHC36 peptide solution. Then, combine the grafted PMAA and His obtained in step (9). 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0128] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 0.5 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0129] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy. Figure 14 The release rates of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting bone titanium-based implant were tested using ultraviolet absorption spectroscopy; the antibacterial properties of the pH-responsive dual controlled-release antibacterial-promoting bone titanium-based implant were tested using the agar plate counting method.

[0130] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0131] Example 5

[0132] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0133] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a 1M ammonium sulfate and 1wt% ammonium fluoride mixture, and adjust the pH of the solution to 5 with sulfuric acid to prepare an electrolyte.

[0134] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 25V, the anodizing time was 0.5h, and the electrode spacing was 2cm.

[0135] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0136] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 450 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 75% of the titanium-based sample.

[0137] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 2mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 24h, and then wash it thoroughly with deionized water.

[0138] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 10mM, the concentration of NHS is 2mM, the molar ratio of EDC to NHS is 5:1, and the amide reaction time is 24h.

[0139] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0140] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 100 mM and the sodium borohydride concentration was 5 mM. The reaction was carried out in the dark for 6 hours, and then thoroughly washed with deionized water.

[0141] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain a 1000 μM HHC36 peptide solution. Then, combine the grafted PMAA and His obtained in step (9). 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0142] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 1 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0143] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy. Figure 15 The release rates of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting bone titanium-based implant were tested using ultraviolet absorption spectroscopy; the antibacterial properties of the pH-responsive dual controlled-release antibacterial-promoting bone titanium-based implant were tested using the agar plate counting method.

[0144] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0145] Example 6

[0146] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0147] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 1M ammonium sulfate and 0.5wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte.

[0148] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 20V, the anodizing time was 1h, and the electrode spacing was 2cm.

[0149] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0150] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 450 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 50% of the titanium-based sample.

[0151] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 2mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 24h, and then wash it thoroughly with deionized water.

[0152] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 50mM, the concentration of NHS is 2mM, the molar ratio of EDC to NHS is 25:1, and the amide reaction time is 24h.

[0153] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0154] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 200 mM and the sodium borohydride concentration was 5 mM. The reaction was carried out in the dark for 6 hours, and then thoroughly washed with deionized water.

[0155] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain a 1000 μM HHC36 peptide solution. Then, combine the grafted PMAA and His obtained in step (9). 50The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0156] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 1 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0157] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy; the release rate of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by ultraviolet absorption spectroscopy; the antibacterial performance of the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by agar plate counting method.

[0158] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0159] Example 7

[0160] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0161] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 2M ammonium sulfate and 1wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte.

[0162] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 18V, the anodizing time was 1h, and the electrode spacing was 2cm.

[0163] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0164] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 500 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 33% of the titanium-based sample.

[0165] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 1 mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 12 h, and then wash it thoroughly with deionized water.

[0166] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 50mM, the concentration of NHS is 5mM, the molar ratio of EDC to NHS is 10:1, and the amide reaction time is 12h.

[0167] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0168] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 500 mM and the sodium borohydride concentration was 10 mM. The reaction was carried out in the dark for 6 hours, and then thoroughly washed with deionized water.

[0169] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain an HHC36 peptide solution with a concentration of 1500 μM. Then, combine the grafted PMAA and His obtained in step (9). 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0170] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 2 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping the kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0171] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy; the release rate of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by ultraviolet absorption spectroscopy; the antibacterial performance of the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by agar plate counting method.

[0172] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0173] Example 8

[0174] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0175] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 5M ammonium sulfate and 2wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte.

[0176] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 15V, the anodizing time was 1h, and the electrode spacing was 2cm.

[0177] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0178] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 600 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 25% of the titanium-based sample.

[0179] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 2mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 48h, and then wash it thoroughly with deionized water.

[0180] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 50mM, the concentration of NHS is 10mM, the molar ratio of EDC to NHS is 5:1, and the amide reaction time is 8h.

[0181] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0182] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 500 mM and the sodium borohydride concentration was 10 mM. The reaction was carried out in the dark for 6 hours, and then thoroughly washed with deionized water.

