Dynamic bionic coating with controllable topological structure as well as preparation method and application of dynamic bionic coating

Abstract

The invention discloses a controllable topological structure dynamic bionic coating as well as a preparation method and application thereof, and belongs to the technical field of medical materials. The preparation method comprises the following steps: firstly, constructing JNHDs by ferroferric oxide nanoparticles and silver nanoparticles in a sodium citrate solution through a photo-reduction reaction, then coating the surface with aminated silicon dioxide through a silanization reaction, then carrying out thiolation on a substrate containing a silicon dioxide layer, and finally, incubating PEGylated SiO2 (at) JNHDs and the thiolated substrate together, so as to obtain the silicon dioxide coated JNHDs. And preparing. The dynamic bionic coating constructed by the invention has long-term effectiveness, can damage a biological membrane through mechanical force under the activation of an external electromagnetic field, promotes the mesenchymal stem cells to be differentiated into osteoblasts by activating mechanical force sensitive calcium ion channels on the surfaces of the mesenchymal stem cells, overcomes the defects that a static bionic material cannot actively respond and dynamically intervene in a'surface competition ', and has a good application prospect. Finally, the purposes of preventing and treating periprosthetic infection and promoting prosthesis-osseointegration are achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a controllable topological structure dynamic biomimetic coating, its preparation method, and its application. Background Technology

[0002] Total joint replacement surgery is currently the most effective treatment for improving motor function and quality of life in patients with end-stage osteoarthritis. However, a serious complication after total joint replacement surgery is periprosthetic infection. Current treatment for periprosthetic infection typically involves more invasive revision surgery, resulting in longer hospital stays and higher medical costs, placing a significant burden on both patients and hospitals. Therefore, effectively preventing and treating periprosthetic infection is a critical challenge that urgently needs to be addressed.

[0003] For periprosthetic infections following total joint replacement surgery, *Staphylococcus epidermidis* and *Staphylococcus aureus* are the main pathogens. Upon contact with the prosthesis, these pathogens secrete extracellular polymers, irreversibly adhering to the prosthesis surface. As the pathogens multiply and the extracellular polymers accumulate, a fortress-like biofilm gradually matures. Biofilm formation is crucial for the persistent nature of periprosthetic infections and severely hinders the integration of the implant with bone tissue (osseous integration). According to the "surface race" theory, pathogens and host cells (such as mesenchymal stem cells) compete to adhere to the prosthesis surface. The first to successfully adhere will "occupy" the prosthesis surface and "resist" the other's adhesion. Ideally, mesenchymal stem cells win the "surface race," forming a protective membrane composed of extracellular matrix and mineral deposits on the prosthesis surface. This prevents pathogen adhesion and biofilm formation, ultimately leading to strong osseous integration.

[0004] Titanium (Ti) and its alloys have been widely used in clinical practice for many years. With the development of nanotechnology, many scholars have devoted themselves to improving the antibacterial properties and promoting cell integration of titanium through the design and modification of nanostructures on the titanium surface, such as the preparation of titanium dioxide nanotube arrays modified with silver nanoparticles. However, most of these strategies are based on static biomimetic materials, whose antibacterial activity is fixed. They cannot actively respond and clear pathogens after bacteria have adhered, nor can they dynamically promote cell behavior to win the "surface race".

[0005] Therefore, how to construct a smart biomimetic material that can respond to external stimuli and whose surface topology and function can be dynamically changed, in order to make up for the technical shortcomings of existing static biomimetic materials, has very important research significance and clinical application value. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a controllable topological structure dynamic biomimetic coating, its preparation method, and its application, thereby solving the problems of existing static biomimetic materials being unable to actively respond, dynamically intervene in the "surface race," and struggling to achieve thorough antibacterial properties and stable osseointegration.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A first aspect of the present invention provides a method for preparing a controllable topological structure dynamic biomimetic coating, comprising the following steps: S1. Janus nanoheteromers were prepared by photoreduction reaction of Fe3O4 nanoparticles, trisodium citrate and silver ion salt solution. S2. The Janus nano-heteromeric dimer obtained in S1 is first subjected to hydrolysis and condensation reaction with ammonia and tetraethyl orthosilicate in a solvent, and then reacted with (3-aminopropyl)triethoxysilane in a solvent to obtain amino-functionalized SiO2@JNHDs. S3. The amino-functionalized SiO2@JNHDs obtained in S2 are reacted with a flexible polymer linker and N,N-diisopropylethylamine in a solvent to prepare PEGylated SiO2@JNHDs. S4. React a substrate with a silica layer on its surface with (3-mercaptopropyl)trimethoxysilane in a solvent to obtain a thiolized substrate; S5. The PEGylated SiO2@JNHDs obtained in S3 and the thiolized substrate obtained in S4 are co-incubated to obtain a dynamic biomimetic coating with controllable topology.

