Multifunctional coating capable of forming long-term stable endogenous electric field to promote antibiosis, preparation method and application thereof

Abstract

The invention discloses a multifunctional coating capable of forming a long-term stable endogenous electric field to promote antibiosis, and a preparation method and application thereof, and belongs to the technical field of biological functional modification. According to the coating, through the elaborately designed Ta / Ag heterostructure, long-term stable generation of an endogenous electric field is achieved, the antibacterial performance is enhanced, osseointegration is promoted, and the problem of drug resistance is avoided; the coating does not need external electrical stimulation, so that the operation process is simplified, and the clinical application risk is reduced; due to the Ta material, the osseointegration capacity is improved, and Ag has broad-spectrum and efficient antibacterial performance. The TC4-Ta / Ag group has excellent charge storage capacity and a stable electric field, and the long-term osseointegration effect is remarkably improved; according to the preparation method of the coating, the coating which is stable in structure and not prone to falling off can be obtained, the long-term stable antibacterial performance is kept, the coating has wide application prospects in the field of titanium alloy bone implants, a new solution is provided for orthopedic surgery and implant treatment, and the treatment effect and life quality of patients are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biofunctional modification, and particularly relates to a multifunctional coating that forms a long-term stable endogenous electric field to promote antibacterial, a preparation method thereof, and an application thereof. Background Art

[0002] Peri-implantitis is a common dental complication that affects approximately 22% of dental implants. Its high incidence not only increases medical costs but also prolongs the recovery time of patients, and may even lead to implant failure. Peri-implant infection can occur at any time point after implant placement, but especially within the initial 4 weeks after implantation. Since the bone integration interface has not been fully formed at this time, the anti-infection ability of the implant is relatively weak, so the infection risk during this period is particularly prominent.

[0003] To address implant-related infections, various strategies have been developed to enhance the antibacterial properties of implant materials. For example, adding antibiotics to the implant surface, but this method may lead to bacterial drug resistance, and over time, its efficacy will gradually decrease or even fail. In addition, the release of antibiotics may also have an adverse effect on surrounding normal tissues. Modifying the implant surface with nanoparticles is also a commonly used method. Due to their unique physicochemical properties, such as large specific surface area and quantum size effect, nanoparticles exhibit good antibacterial properties, but nanoparticles may be toxic to human cells and tissues, especially at high concentrations or with long-term exposure. This toxic effect may limit the widespread application of nanoparticles in implant surface modification. In addition, although biomacromolecules can be used for antibacterial, their instability in vitro or in vivo may lead to degradation, thus affecting their effects. In recent years, the application of exogenous electric fields has shown potential in the antibacterial field. Exogenous electric fields can achieve antibacterial effects by interfering with bacterial signal transduction, affecting cell membrane permeability, etc. However, the application of exogenous electric fields also faces many challenges. First, the operation of exogenous electric fields is relatively complex and requires professional equipment and operating skills. Second, the application of exogenous electric fields is device-dependent and it is difficult to achieve stable long-term in vivo application. In addition, exogenous electric fields may cause damage to surrounding normal cells and tissues, leading to unnecessary complications.

[0004] Although there have been studies exploring the use of endogenous electric fields to promote tissue regeneration and antibacterial, there are currently no research reports on functional coatings that rely on their own potential differences to form endogenous electric fields and related reports. Endogenous electric fields refer to electric fields generated by organisms themselves, which play an important role in maintaining physiological balance, promoting cell proliferation and differentiation, etc. Therefore, developing a multifunctional coating that can form a long-term stable endogenous electric field and promote antibacterial is of great significance for improving the anti-infection ability of implants and promoting bone integration. Summary of the Invention

[0005] In view of the technical status quo that there is a problem in the existing technology that it is impossible to simultaneously achieve multiple functions of long-term stable endogenous electric field generation, efficient antibacterial, and enhanced bone integration, the object of the present invention is to provide a multifunctional coating that forms a long-term stable endogenous electric field to promote antibacterial, a preparation method thereof, and an application thereof.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a preparation method of a multifunctional coating that forms a long-term stable endogenous electric field to promote antibacterial, including: sputtering a tantalum film on a processed medical metal matrix to obtain a base layer of the coating; sputtering a silver film on the base layer to obtain a Ta / Ag double-layer coating; immersing the Ta / Ag double-layer coating in simulated body fluid, cleaning, and drying to obtain a multifunctional coating that can form a long-term stable endogenous electric field to promote antibacterial.