[0183] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain an HHC36 peptide solution with a concentration of 1500 μM. Then, combine the grafted PMAA and His obtained in step (9). 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0184] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 2 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping the kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0185] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy; the release rate of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by ultraviolet absorption spectroscopy; the antibacterial performance of the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by agar plate counting method.

[0186] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0187] Example 9

[0188] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0189] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 1M ammonium sulfate and 2wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte.

[0190] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 18V, the anodizing time was 1h, and the electrode spacing was 2cm.

[0191] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0192] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 450 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 66% of the titanium-based sample.

[0193] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 5mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 8h, and then wash it thoroughly with deionized water.

[0194] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 50mM, the concentration of NHS is 25mM, the molar ratio of EDC to NHS is 2:1, and the amide reaction time is 12h.

[0195] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0196] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 500 mM and the sodium borohydride concentration was 5 mM. The reaction was carried out in the dark for 12 h, and then thoroughly washed with deionized water.

[0197] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain an HHC36 peptide solution with a concentration of 2500 μM. Then, combine the grafted PMAA and His obtained in step (9). 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0198] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 2.5 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0199] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy; the release rate of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by ultraviolet absorption spectroscopy; the antibacterial performance of the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by agar plate counting method.

[0200] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0201] Example 10

[0202] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0203] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 1M ammonium sulfate and 0.5wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte.

[0204] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 20V, the anodizing time was 0.5h, and the electrode spacing was 2cm.

[0205] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0206] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 400 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 75% of the titanium-based sample.

[0207] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 5mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 12h, and then wash it thoroughly with deionized water.

[0208] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 100mM, the concentration of NHS is 25mM, the molar ratio of EDC to NHS is 4:1, and the amide reaction time is 24h.

[0209] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0210] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 100 mM and the sodium borohydride concentration was 5 mM. The reaction was carried out in the dark for 12 h, and then thoroughly washed with deionized water.

[0211] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain a 1000 μM HHC36 peptide solution. Then, combine the grafted PMAA and His obtained in step (9). 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0212] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 1 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0213] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy; the release rate of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by ultraviolet absorption spectroscopy; the antibacterial performance of the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by agar plate counting method.

[0214] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0215] Example 11

[0216] (1) The titanium-based implant was ultrasonically cleaned for 10 min each with acetone, ethanol and deionized water, and then treated with hydrofluoric acid at a concentration of 4% by volume for 1 min.

[0217] (2) Dissolve ammonium sulfate and ammonium fluoride in deionized water to obtain a mixture of 1M ammonium sulfate and 0.4wt% ammonium fluoride, and adjust the pH of the solution to 5 using sulfuric acid to prepare an electrolyte.

[0218] (3) Using the titanium-based implant treated with hydrofluoric acid in step (1) as the anode and the platinum sheet as the cathode, anodizing was performed using an electrochemical workstation in constant voltage mode, wherein the voltage was 20V, the anodizing time was 0.5h, and the electrode spacing was 2cm.

[0219] (4) Rinse the titanium-based implant with titanium dioxide nanotube structure on the surface after anodizing with deionized water and dry it in a drying oven at 60°C.

[0220] (5) The specific polyvinyl alcohol modified ink is fully dissolved to 450 mg / mL by anhydrous DMSO, and then the ink is covered on the titanium-based surface in step (4) by planar 3D printing technology. By setting the printing air pressure and printing spacing, the ink can cover 50% of the titanium-based sample.

[0221] (6) Dissolve dopamine in Tris / HCl buffer solution (pH=8.5, 1M) with a dopamine solution concentration of 5mg / mL, and immerse the dried titanium-based implant with titanium dioxide nanotube structure in the dopamine solution for 24h, and then wash it thoroughly with deionized water.

[0222] (7) Dissolve PMAA in MES buffer solution (pH = 6.0, 0.1M), soak the product washed in step (6) in MES buffer solution, and add EDC / HNS to carry out amide reaction. The concentration of PMAA is 100mM, the concentration of NHS is 5mM, the molar ratio of EDC to NHS is 25:1, and the amide reaction time is 24h.