[0008] The beneficial effects of this invention are as follows: By constructing Fe3O4-Ag Janus nanoparticles with an asymmetric structure, this invention integrates the magnetic responsiveness of Fe3O4 and the inherent antibacterial properties of Ag. Utilizing a silica shell for coating and amino functionalization provides excellent biocompatibility and active sites for further chemical modification. The nanoparticles are "anchored" to the Ti substrate surface via long-chain flexible PEG linkers, creating a unique "controllable topology." Under the influence of an external magnetic field, the magnetic Janus nanoparticles deflect and move, thereby dynamically altering the microstructure and physical stimulation of the material surface. This dynamic property can mechanically interfere with and disrupt the structure of early bacterial biofilms, while simultaneously providing dynamic mechanical signals to mesenchymal stem cells, activating their mechanosensitive ion channels (such as Piezo1), promoting osteogenic differentiation, and thus dynamically intervening in and helping cells win the "surface race."

[0009] Furthermore, S1 specifically includes the following steps: S101. Disperse Fe3O4 nanoparticles in a trisodium citrate solution and stir until the dispersion is uniform. S102. Add silver ion salt solution to the uniformly dispersed system obtained in S101 and continue stirring. S103. After stirring S102, the resulting mixture was irradiated with ultraviolet light to obtain Janus nano-heterogeneous dimers.

[0010] Furthermore, the stirring temperature in S101 is 50-70℃; The stirring temperature in S102 is 50-70℃, and the time is 1-3 h; the silver ion salt includes AgNO3; The UV irradiation time in S103 is 20-40 min.

[0011] Furthermore, in S101, the volume ratio of Fe3O4 nanoparticles to trisodium citrate solution is 0.1-1:10-30; the concentration ratio of trisodium citrate solution is 20%-30% w / v.

[0012] Furthermore, the silver ion salt solution in S102 is an AgNO3 solution with a concentration of 5-20 mmol / L; the volume ratio of the silver ion salt solution to the trisodium citrate solution is 0.1-0.5:10-30.

[0013] Furthermore, S2 specifically includes the following steps: S201. Janus nano-heterodimer was dispersed in an ethanol solution containing ammonia, and tetraethyl orthosilicate was added to carry out a hydrolysis-condensation reaction to obtain SiO2@JNHDs. S202. SiO2@JNHDs were reacted with (3-aminopropyl)triethoxysilane in ethanol to prepare amino-functionalized SiO2@JNHDs.

[0014] Furthermore, the hydrolysis-condensation reaction in S201 was carried out at room temperature for 1-3 hours. The reaction in S202 was carried out at room temperature for 10-36 hours.

[0015] Furthermore, in S201, the mass-to-volume ratio of Janus nanoheterodimer, ethanol solution containing ammonia, and tetraethyl orthosilicate is 100-500 μg: 500-700 mL: 0.1-0.3 μL; and the volume ratio of ammonia, water, and ethanol in the ethanol solution containing ammonia is 10-50: 50-100: 400-600.

[0016] Furthermore, the mass-to-volume ratio of (3-aminopropyl)triethoxysilane, ethanol in S202 and Janus nanoheterodimer in S201 is 5-20 μL: 400-600 μL: 100-500 μg.

[0017] Furthermore, the flexible polymer linker in S3 is polyethylene glycol with N-hydroxysuccinimide ester and maleimide groups at both ends, with a molecular weight of 3000-10000 Da; the reaction temperature is room temperature and the reaction time is 10-36 h.

[0018] The beneficial effects of adopting the above-mentioned further technical solution are as follows: The present invention uses polyethylene glycol with a molecular weight of 3000-10000 Da, which has N-hydroxysuccinimide ester and maleimide groups at both ends, as a flexible polymer linker to ensure that it has sufficient length and flexibility, providing space for the dynamic movement of nanoparticles.

[0019] Furthermore, the mass-to-volume ratio of the flexible polymer linker, N,N-diisopropylethylamine, and solvent in S3 is 1-10 mg: 1-3 μL: 400-600 μL; the solvent is water.

[0020] Furthermore, S4 specifically includes the following steps: S401. First, immerse the substrate in a suspension of hydroxyapatite nanoparticles, and then dry it at room temperature to obtain a substrate containing a layer of hydroxyapatite nanoparticles. S402: The substrate containing hydroxyapatite nanoparticles obtained in S401 is first dispersed in ammonia and tetraethyl orthosilicate in a solvent to carry out a hydrolysis and condensation reaction to obtain a substrate containing silica. S403: The substrate containing the silica layer obtained in S402 is subjected to thiolation treatment with (3-mercaptopropyl)trimethoxysilane in a solvent to obtain a thiolated substrate. The substrate is Ti or titanium alloy.

[0021] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: By depositing a layer of hydroxyapatite nanoparticles before constructing the silica layer, the present invention effectively enhances the biological activity and subsequent modification effect.

[0022] Furthermore, the concentration of the hydroxyapatite nanoparticle suspension in S401 was 0.5-2 mg / mL, and the drying time at room temperature was 12-36 h.

[0023] Further, in S402, the substrate containing the hydroxyapatite nanoparticle layer is first dispersed in a mixed solution of ammonia, water and ethanol in a volume ratio of 10-50:50-100:400-600, and shaken for 10-20 min; then tetraethyl orthosilicate is added (0.1-0.3 μL per well of a 24-well plate), and shaken for another 1-3 h.