[0007] For the processed medical metal matrix, the surface of the medical metal substrate is mechanically polished, cleaned, and dried to obtain the processed medical metal matrix; the medical metal matrix is titanium or a titanium alloy.

[0008] The conditions of the mechanical polishing treatment are: surface polishing is carried out using diamond polishing paste, the polishing time is 10 to 15 minutes, the polishing pressure is 40 to 60 N, the polishing speed is 100 to 150 rpm, and the surface roughness after polishing is Ra < 0.1 µm.

[0009] The thickness of the tantalum film is 200 to 300 nm, and the thickness of the silver film is 200 to 300 nm.

[0010] The conditions for sputtering the tantalum film are: the sputtering power is 100 W to 200 W, the sputtering atmosphere is argon, the sputtering pressure is 0.3 Pa to 0.5 Pa, and the sputtering time is 20 min to 30 min.

[0011] Further, the conditions for sputtering the tantalum film are: the sputtering power is 150 W, the sputtering atmosphere is argon, the sputtering pressure is 0.4 Pa, and the sputtering time is 25 min.

[0012] The conditions for sputtering the silver film are: the sputtering power is 150 W to 250 W, the sputtering atmosphere is argon, the sputtering pressure is 0.2 Pa to 0.4 Pa, and the sputtering time is 20 min to 40 min.

[0013] Further, the conditions for sputtering the silver film are: the sputtering power is 200 W, the sputtering atmosphere is argon, the sputtering pressure is 0.3 Pa, and the sputtering time is 30 min.

[0014] The soaking conditions are to soak the coating sample in simulated body fluid at a soaking temperature of 36-38°C and a soaking time of 20-28 h, and the simulated body fluid is replaced every 4 hours for the sample.

[0015] The composition of the simulated body fluid is as follows: sodium chloride (NaCl) 9 g / L, calcium chloride (CaCl 2 ), 0.2 g / L, magnesium chloride (MgCl 2 ), 0.1 g / L, sodium dihydrogen phosphate (NaH 2 PO 4 ), 0.4 g / L, potassium chloride (KCl) 0.4 g / L, and the pH value is 7.4.

[0016] Furthermore, the soaking conditions are to soak the coating sample in simulated body fluid at a soaking temperature of 37°C and a soaking time of 24 h.

[0017] The multifunctional coating obtained by the above preparation method can form a long-term stable endogenous electric field to promote antibacterial effect.

[0018] The multifunctional coating is a bilayer structure composed of a tantalum bottom layer and a silver top layer. The tantalum bottom layer serves as the base layer of the coating and is directly sputtered on the surface of the medical metal matrix; the silver top layer is sputtered on the tantalum bottom layer, which can release silver ions to form an endogenous electric field to inhibit the growth and reproduction of bacteria.

[0019] The application of the above multifunctional coating that can form a long-term stable endogenous electric field to promote antibacterial effect in the preparation of dental implants, bone connecting plates, bone reconstruction porous scaffolds, intramedullary prostheses, internal fixation scaffolds, external fixation scaffolds or joint prostheses.

[0020] Compared with the prior art, the present invention has the following beneficial effects: A multifunctional coating provided by the present invention for forming a long-term stable endogenous electric field to promote antibacterial effect realizes the generation of a long-term stable endogenous electric field, improves the local charge microenvironment, enhances the antibacterial property and promotes bone integration, avoids the drug resistance problem that may occur in traditional antibacterial strategies, and provides a new and continuously effective antibacterial means; without external electrical stimulation, it overcomes the limitations such as the operation complexity, equipment dependence and potential damage to normal cells of the exogenous electric field technology, simplifies the operation process and reduces the risks in long-term clinical applications; by using Ta material, the bone integration ability is improved. Ta has excellent biocompatibility and chemical stability and can be retained in the host tissue for a long time, which helps to promote the combination of the implant and bone tissue, making the implant have better clinical application prospects; Ag in the coating has broad-spectrum and high-efficiency antibacterial properties. It can inhibit the metabolic process by binding to the enzymes in bacteria, promote the generation of reactive oxygen species, and cause damage to bacterial DNA, proteins and lipids, providing a new solution for preventing implant infections; the TC4-Ta / Ag group has superior charge storage ability and a stable electric field, which can significantly improve the long-term bone integration effect and shows better effects in a complex in-vivo environment compared with traditional strategies, providing strong support for the long-term antibacterial and healing promotion of implants in clinical applications.