[0223] (8) Remove the ink from the surface covered in step (7) by ultrasonication with anhydrous ethanol for 5 minutes, and then clean it with deionized water and dry it in a drying oven at 60°C.

[0224] (9) His 50 The peptide was dissolved in anhydrous ethanol, and sodium borohydride was added as a catalyst. The His50 concentration was 100 mM and the sodium borohydride concentration was 5 mM. The reaction was carried out in the dark for 6 hours, and then thoroughly washed with deionized water.

[0225] (10) Dissolve the HHC36 peptide in an ethanol solution to obtain a 1000 μM HHC36 peptide solution. Then, combine the grafted PMAA and His obtained in step (9). 50 The titanium-based implant was then placed in a vacuum desiccator, and 50 μL of HHC36 peptide solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. This process of adding HHC36 peptide solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then rinsed with deionized water and dried with nitrogen.

[0226] (11) Kaempferol was dissolved in an ethanol solution (pH=5) to obtain a kaempferol solution concentration of 1 mg / mL. The titanium-based implant from step (10) was placed in a vacuum desiccator, and 50 μL of kaempferol solution was dropped onto its surface. The solution was then drawn into the titanium dioxide nanotubes using a vacuum method. The process of dropping kaempferol solution and drawing the solution into the titanium dioxide nanotubes was repeated 20 times. The implant was then washed with deionized water and dried with nitrogen gas to finally obtain a pH-responsive dual-controlled-release antibacterial-bone-promoting titanium-based implant.

[0227] (12) The morphology of the prepared nanotubes was observed by field emission scanning electron microscopy; the release rate of HHC36 peptide and kaempferol in the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by ultraviolet absorption spectroscopy; the antibacterial performance of the pH-responsive dual controlled-release antibacterial-promoting titanium-based implant was tested by agar plate counting method.

[0228] The results showed that the release rate of HHC36 antimicrobial peptides was significantly higher at pH=5 than at pH=7.4, while the release rate of kaempferol was significantly lower at pH=5 than at pH=7.4; and that antimicrobial drugs were released burstily at pH=5 simulating a bacterial infection microenvironment, while they were released slowly at pH=7.4 simulating a normal microenvironment.

[0229] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant, characterized in that: Includes the following steps: (1) Using a titanium-based implant as the anode and a platinum sheet as the cathode, after anodizing under constant voltage mode, the implant is thoroughly rinsed and dried to obtain a titanium-based implant with a titanium dioxide nanotube structure on the surface. (2) The surface of the titanium-based implant obtained in step (1) is partially covered by the polyvinyl butyral ink pattern using 3D printing technology, resulting in a titanium-based implant with part of the surface covered. (3) Prepare a dopamine solution under alkaline conditions, immerse the titanium-based implant obtained in step (2) in the aforementioned dopamine solution, incubate in the dark, wash and dry to obtain a titanium-based implant with amino reactive groups introduced on the surface of the nanotube; (4) Prepare a PMAA solution in an aqueous solution, and immerse the titanium-based implant obtained in step (3) into the aforementioned PMAA solution. Under the condition of introducing a catalyst, carry out an amide reaction, wash, and dry to obtain a titanium-based implant grafted with PMAA. (5) removing the ink pattern on the surface of the titanium-based implant obtained in step (4) with ethanol to expose the remaining surface; mixing the His 50 polypeptide with the titanium-based implant whose remaining surface is exposed as mentioned above, adding a catalyst, and reacting in the dark to obtain a titanium-based implant grafted with PMAA and His 50 polypeptide at the same time; and (6) removing the titanium-based implant grafted with PMAA and His (6) HHC36 antibacterial peptide solution was dropped onto the surface of the titanium-based implant obtained in step (5), and the HHC36 peptide solution was absorbed into the titanium dioxide nanotube to obtain a titanium-based implant loaded with HHC36 peptide. The steps of dropping HHC36 peptide solution and absorbing it into the titanium dioxide nanotube were repeated. Then, kaempferol solution was dropped onto the surface, and the steps of dropping kaempferol solution and absorbing it into the titanium dioxide nanotube were repeated. The implant was washed and dried to finally obtain a pH-responsive dual controlled-release antibacterial-osteogenic titanium-based implant loaded with HHC36 antibacterial peptide and kaempferol osteogenic molecules.