[0024] Furthermore, the volume ratio of (3-mercaptopropyl)trimethoxysilane to solvent in S403 is 10-50:450-500; the thiolation treatment is carried out at room temperature for 20-28 hours.

[0025] Furthermore, the incubation temperature in S5 is room temperature, and the incubation time is 12-20 h.

[0026] The beneficial effects of adopting the above-mentioned further technical solution are as follows: The present invention performs a thiol-maleimide click chemical reaction by co-incubating PEGylated nanoparticles and thiolized substrates at room temperature, so that the maleimide groups at the end of PEG react with the thiol groups on the substrate surface in a highly efficient and specific manner to form a stable thioether covalent bond.

[0027] In a second aspect, the present invention provides a controllable topological structure dynamic biomimetic coating, which is prepared by the above-described preparation method.

[0028] A third aspect of the present invention provides the application of the above-described controllable topological structure dynamic biomimetic coating in the preparation of orthopedic implants.

[0029] The present invention has the following beneficial effects: 1. Dynamic Intervention in the "Surface Race": This invention abandons the traditional static coating approach and creatively constructs an interface with a dynamically changing surface topology. Under external magnetic field stimulation, grafted Janus nanoparticles can generate micron / nanoscale mechanical movements. This dynamic signal can preferentially inhibit bacterial adhesion and biofilm formation, while selectively promoting the adhesion, spreading, and osteogenic differentiation of mesenchymal stem cells, thereby intelligently intervening in and helping host cells win the "surface race."

[0030] 2. Multiple antibacterial mechanisms: This invention combines the chemical antibacterial properties of Ag nanoparticles with the physical antibacterial properties of Fe3O4 nanoparticles. Dynamic mechanical movement can directly disrupt the established biofilm structure, overcoming the shortcomings of static materials in being ineffective against mature biofilms, thus achieving "integrated prevention and treatment".

[0031] 3. Promote osteogenic integration: This invention uses the dynamic mechanical force generated by magnetic field control to activate the mechanosensitive ion channel Piezo1 on the surface of mesenchymal stem cells, triggering calcium ion influx and thereby regulating the expression of downstream osteogenic-related genes, thus actively promoting osteogenic integration from a biological mechanism perspective.

[0032] 4. Stable Construction: This invention uses an advanced click chemistry method to covalently graft nanoparticles onto the substrate through flexible long chains, resulting in a strong connection and good stability. This avoids the risk of nanoparticles falling off during long-term use or rinsing with bodily fluids, ensuring the durability of the coating function. Attached Figure Description

[0033] Figure 1 A scanning electron microscope image of a cicada's wing; Figure 2 The diagram shows the construction and working principle of the controllable topological dynamic biomimetic coating, where a is a schematic diagram of the synthesis of the controllable topological dynamic biomimetic coating, and b is a schematic diagram of the working principle of the controllable topological dynamic biomimetic coating under an applied electromagnetic field. Figure 3 The images show the physical characterization properties of Fe3O4-Ag Janus nanoheterodimers. In the images, A is a transmission electron microscope image, BD are the elemental distribution maps of Fe, Ag and O respectively, E is a fusion map of all elemental distributions, F is an EDS analysis map, G is a nanoparticle size map, and H is a paramagnetic characterization result map. Figure 4 The diagram shows the physical characterization properties of the controllable topological structure dynamic biomimetic coating. In the diagram, A is a scanning electron microscope image, B is an atomic force microscope image, C is an elemental distribution map, D is a roughness distribution map, and E is an EDS analysis map. Figure 5 The diagram shows the pulsed electromagnetic field device and the electromagnetic field intensity distribution. In the diagram, A is the electromagnetic field waveform generator, B is the electromagnetic field signal amplifier, C is the Helmholtz coil used to generate a uniform magnetic field, D is the solenoid used to generate a non-uniform magnetic field, and E and F are the magnetic field distribution simulation diagrams of the Helmholtz coil and the solenoid, respectively. Figure 6 The image shows the test results of the bactericidal ability of the controllable topology dynamic biomimetic coating. In the image, A is the staining test result of live / dead bacteria, B is the scanning electron microscope image, and C is the plate antibacterial test result. Figure 7 The images show the bone regeneration capacity of the controllable topological structure dynamic biomimetic coating. In the images, A is the Western blot result, B is the alizarin red staining image, and C is the ALP staining image. Figure 8 The image shows the Western blot results of the expression of the mechanosensitive calcium channel piezo1 in mesenchymal stem cells. Figure 9 The figure shows the experimental characterization results of the ability of a controllable topological structure dynamic biomimetic coating to activate piezo1 and promote osteogenic differentiation under the action of an electromagnetic field. In the figure, A is the experimental group and B is the control group. Figure 10 These are images of the results from animal experiments, where A is an image and B is a quantitative analysis graph. Figure 11 The images show the histological staining results of animal experiments. In the images, A represents the H&E staining result, B represents the Masson staining result, and C represents the Gram staining result. Detailed Implementation