[0021] The preparation method of the multifunctional coating provided by the present invention for forming a long-term stable endogenous electric field to promote antibacterial effect realizes excellent antibacterial properties, biocompatibility and mechanical properties through a carefully designed Ta / Ag heterostructure and optimized sputtering process parameters; by optimizing the sputtering process parameters and adjusting the ratio of Ta / Ag, a coating with a stable structure and not easy to fall off can be obtained, and the coating can maintain stable antibacterial properties during long-term use; Ta is a metal with good biocompatibility and serves as the bottom layer in the coating, which helps to improve the overall biocompatibility. The Ag layer is on the surface of the coating, which can reduce the chance of direct contact between silver ions and biological tissues, thus reducing the risk of allergic reactions.

[0022] Application of a multifunctional coating capable of forming a long-term stable endogenous electric field to promote antibacterial activity. The multifunctional Ta / Ag coating provided by the present invention, which can form a long-term stable endogenous electric field to promote antibacterial activity, achieves excellent antibacterial performance, bone integration ability, and long-term effects through a carefully designed coating structure and optimized preparation process. Compared with traditional antibacterial strategies that rely on the release of antibacterial agents, the endogenous electric field antibacterial method of the present invention avoids the emergence of drug resistance problems and provides a reliable guarantee for long-term antibacterial activity; the TC4-Ta / Ag coating has superior charge storage capacity and a stable electric field, and can maintain long-term stable performance in a complex in vivo environment; the long-term antibacterial effect and stable bone integration ability reduce the occurrence of complications such as implant infection, loosening, and shedding, improving the treatment effect and quality of life of patients. This coating has broad application prospects in the field of titanium alloy bone implants and is expected to provide new solutions and breakthroughs for orthopedic surgery and implant treatment. Description of the Drawings

[0023] Figure 1 Surface morphology and elemental analysis of the titanium alloy implant with the Ta / Ag multifunctional coating of Example 1 of the present invention; among them, TC4-Ta / Ag is Example 1, TC4-Ta-Ag is Comparative Example 1, and TC4-Ag / Ta is Comparative Example 2; Figure 2 Tomographic morphology of the titanium alloy implant with the Ta / Ag multifunctional coating of Example 1 of the present invention; among them, TC4-Ta / Ag is Example 1, TC4-Ta-Ag is Comparative Example 1, and TC4-Ag / Ta is Comparative Example 2; Figure 3 Statistical chart of the potential difference between the titanium alloy implant with the Ta / Ag multifunctional coating of Example 1 of the present invention and the control sample; Figure 4 Patch clamp analysis of the effects of the Ta / Ag multifunctional coating of Example 1 of the present invention and the control sample on the surface cell membrane potential; CON is the blank control group, TC4 is the titanium alloy, TC4-Ta / Ag is Example 1, TC4-Ta-Ag is Comparative Example 1, and TC4-Ag / Ta is Comparative Example 2; Figure 5 Effects of the Ta-Ag multifunctional coating of Example 1 of the present invention and the control on bacterial morphology and antibacterial rate; among them, CON is the blank control group, TC4 is the titanium alloy, TC4-Ta / Ag is Example 1, TC4-Ta-Ag is Comparative Example 1, TC4-Ag / Ta is Comparative Example 2, and the bacteria are Escherichia coli E. coli and Staphylococcus aureus S. aureus ; Figure 6To analyze the effect of the Ta-Ag multifunctional coating in Example 1 of the present invention on bone integration ability after implantation in vivo by VG staining, TC4 is titanium alloy, TC4-Ta / Ag is Example 1, TC4-Ta-Ag is Comparative Example 1, and TC4-Ag / Ta is Comparative Example 2; Figure 7 To analyze the biotoxicity effect of the Ta-Ag multifunctional coating in Example 1 of the present invention and the control on the whole body organs after implantation in vivo by HE staining, no obvious inflammatory cells or lesions were found in the heart, liver, spleen, lungs, kidneys, and testes, indicating that the material has no obvious toxicity to the whole body organs. Detailed implementation manners