2. The method for preparing the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant according to claim 1, characterized in that: The titanium-based implant described in step (1) is first treated with a hydrofluoric acid solution with a concentration of 1-5% by volume for 1-5 minutes and then used as an anode for anodizing. The electrolyte for anodic oxidation mentioned in step (1) is a mixture of (NH4)2SO4 and NH4F, wherein the concentration of (NH4)2SO4 is 0.5-5 mol / L; the concentration of NH4F is 0.1-2 wt.%; and the pH is 5±1. The parameters for anodizing in step (1) are: voltage 10-40V, electrode spacing 2-5cm, and time 0.5-3h.

3. The method for preparing the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant according to claim 1, characterized in that: The ink mentioned in step (2) is obtained by dissolving polyvinyl butyral in DMSO, with a concentration of 100-800 mg / mL; The polyvinyl butyral ink described in step (2) is dissolved in anhydrous DMSO at a concentration of 200–500 mg / mL. The coverage mentioned in step (2) refers to coverage of 25% to 75%.

4. The method for preparing the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant according to claim 1, characterized in that: The preparation of dopamine solution in step (3) is as follows: Dopamine is dissolved in Tris / HCl buffer solution with pH = 8.5±1 and 1±0.5 mol / L, and the concentration of dopamine solution is 1 to 5 mg / mL; The incubation time in step (3) is 12 to 48 hours.

5. The method for preparing the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant according to claim 1, characterized in that: The preparation of PMAA solution in step (4) is as follows: PMAA is dissolved in MES buffer solution with pH = 6.0 ± 0.5 and 0.1 ± 0.05 mol / L. The amount of PMAA used is based on its concentration in the reaction system of 0.5 to 100 mmol / L. The catalysts mentioned in step (4) are EDC and NHS. The amount of NHS used is based on its concentration in the reaction system of 1 to 25 mmol / L, and the molar ratio of EDC to NHS is 1 to 25:

1. The reaction time for the amide in step (4) is 4 to 36 hours.

6. The method for preparing the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant according to claim 1, characterized in that: The ink pattern removal described in step (5) is achieved by ultrasonic treatment with anhydrous ethanol; His mentioned in step (5) 50 The polypeptides were dissolved in anhydrous ethanol at concentrations of 10–500 mmol / L. The catalyst mentioned in step (5) is NaBH4, and its amount is calculated based on its concentration in the reaction system as 1 to 10 mM. The light-protected reaction time described in step (5) is 1 to 12 hours.

7. The method for preparing the pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant according to claim 1, characterized in that: The HHC36 antibacterial peptide solution and kaempferol solution mentioned in step (6) are both dissolved in ethanol, and the concentrations are both 50-1000 μmol / L; The repeated steps described in step (6) are all performed 1 to 50 times; The process of drawing the titanium dioxide nanotubes into the air in step (6) is achieved by vacuuming, and the equipment used for vacuuming is a vacuum dryer. The cleaning described in step (6) is performed using deionized water; The drying process described in step (6) involves using nitrogen gas.

8. The method for preparing a pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant according to any one of claims 1-7, characterized in that: The PMAA mentioned in step (4) and the His mentioned in step (5) 50 The structural formulas of the HHC36 polypeptide and kaempferol mentioned in step (6) are shown in Formula I, Formula II, Formula III, and Formula V, respectively: Formula I: ; Formula II: ; Formula III: ; Formula V: .

9. A pH-responsive dual-controlled-release antibacterial-osteogenic titanium-based implant, characterized in that: It is obtained by the preparation method described in any one of claims 1 to 8.

10. The application of the pH-responsive dual controlled-release antibacterial-osteogenic titanium-based implant as described in claim 9 in the preparation of bone repair materials.

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