[0034] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] Example 1: A method for preparing a controllable topological structure dynamic biomimetic coating includes the following steps: Preparation of S1, Janus nanoheteromeric dimers Specifically, it includes the following steps: S101. Separate 500 μL of Fe3O4 nanoparticles into 20 mL of trisodium citrate solution (25% w / v, dissolved in ddH2O) and stir at 60℃ to disperse evenly. S102. Add 250 μL of 10 mmol / L AgNO3 solution to the uniformly dispersed mixture obtained in S101, and continue stirring at 60℃ for 2 h. S103. After stirring S102, the resulting mixture was exposed to ultraviolet light for 30 min to obtain Janus nanoheterodimers (JNHDs).

[0036] Preparation of S2 and amino-functionalized SiO2@JNHDs Specifically, it includes the following steps: S201. 250 μg JNHDs were dispersed in 500 μL of ethanol containing 25 μL NH3·H2O and 75 μL ddH2O. The suspension was then stirred vigorously for 10 min. Next, 0.2 μL of tetraethyl orthosilicate (TEOS) was added to the suspension and stirred for 2 h. Finally, the product was washed three times with ethanol to obtain SiO2@JNHDs. S202. SiO2@JNHDs were dispersed in 500 μL of ethanol containing 10 μL of (3-aminopropyl)triethoxysilane (APTES), and then stirred for 24 hours. Finally, the product was washed three times with ddH2O to obtain amino-functionalized SiO2@JNHDs.

[0037] Preparation of S3 and PEGylated SiO2@JNHDs Specifically, the following steps are included: Amino-functionalized SiO2@JNHDs were stirred for 5 min in 500 μL ddH2O containing 5 mg of a long flexible linker [maleimide-polyethylene glycol-succinimide carboxymethyl ester]. Then, 2 μL of N,N-diisopropylethylamine (DIPEA) was added and stirred for 24 hours. Finally, the mixture was washed three times with ddH2O to obtain PEGylated SiO2@JNHDs.

[0038] S4, Preparation of Thiolized Substrates Specifically, it includes the following steps: S401. A pure Ti substrate (purity: 99.9%) was placed in a 24-well plate and immersed in a 500 μL suspension of hydroxyapatite nanoparticles (nHA) (1 mg / mL). The sample was then dried at room temperature for 24 h to obtain a substrate containing a hydroxyapatite nanoparticle layer. S402. The substrate containing hydroxyapatite nanoparticles obtained in S401 was first dispersed in 500 μL of ethanol containing 25 μL NH3·H2O and 75 μL ddH2O. Then, the 24-well plate was shaken for 15 min. After that, 0.2 μL of TEOS was added to each well, and the 24-well plate was shaken for another 2 h. Finally, the product was washed three times with ethanol to obtain the substrate containing silica. S403. Immerse each sample in a mixture of 475 μL ethanol and 25 μL (3-mercaptopropyl)trimethoxysilane (MPTMS) for 24 h, and finally rinse the sample with ddH2O to obtain a thiolized substrate.

[0039] S5. Preparation of controllable topological structure dynamic biomimetic coatings The product obtained from S405 was treated with 500 μL of PEGylated SiO2@JNHDs (diluted with ddH2O 1:10) for 16 h to obtain a controllable topological dynamic biomimetic coating (MMTS).

[0040] Example 2: Preparation of S1, Janus nanoheteromeric dimers Specifically, it includes the following steps: S101. Separate 600 μL of Fe3O4 nanoparticles into 30 mL of trisodium citrate solution (20% w / v, dissolved in ddH2O) and stir at 60℃ to disperse evenly. S102. Add 300 μL of 10 mmol / L AgNO3 solution to the uniformly dispersed mixture obtained in S101, and continue stirring at 60℃ for 2 h. S103. After stirring S102, the resulting mixture was exposed to ultraviolet light for 30 min to obtain Janus nanoheterodimers (JNHDs).

[0041] Preparation of S2 and amino-functionalized SiO2@JNHDs Specifically, it includes the following steps: S201. 300 μg JNHDs were dispersed in 500 μL of ethanol containing 30 μL NH3·H2O and 70 μL ddH2O. The suspension was then stirred vigorously for 10 min. Next, 0.3 μL of tetraethyl orthosilicate (TEOS) was added to the suspension and stirred for 2 h. Finally, the product was washed three times with ethanol to obtain SiO2@JNHDs. S202. SiO2@JNHDs were dispersed in 500 μL of ethanol containing 15 μL of (3-aminopropyl)triethoxysilane (APTES), and then stirred for 24 hours. Finally, the product was washed three times with ddH2O to obtain amino-functionalized SiO2@JNHDs.

[0042] Preparation of S3 and PEGylated SiO2@JNHDs Specifically, the following steps are included: Amino-functionalized SiO2@JNHDs were stirred for 5 min in 500 μL ddH2O containing 7 mg of a long flexible linker [maleimide-polyethylene glycol-succinimide carboxymethyl ester]. Then, 3 μL of N,N-diisopropylethylamine (DIPEA) was added and stirred for 24 hours. Finally, the mixture was washed three times with ddH2O to obtain PEGylated SiO2@JNHDs.