[0024] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0025] Example 1 This example provides a multifunctional coating that forms a long-term stable endogenous electric field to promote antibacterial properties. The specific preparation process is as follows: (1) The surface of the titanium alloy substrate was mechanically polished. Diamond polishing paste was used for surface polishing. The polishing time was 10 minutes, the polishing pressure was 50 N, and the polishing speed was 100 rpm to make its surface smooth and flat. The surface roughness after polishing was Ra < 0.1 µm; (2) The polished titanium alloy substrate was placed in a vacuum sputtering chamber. A Ta film was sputtered on the surface of the titanium alloy substrate using a DC magnetron sputtering source. The sputtering power was 150 W, the sputtering atmosphere was argon, the sputtering pressure was 0.4 Pa, and the sputtering time was 25 minutes. The thickness of the formed Ta layer was about 250 nm; (3) An Ag film was continuously sputtered on the surface of the Ta layer. The sputtering power was 200 W, the sputtering atmosphere was argon, the sputtering pressure was 0.3 Pa, and the sputtering time was 30 minutes. The thickness of the formed Ag layer was about 250 nm, so as to obtain a Ta / Ag multifunctional coating on the surface of the titanium alloy substrate; (4) Immerse the titanium alloy substrate with sputtered Ta / Ag bilayer coating in a simulated body fluid (SBF) solution at a temperature of 37 °C for 24 h to form a bioactive coating on the surface of the Ta / Ag coating; replace the simulated body fluid every 4 h; the composition of the simulated body fluid is: sodium chloride (NaCl) 9 g / L, calcium chloride (CaCl 2 ) 0.2 g / L, magnesium chloride (MgCl 2 ) 0.1 g / L, sodium dihydrogen phosphate (NaH 2 PO 4 ) 0.4 g / L, potassium chloride (KCl) 0.4 g / L, and the pH value is adjusted to 7.4.

[0026] (5) Take out the immersed sample and dry it naturally at room temperature.

[0027] Example 2 This example provides a multifunctional coating that forms a long-term stable endogenous electric field to promote antibacterial, and the specific preparation process is as follows: (1) Mechanically polish the surface of the titanium alloy substrate, use diamond polishing paste for surface polishing, the polishing time is 10 minutes, the polishing pressure is 50 N, and the polishing speed is 100 rpm to make its surface smooth and flat. The surface roughness after polishing is Ra < 0.1 µm; (2) Place the polished titanium alloy substrate in a vacuum sputtering chamber, use a DC magnetron sputtering source, sputter a Ta film on the surface of the titanium alloy substrate, the sputtering power is 100 W, the sputtering atmosphere is argon, the sputtering pressure is 0.3 Pa, and the sputtering time is 20 minutes. The thickness of the formed Ta layer is about 200 nm; (3) Continue to sputter an Ag film on the surface of the Ta layer, the sputtering power is 150 W, the sputtering atmosphere is argon, the sputtering pressure is 0.2 Pa, and the sputtering time is 20 minutes. The thickness of the formed Ag layer is about 220 nm, so as to obtain a Ta / Ag multifunctional coating on the surface of the titanium alloy substrate; (4) Immerse the titanium alloy substrate with sputtered Ta / Ag bilayer coating in a simulated body fluid (SBF) solution at a temperature of 35 °C for 20 h to form a bioactive coating on the surface of the Ta / Ag coating; replace the simulated body fluid every 4 h; the composition of the simulated body fluid is: sodium chloride (NaCl) 9 g / L, calcium chloride (CaCl 2 ) 0.2 g / L, magnesium chloride (MgCl 2 ) 0.1 g / L, sodium dihydrogen phosphate (NaH 2 PO 4 ) 0.4 g / L, potassium chloride (KCl) 0.4 g / L, and the pH value is adjusted to 7.4.

[0028] (5) Take out the immersed sample and dry it naturally at room temperature.

[0029] Example 3 This example provides a multifunctional coating that forms a long-term stable endogenous electric field to promote antibacterial properties. The specific preparation process is as follows: (1)The surface of the titanium alloy substrate is mechanically polished using diamond polishing paste. The polishing time is 10 minutes, the polishing pressure is 50 N, and the polishing speed is 100 rpm to make its surface smooth and flat. The surface roughness after polishing is Ra < 0.1 µm; (2)The polished titanium alloy substrate is placed in a vacuum sputtering chamber. Using a DC magnetron sputtering source, a Ta film is sputtered on the surface of the titanium alloy substrate. The sputtering power is 200 W, the sputtering atmosphere is argon, the sputtering pressure is 0.5 Pa, and the sputtering time is 30 minutes. The thickness of the formed Ta layer is about 280 nm; (3)An Ag film is continuously sputtered on the surface of the Ta layer. The sputtering power is 250 W, the sputtering atmosphere is argon, the sputtering pressure is 0.4 Pa, and the sputtering time is 40 minutes. The thickness of the formed Ag layer is about 300 nm, thereby obtaining a Ta / Ag multifunctional coating on the surface of the titanium alloy substrate; (4)The titanium alloy substrate sputtered with the Ta / Ag double-layer coating is immersed in a simulated body fluid (SBF) solution at a temperature of 38 °C for 26 h to form a bioactive coating on the surface of the Ta / Ag coating; the simulated body fluid is replaced every 4 hours; the composition of the simulated body fluid is: sodium chloride (NaCl) 9 g / L, calcium chloride (CaCl 2 )0.2 g / L, magnesium chloride (MgCl 2 )0.1 g / L, sodium dihydrogen phosphate (NaH 2 PO 4 )0.4 g / L, potassium chloride (KCl) 0.4 g / L, and the pH value is adjusted to 7.4.

[0030] (5)The soaked sample is taken out and naturally dried at room temperature.

[0031] Comparative Example 1 In the comparative example, based on Example 1, Ta and Ag are sputtered simultaneously to obtain a multifunctional coating. The specific process is as follows: (1)The surface of the titanium alloy substrate is mechanically polished using diamond polishing paste. The polishing time is 10 minutes, the polishing pressure is 50 N, and the polishing speed is 100 rpm to make its surface smooth and flat. The surface roughness after polishing is Ra < 0.1 µm; (2)Place the polished titanium alloy substrate in a vacuum sputtering chamber. Using a DC magnetron sputtering source, simultaneously sputter a Ta-Ag film on the surface of the titanium alloy substrate. The sputtering power is 200 W, the sputtering atmosphere is argon, the sputtering pressure is 0.4 Pa, and the sputtering time is 25 minutes. The thickness of the formed Ta-Ag layer is approximately 260 nm; (3)Immerse the titanium alloy substrate with the sputtered Ta-Ag coating in a simulated body fluid (SBF) solution at a temperature of 37 °C for 24 h to form a bioactive coating on the surface of the Ta-Ag coating; replace the simulated body fluid every 4 hours; The composition of the simulated body fluid is: sodium chloride (NaCl) 9 g / L, calcium chloride (CaCl 2 ), 0.2 g / L, magnesium chloride (MgCl 2 ), 0.1 g / L, sodium dihydrogen phosphate (NaH 2 PO 4 ), 0.4 g / L, potassium chloride (KCl) 0.4 g / L, and the pH value is adjusted to 7.4.

[0032] (4)Take out the soaked sample and let it dry naturally at room temperature.

[0033] Comparative Example 2 On the basis of Example 1, in the comparative example, first sputter a layer of Ag film on the titanium alloy substrate, and then sputter a layer of Ta film to obtain a multifunctional coating. The specific process is as follows: (1)Mechanically polish the surface of the titanium alloy substrate. Use diamond polishing paste for surface polishing. The polishing time is 10 minutes, the polishing pressure is 50 N, and the polishing speed is 100 rpm to make its surface smooth and flat. The surface roughness after polishing is Ra < 0.1 µm; (2)Place the polished titanium alloy substrate in a vacuum sputtering chamber. Using a DC magnetron sputtering source, sputter a layer of Ag film on the surface of the titanium alloy substrate. The sputtering power is 200 W, the sputtering atmosphere is argon, the sputtering pressure is 0.3 Pa, and the sputtering time is 30 minutes. The thickness of the formed Ag layer is approximately 250 nm, thereby obtaining an Ag coating on the surface of the titanium alloy substrate; (3)Continue to sputter a Ta film on the surface of the Ag layer. The sputtering power is 150 W, the sputtering atmosphere is argon, the sputtering pressure is 0.4 Pa, and the sputtering time is 25 minutes. The thickness of the formed Ta layer is approximately 250 nm, thereby obtaining an Ag / Ta multifunctional coating on the surface of the titanium alloy substrate; (4)Immerse the titanium alloy substrate with the sputtered Ag coating in a simulated body fluid (SBF) solution at a temperature of 37 °C for 24 h to form a bioactive coating on the surface of the Ag coating; replace the simulated body fluid every 4 hours; The composition of the simulated body fluid is: sodium chloride (NaCl) 9 g / L, calcium chloride (CaCl 2)0.2 g / L of magnesium chloride (MgCl 2 )0.1 g / L of sodium dihydrogen phosphate (NaH 2 PO 4 )0.4 g / L, 0.4 g / L of potassium chloride (KCl), and the pH value was adjusted to 7.4.