[0043] S4, Preparation of Thiolized Substrates Specifically, it includes the following steps: S401. A pure Ti substrate (purity: 99.9%) was placed in a 24-well plate and immersed in a 500 μL suspension of hydroxyapatite nanoparticles (nHA) (1 mg / mL). The sample was then dried at room temperature for 24 h to obtain a substrate containing a hydroxyapatite nanoparticle layer. S402. The substrate containing hydroxyapatite nanoparticles obtained in S401 was first dispersed in 500 μL of ethanol containing 30 μL NH3·H2O and 70 μL ddH2O. Then, the 24-well plate was shaken for 15 min. After that, 0.3 μL of TEOS was added to each well, and the 24-well plate was shaken for another 2 h. Finally, the product was washed three times with ethanol to obtain the substrate containing silica. S403. Immerse each sample in a mixture of 470 μL ethanol and 30 μL (3-mercaptopropyl)trimethoxysilane (MPTMS) for 24 h, and finally rinse the sample with ddH2O to obtain a thiolized substrate.

[0044] S5. Preparation of controllable topological structure dynamic biomimetic coatings The product obtained from S405 was treated with 500 μL of PEGylated SiO2@JNHDs (diluted with ddH2O 1:10) for 16 h to obtain a controllable topological dynamic biomimetic coating (MMTS).

[0045] Example 3: Preparation of S1, Janus nanoheteromeric dimers Specifically, it includes the following steps: S101. Separate 400 μL of Fe3O4 nanoparticles into 10 mL of trisodium citrate solution (30% w / v, dissolved in ddH2O) and stir at 60℃ to disperse evenly. S102. Add 200 μL of 10 mmol / L AgNO3 solution to the uniformly dispersed mixture obtained in S101, and continue stirring at 60℃ for 2 h. S103. After stirring S102, the resulting mixture was exposed to ultraviolet light for 30 min to obtain Janus nanoheterodimers (JNHDs).

[0046] Preparation of S2 and amino-functionalized SiO2@JNHDs Specifically, it includes the following steps: S201. 200 μg JNHDs were dispersed in 500 μL of ethanol containing 20 μL NH3·H2O and 80 μL ddH2O. The suspension was then stirred vigorously for 10 min. Next, 0.1 μL of tetraethyl orthosilicate (TEOS) was added to the suspension and stirred for 2 h. Finally, the product was washed three times with ethanol to obtain SiO2@JNHDs. S202. SiO2@JNHDs were dispersed in 500 μL of ethanol containing 5 μL of (3-aminopropyl)triethoxysilane (APTES), and then stirred for 24 hours. Finally, the product was washed three times with ddH2O to obtain amino-functionalized SiO2@JNHDs.

[0047] Preparation of S3 and PEGylated SiO2@JNHDs Specifically, the following steps are included: Amino-functionalized SiO2@JNHDs were stirred for 5 min in 500 μL ddH2O containing 3 mg of long flexible linker [maleimide-polyethylene glycol-succinimide carboxymethyl ester], then 1 μL of N,N-diisopropylethylamine (DIPEA) was added and stirred for 24 hours. Finally, the mixture was washed three times with ddH2O to obtain PEGylated SiO2@JNHDs.

[0048] S4, Preparation of Thiolized Substrates Specifically, it includes the following steps: S401. A pure Ti substrate (purity: 99.9%) was placed in a 24-well plate and immersed in a 500 μL suspension of hydroxyapatite nanoparticles (nHA) (1 mg / mL). The sample was then dried at room temperature for 24 h to obtain a substrate containing a hydroxyapatite nanoparticle layer. S402. The substrate containing hydroxyapatite nanoparticles obtained in S401 was first dispersed in 500 μL of ethanol containing 20 μL NH3·H2O and 80 μL ddH2O. Then, the 24-well plate was shaken for 15 min. After that, 0.1 μL of TEOS was added to each well, and the 24-well plate was shaken for another 2 h. Finally, the product was washed three times with ethanol to obtain the substrate containing silica. S403. Immerse each sample in a mixture of 480 μL ethanol and 20 μL (3-mercaptopropyl)trimethoxysilane (MPTMS) for 24 h, and finally rinse the sample with ddH2O to obtain a thiolized substrate.

[0049] S5. Preparation of controllable topological structure dynamic biomimetic coatings The product obtained from S405 was treated with 500 μL of PEGylated SiO2@JNHDs (diluted with ddH2O 1:10) for 16 h to obtain a controllable topological dynamic biomimetic coating (MMTS).

[0050] Experimental example: 1. Microscopic morphological characteristics of cicada wings The cicada wings were characterized by scanning electron microscopy, and the experimental results are as follows: Figure 1 As shown. In order to conduct biomimetic research on the nanostructure of cicada wings, which have a natural antibacterial morphology, the present invention used scanning electron microscopy to observe the nanostructure of cicada wings, showing that the surface of cicada wings is a densely distributed dot-like protrusion structure.