[0034] (5) Take out the soaked samples and dry them naturally at room temperature.

[0035] II. Performance Test (1) Bone integration ability test of titanium alloy implants Perform performance tests on Examples 1 - 3 and Comparative Examples 1 - 2 of the present invention. Implant implants with Ta / Ag coatings of different spatial layouts into a rat infected bone defect model to construct a rat infected bone defect model. The specific method is as follows: Forty 5 - week - old male Sprague - Dawley (SD) rats with an average body weight of 250 ± 30 g were randomly divided into five groups: TC4 group, Ta - Ag group, Ag / Ta group, Ta / Ag group, and blank control group, with 5 rats in each group. The animal experiment protocol has been approved by the Animal Ethics Committee of Xi'an Jiaotong University (Xi'an, China). To prepare the implants, Staphylococcus aureus (S. aureus) (2 × 10³ colony - forming units, 20 μL PBS solution) was evenly coated on the surface of the implants, and the implants were incubated in a humid environment at 37°C for 4 hours to promote bacterial attachment. The experimental operations were carried out under sterile conditions, and each rat received two implants in the distal femur. Before the start of the surgical procedure, anesthesia was performed by intraperitoneal injection of 10% chloral hydrate (0.1 mL / 100 g, #P3761, Sigma - Aldrich, USA). A 10 - mm longitudinal incision was made along the medial side of the knee joint, and the patella was displaced laterally to expose the extensor system. After the knee joint was flexed, a low - speed hand drill was used to drill a hole with a diameter of 2 mm and a depth of 10 mm from the intercondylar notch. Normal saline was used for cooling and irrigation throughout the surgical process. Implants from different groups were inserted into the femoral medullary cavity through the distal femoral epiphysis until the end of the implant was located below the articular surface. Subsequently, the patella was reduced, and analgesic drugs were provided through drinking water for 3 days after the operation.

[0036] Eight weeks after the operation, the rats were sacrificed with chloral hydrate according to the group (5 rats in each group), and their femurs were collected for further analysis.

[0037] Observe the promoting effect of Ta / Ag coatings with different spatial layouts on the bone integration ability of titanium alloy implants by hard tissue sectioning and VG staining. The experimental steps are as follows: First, the femur was fixed in a 4% phosphate formaldehyde solution for 48 hours. After fixation, the samples were dehydrated through a series of ethanol solutions with gradually increasing concentrations (70%, 80%, 85%, 90%, 95%, 100%, v / v). After dehydration was completed, the samples were embedded in PMMA resin, which could provide sufficient support to ensure the integrity of the tissue during sectioning. During the preparation of the resin-embedded samples, a Leica SP1600 microtome (Leica, Germany) was used to cut the samples into 150 µm thick sections. These sections were further polished to a final thickness of 20 µm to ensure clarity during microscopic observation and visibility of fine structures. To observe the structure and distribution of mineralized bone tissue, Van Gieson’s staining method was adopted. This staining method could stain the mineralized bone tissue red, thus clearly showing the bone formation around the implant. Histological observation of the sections was performed using an optical microscope (Olympus, WILD MP5, Japan).

[0038] To quantitatively analyze the degree of bone-implant contact, BIOQUANT OSTEO software (USA) was used for histological measurement analysis. This software could accurately measure the contact area between the bone tissue and the implant surface and calculate the bone-implant contact index (BIC), which is widely used to evaluate the bone integration effect of implants. By comparing with different groups, the effects of different implant surface treatment methods on bone integration could be evaluated, thus providing a theoretical basis for the optimization of implant materials.

[0039] (2) Safety assessment test The in vivo safety assessment of Ta / Ag-coated implants with different spatial layouts: After the experiment, important tissue organs such as the kidneys, liver, lungs, and heart were retained. Pathological detection was performed through pathological sections, and H&E staining was used to detect the inflammatory infiltration in each tissue organ to evaluate whether there was immune damage in each tissue organ.