[0051] 2. Schematic diagram of the construction and working principle of controllable topological dynamic biomimetic coating. A schematic diagram of the synthesis of the controllable topological structure dynamic biomimetic coating in this invention is shown below. Figure 2As shown in Figure a, core-shell structured nanoparticles were first synthesized. The core of these nanoparticles was a magnetic-noble metal Janus heterodimer, and the shell was silicon dioxide. Then, the amino-functionalized core-shell structured nanoparticles were surface-PEGylated using a flexible polymeric linker to obtain PEGylated nanoparticles. Next, a thiolized titanium substrate was synthesized. Finally, the PEGylated nanoparticles and the thiolized titanium substrate were coupled together through a chemical reaction, allowing the PEGylated nanoparticles to be covalently grafted onto the substrate surface via a flexible polymeric linker, forming a controllable topological structure dynamic biomimetic coating.

[0052] Figure 2 Figure b illustrates the principle that the controllable topological structure dynamic biomimetic coating prepared in this invention causes mechanical vibration of the coating on the surface of the prosthesis under the action of an external electromagnetic field, thereby exerting bactericidal and bone-promoting effects.

[0053] 3. Physical characterization of Fe3O4-Ag Janus nanoheterodimers (JNHDs) The JNHDs prepared in Example 1 were characterized by transmission electron microscopy, elemental distribution, particle size analysis, and paramagnetism. The experimental results are as follows: Figure 3 As shown.

[0054] like Figure 3 As shown in Figures B-F, the main elemental distribution in JNHDs is Fe, Ag, and O. Figure 3 The figure shows that the diameter of Fe3O4 nanoparticles is approximately 17 mm, and the diameter of Ag nanoparticles is approximately 5 mm. Figure 3 The H-plot shows that, in the absence of an external magnetic field, the nanoparticles are uniformly dispersed in the droplet. With and without a central magnetic field (2), the Fe3O4 nanoparticles and JNHDs exhibit similar paramagnetism, indicating that the nano-heterogeneous dimer prepared in this invention possesses good paramagnetism. These results demonstrate that Fe3O4 nanoparticles and Ag nanoparticles successfully connect to form a nano-heterogeneous dimer, exhibiting good paramagnetism.

[0055] 4. Physical characterization of controllable topological structure dynamic biomimetic coating The controllable topological structure dynamic biomimetic coating prepared in Example 1 was characterized by scanning electron microscopy, atomic force microscopy, elemental distribution, and surface roughness distribution. The experimental results are as follows: Figure 4 As shown.

[0056] like Figure 4 As shown in Figures A and B, the controllable topological structure dynamic biomimetic coating prepared in Example 1 exhibits a nanomorphic morphology of densely distributed dot-like protrusions resembling cicada wings; as shown in Figures A and B. Figure 4 As shown in Figure C, the coating surface contains elements such as Fe, Ag, O, Si, N, and Ti; Figure 4 As shown in Figure D, the connection of the nano-heteromeric dimers gives the smooth titanium plate surface a certain degree of roughness; as... Figure 4 As shown in Figure E, EDS analysis of the coating surface confirms that the elemental distribution of the coating surface is in line with expectations.

[0057] 5. Characterization of the bactericidal ability of controllable topological structure dynamic biomimetic coating This invention designs a pulsed electromagnetic field generating device, such as... Figure 5 As shown in Figures A and D, where A represents the electromagnetic field waveform generator, B represents the electromagnetic field signal amplifier, C represents the Helmholtz coil used to generate a uniform magnetic field, and D represents the solenoid used to generate a non-uniform magnetic field. The simulated magnetic field distribution of the Helmholtz coil and solenoid is shown below. Figure 5 As shown in Figures E and F.

[0058] To test the antibacterial ability of the controllable topological structure dynamic biomimetic coating under electromagnetic field activation, this invention sets up four groups: titanium plate group (Ti), controllable topological structure dynamic biomimetic coating group (MMTS), titanium plate + electromagnetic field group (Ti+EMF), and controllable topological structure dynamic biomimetic coating + electromagnetic field group (MMTS+EMF). The electromagnetic field is generated through the above-mentioned... Figure 5 The equipment was used to characterize the antibacterial properties of Staphylococcus aureus (S. Aureus) and Escherichia coli (E. coli), including fluorescence staining, electron microscopy characterization and plate inhibition test.

[0059] Experimental results are as follows Figure 6 As shown. Figure 6 The live / dead bacteria staining results in Figure A show that, under the action of electromagnetic field (EMF), the surface of the controllable topological structure dynamic biomimetic coating + electromagnetic field group (MMTS+EMF) has a large number of dead bacteria (red fluorescence), while the surfaces of other control groups still have a large number of live bacteria (green fluorescence). Figure 6 Figure B shows that the bacterial morphology on the MMTS+EMF surface has shrunk and broken, indicating that it can kill bacteria on the surface by destroying the bacterial walls and membranes. Figure 6 As shown in Figure C, the bacterial colony count of Staphylococcus aureus floating around the prosthesis was significantly lower in the MMTS+EMF group compared to other groups. These experiments demonstrate that the controllable topological structure dynamic biomimetic coating can exert a controllable and effective bactericidal effect under the activation of an electromagnetic field.

[0060] 6. Characterization of the bone regeneration-promoting ability of controllable topological structure dynamic biomimetic coating To test the ability of a controllable topological dynamic biomimetic coating to promote osteogenic differentiation of mesenchymal stem cells under electromagnetic field activation, this invention set up six groups: a titanium plate group (Ti, for control), a conventional biomimetic coating group (UMTS, without flexible polymeric linker), a controllable topological dynamic biomimetic coating group (MMTS, containing flexible polymeric linker), a titanium plate + electromagnetic field group (Ti + EMF, for control), a conventional biomimetic coating group + electromagnetic field (UMTS + EMF, where the surface of the material without flexible polymeric linker can be explosively released and lost under EMF activation), and a controllable topological dynamic biomimetic coating + electromagnetic field group (MMTS + EMF, where the surface of the material containing flexible polymeric linker is still linked to the prosthesis surface under EMF activation, generating local periodic micro-vibrations to prevent explosive release and loss). The electromagnetic field is used through the above... Figure 5 The equipment used in the study produced the protein, and the characterization methods included Western blot, alizarin red staining, and alkaline phosphatase (ALP) staining.

[0061] Experimental results are as follows Figure 7 As shown. Figure 7 As shown in Figure A, the Western blot results reveal that MMTS+EMF significantly upregulated the levels of proteins related to mesenchymal stem cell adhesion (Itgα5 and Itgβ3) and osteogenic differentiation (OPN, RUNX2, BMP2). Figure 7 Alizarin red staining in Figure B and ALP staining in Figure C revealed that MMTS+EMF significantly promoted osteogenic differentiation and mineral deposition of mesenchymal stem cells. These results indicate that a controllable topological structure dynamic biomimetic coating, activated by an electromagnetic field, can exert a controllable and effective effect in promoting osteogenic differentiation and new bone formation.

[0062] 7. The effect of controllable topological structure dynamic biomimetic coating on activating piezo1 under the action of electromagnetic field, thereby promoting osteogenic differentiation ability. This invention uses Western blot to detect the protein expression level of piezo1, a mechanosensitive calcium ion channel, in mesenchymal stem cells on days 3, 7, 14, and 28 of osteogenic differentiation.

[0063] Experimental results are as follows Figure 8 As shown, the results indicate that the protein expression level of piezo1 was highest on days 7 and 14 of osteogenic differentiation, suggesting that piezo1 plays an important role in the mid-stage of osteogenic differentiation and has a decisive role in the later-stage new bone formation.

[0064] This invention uses the Fluo4-AM probe to label intracellular calcium ions, thereby observing whether the mechanosensitive calcium ion channel piezo1 can be activated by a controllable topological dynamic biomimetic coating. Calcium ion influx was characterized according to the groupings in the bone regeneration capacity characterization experiment of the controllable topological dynamic biomimetic coating, with cells pre-incubated with the piezol inhibitor Dookul serving as a control group.

[0065] Experimental results are as follows Figure 9 As shown, the fluorescence signal in the MMTS+EMF group increased rapidly after EMF activation. Figure 9 Figure A shows that MMTS+EMF can activate calcium ion channels, thereby causing calcium ion influx into cells. In the control group, although MMTS+EMF can increase calcium ion influx, the influx intensity is significantly reduced. Figure 9 (Figure B in the middle). The above results indicate that MMTS+EMF can indeed activate piezo1 to induce calcium ion influx. Calcium ions are not only important osteogenic signaling molecules, but also the main raw materials for new bone formation. Therefore, the activation of piezo1 by MMTS+EMF to induce calcium ion influx is the main molecular signaling mechanism by which it promotes new bone formation.

[0066] 8. Animal experiments A rat tibial infection model was constructed and grouped as follows: Each group of implanted prostheses contained different coatings and were pre-soaked in 1×10 6 Staphylococcus aureus (S. aureus) suspension at a concentration of CFU / mL was incubated for 30 minutes to allow pathogenic bacteria to adhere to the surface, thus constructing an in vivo model of periprosthetic infection. The groups included: titanium plate (Ti), conventional biomimetic coating (UMTS), controllable topological dynamic biomimetic coating (MMTS), titanium plate + electromagnetic field (Ti+EMF), conventional biomimetic coating + electromagnetic field (UMTS+EMF), and controllable topological dynamic biomimetic coating + electromagnetic field (MMTS+EMF).

[0067] Micro-CT scans were used to detect tibial infection and bone regeneration in each group of rats. The experimental results are as follows: Figure 10 As shown.