[0040] The steps of H&E staining were as follows: 1) Dewax the ventricular paraffin sections with xylene; 2) Wash the sections successively with absolute ethanol and 85% ethanol; 3) Wash the sections with distilled water; 4) Stain with hematoxylin for 6 minutes; 5) Wash off the hematoxylin with distilled water; 6) Wash the sections again with double-distilled water after differentiation with hydrochloric acid ethanol; 7) After washing with PBS, stain the sections with eosin solution for about 3 minutes; 8) Differentiate according to the ethanol gradient of 85%-95%-100%; 9) Treat the sections successively with carbolic acid xylene and xylene (I), xylene (II), xylene (III); 10) Mount the sections with neutral gum.

[0041] See the appendix Figure 1, the morphologies of the coatings obtained in Example 1, Comparative Example 1, and Comparative Example 2 were observed. The Ta / Ag coatings of all experimental groups were uniformly and stably attached to the surface of the titanium alloy, and no obvious coating defects were observed, indicating that the sputtering process can effectively prepare coatings with good quality. The elemental composition of the coating surface was detected by X-ray photoelectron spectroscopy, and the presence and uniform distribution of Ta and Ag elements in the coating were confirmed. In this experiment, by comparing the surface morphologies and elemental compositions of Ta / Ag coatings prepared under different sputtering conditions, the effectiveness and reliability of the sputtering process were verified. These results provide strong support for further optimizing the coating preparation of titanium alloy implants.

[0042] See the appendix Figure 2 , to investigate the influence of the three kinds of coated titanium alloy implants in Example 1, Comparative Example 1, and Comparative Example 2 on the tomographic morphology, and then evaluate the overall stability, compactness, and mechanical properties of the coating. The scanning electron microscope (SEM) was used to observe and analyze the tomographic morphology of the coating. All three coatings showed a uniform structure, and no obvious peeling phenomenon was observed, indicating that the overall stability of the coating is good, and the thickness and structural differences of the coating have little effect on the overall performance.

[0043] See the appendix Figure 3 , to investigate the potential difference statistics of the three kinds of coated titanium alloy implants in Example 1, Comparative Example 1, and Comparative Example 2. The atomic force microscope (AFM) scanning Kelvin probe microscopy (SKPM) mode was used to measure the potential difference on the surface of the titanium alloy implant, and then analyze how this potential difference change affects the antibacterial ability and osseointegration ability of the implant. There are significant differences in the potential differences between the coatings. By changing the spatial layout of the Ta / Ag coating on the surface of the titanium alloy, the potential difference on its surface can be effectively regulated, thereby affecting its biological properties and clinical application effects. See the appendix Figure 4 , the patch clamp technique was used to analyze the influence of Ta / Ag coatings with different spatial layouts on the surface cell membrane potential of BMSCs (bone marrow mesenchymal stem cells), so as to evaluate the influence of these coatings on cell biological activity. The membrane potential of BMSCs in the TC4-Ta / Ag group was significantly depolarized. Compared with the control group, the change in its membrane potential was the most obvious. The fluorescence probe experiment also supported the results of the patch clamp experiment, showing that the TC4-Ta / Ag group had the strongest change in the membrane potential of BMSCs; indicating that the TC4-Ta / Ag coating combination showed significant advantages in regulating the membrane potential of BMSCs, so it has better biological activity, which is of great significance for the research and application of biomaterial surface modification.

[0044] See the appendix Figure 5, the coatings of Example 1, Comparative Example 1 and Comparative Example 2 were co-cultured with E. coli and S. aureus to observe the antibacterial effect of the coatings. The morphological effects of the coatings on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were analyzed by scanning electron microscopy. The bacterial morphology of the control group (CON) was normal and evenly distributed. The bacterial morphology of the TC4 group was similar to that of the control group, indicating that the TC4 coating itself had no significant effect on the bacterial morphology; the bacterial morphology of the TC4-Ta-Ag and TC4-Ta / Ag groups was significantly smaller and more evenly distributed, indicating that these coatings had a significant inhibitory effect on the growth and morphology of bacteria; antibacterial rate data analysis: The antibacterial rates of the TC4-Ta-Ag and TC4-Ta / Ag groups against Escherichia coli ( E. coli ), and Staphylococcus aureus ( S. aureus ) were significantly higher than those of other groups; for Escherichia coli, the antibacterial rates of the TC4-Ta-Ag and TC4-Ta / Ag groups were close to 100%, indicating that these two coatings had a strong inhibitory effect on Escherichia coli; for Staphylococcus aureus, the antibacterial rate of the TC4-Ta / Ag group was also close to 100%, while the TC4-Ta-Ag group also showed a high antibacterial rate, but slightly lower than that of the TC4-Ta / Ag group. It is shown that the TC4-Ta-Ag and TC4-Ta / Ag coatings exhibit excellent performance in inhibiting the growth of Escherichia coli and Staphylococcus aureus. These coatings can inhibit the growth and reproduction of bacteria by changing the surface properties of bacteria, destroying the bacterial structure or interfering with its metabolic process, etc.; the coating combinations with excellent antibacterial properties were screened out as the TC4-Ta-Ag and TC4-Ta / Ag coatings.