[0068] like Figure 10 As shown in Figure A, the dead bone and bony shell indicated by the red arrows are still present in the Ti group and the Ti+EMF group, indicating the successful establishment of the animal model. In contrast, the prosthesis and newly formed bone tissue indicated by the green arrows in the MMTS group have formed a tight connection, indicating the formation of good prosthesis-bone integration. Figure 10In the quantitative analysis of the B-plot, the MMTS+EMF group did indeed significantly increase bone volume fraction (BV / TV), trabecular bone number (TB.N), and reduce trabecular bone separation (TbSp).

[0069] H&E staining, Masson staining, and Gram staining were performed around the prosthesis. The experimental results are as follows: Figure 11 As shown.

[0070] The results showed that bacterial colonies remained around the prosthesis (in the cavity) in both the Ti and Ti+EMF groups, but no significant new bone formation was observed. However, significant new bone formation was observed in the MMTS+EMF group, and no bacterial colonies were found. These experiments demonstrate that, in animal experiments, MMTS+EMF can significantly kill bacteria around the prosthesis and promote prosthesis-osseointegration, playing an important role in preventing periprosthetic infection and promoting osseointegration.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a controllable topological structure dynamic biomimetic coating, characterized in that, Includes the following steps: S1. Janus nanoheteromers were prepared by photoreduction reaction of Fe3O4 nanoparticles, trisodium citrate and silver ion salt solution. S2. The Janus nano-heteromeric dimer obtained in S1 is first subjected to hydrolysis and condensation reaction with ammonia and tetraethyl orthosilicate in a solvent, and then reacted with (3-aminopropyl)triethoxysilane in a solvent to obtain amino-functionalized SiO2@JNHDs. S3. The amino-functionalized SiO2@JNHDs obtained in S2 are reacted with a flexible polymer linker and N,N-diisopropylethylamine in a solvent to prepare PEGylated SiO2@JNHDs. S4. React a substrate with a silica layer on its surface with (3-mercaptopropyl)trimethoxysilane in a solvent to obtain a thiolized substrate; S5. The PEGylated SiO2@JNHDs obtained in S3 and the thiolized substrate obtained in S4 are co-incubated to obtain a dynamic biomimetic coating with controllable topology.

2. The method for preparing a controllable topological structure dynamic biomimetic coating according to claim 1, characterized in that, S1 specifically includes the following steps: S101. Disperse Fe3O4 nanoparticles in a trisodium citrate solution and stir until the dispersion is uniform. S102. Add silver ion salt solution to the uniformly dispersed system obtained in S101 and continue stirring. S103. After stirring S102, the resulting mixture was irradiated with ultraviolet light to obtain Janus nano-heterogeneous dimers.

3. The method for preparing a controllable topological structure dynamic biomimetic coating according to claim 2, characterized in that, The stirring temperature in S101 is 50-70℃; The stirring in S102 is continued at a temperature of 50-70℃ for 1-3 hours; the silver ion salt includes AgNO3. The ultraviolet light irradiation time in S103 is 20-40 min.

4. The method for preparing a controllable topological structure dynamic biomimetic coating according to claim 1, characterized in that, S2 specifically includes the following steps: S201. Janus nano-heterodimer was dispersed in an ethanol solution containing ammonia, and tetraethyl orthosilicate was added to carry out a hydrolysis-condensation reaction to obtain SiO2@JNHDs. S202. SiO2@JNHDs were reacted with (3-aminopropyl)triethoxysilane in ethanol to prepare amino-functionalized SiO2@JNHDs.

5. The method for preparing a controllable topological structure dynamic biomimetic coating according to claim 4, characterized in that, The hydrolysis-condensation reaction in S201 is carried out at room temperature for 1-3 hours. The reaction in S202 is carried out at room temperature for 10-36 hours.

6. The method for preparing a controllable topological structure dynamic biomimetic coating according to claim 1, characterized in that, The flexible polymer linker in S3 is polyethylene glycol with N-hydroxysuccinimide ester and maleimide groups at both ends, with a molecular weight of 3000-10000 Da; the reaction temperature is room temperature and the reaction time is 10-36 h.

7. The method for preparing a controllable topological structure dynamic biomimetic coating according to claim 1, characterized in that, S4 specifically includes the following steps: S401. First, immerse the substrate in a suspension of hydroxyapatite nanoparticles, and then dry it at room temperature to obtain a substrate containing a layer of hydroxyapatite nanoparticles. S402: The substrate containing hydroxyapatite nanoparticles obtained in S401 is first dispersed in ammonia and tetraethyl orthosilicate in a solvent to carry out a hydrolysis and condensation reaction to obtain a substrate containing silica. S403: The substrate containing the silica layer obtained in S402 is subjected to thiolation treatment with (3-mercaptopropyl)trimethoxysilane in a solvent to obtain a thiolated substrate. The substrate is Ti or a titanium alloy.

8. The method for preparing a controllable topological structure dynamic biomimetic coating according to claim 1, characterized in that, The incubation temperature in S5 is room temperature, and the incubation time is 12-20 h.

9. A controllable topological structure dynamic biomimetic coating, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The application of the controllable topological structure dynamic biomimetic coating of claim 9 in the preparation of orthopedic implants.

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