[0045] See attached Figure 6, the effect of bone integration was evaluated by measuring the ratio of the length of newly formed bone in direct contact with the material surface to the cross-sectional length of the material (BIC). The BIC value of the TC4 group was 0.17±0.012, indicating relatively weak bone integration ability; the BIC value of the TC4-Ta-Ag group was 0.42±0.023, showing an improvement compared to the TC4 group; the BIC value of the TC4-Ag / Ta group was 0.36±0.015, and its bone integration ability was between that of the TC4 group and the TC4-Ta-Ag group; the BIC value of the TC4-Ta / Ag group was 0.78±0.018, significantly higher than that of other groups, indicating that it could better promote bone integration. This result shows that the TC4-Ta / Ag group has the best bone integration ability because the material in this group can form a stable endogenous electric field with the cells on the surface, thus having a long-term stable antibacterial ability, and the final biological performance is better bone integration ability, which is of great significance for the development of medical implant materials with excellent biocompatibility and antibacterial properties. Through the above VG staining experiment, sputtering tantalum first and then silver (TC4-Ta / Ag) has excellent antibacterial ability in an inflammatory environment due to the formation of an endogenous electric field between the osteoblast cell membranes in vivo, and finally shows the best bone integration ability.

[0046] See the appendix Figure 7 , no obvious inflammatory cells or lesions were found in the heart, liver, spleen, lungs, kidneys, and testes of each group, indicating that the titanium alloy bone implant with Ta / Ag coating did not cause obvious damage to the important tissues and organs, and the in vivo safety of this implant was good, and it may have great clinical application prospects and value.

[0047] The above content is only to illustrate the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a multifunctional coating that forms a long-term stable endogenous electric field to promote antibacterial properties, characterized in that: include: sputtering a tantalum film on the treated medical metal substrate to obtain a base layer of the coating; A silver film is sputtered on the base layer to obtain a Ta / Ag double-layer coating; The Ta / Ag double-layer coating was immersed in simulated body fluid, washed, and dried to obtain a multifunctional coating that forms a long-term stable endogenous electric field to promote antibacterial properties.

2. The method for preparing a multifunctional coating for forming a long-term stable endogenous electric field to promote antibacterial properties according to claim 1, characterized in that: The treated medical metal matrix is ​​obtained by mechanically polishing the surface of the medical metal substrate, cleaning it, and drying it; the medical metal matrix is ​​titanium or titanium alloy.

3. The method for preparing a multifunctional coating for forming a long-term stable endogenous electric field to promote antibacterial properties according to claim 2, characterized in that: The mechanical polishing treatment conditions are: polishing time is 10-15 minutes, polishing pressure is 40-60N, polishing speed is 100-150rpm, and the surface roughness after polishing is Ra < 0.1 µm.

4. The method for preparing a multifunctional coating for forming a long-term stable endogenous electric field to promote antibacterial properties according to claim 1, characterized in that: The thickness of the tantalum film is 200-300 nm, and the thickness of the silver film is 200-300 nm.

5. The method for preparing a multifunctional coating for forming a long-term stable endogenous electric field to promote antibacterial properties according to claim 1, characterized in that: The conditions for sputtering the tantalum film are: sputtering power is 100 W~200 W, sputtering atmosphere is argon, sputtering pressure is 0.3 Pa~0.5 Pa, and sputtering time is 20 min~30 min.

6. The method for preparing a multifunctional coating for forming a long-term stable endogenous electric field to promote antibacterial properties according to claim 1, characterized in that: The conditions for sputtering the silver film are: sputtering power is 150 W to 250 W, sputtering atmosphere is argon, sputtering pressure is 0.2 Pa to 0.4 Pa, and sputtering time is 20 min to 40 min.

7. The method for preparing a multifunctional coating for forming a long-term stable endogenous electric field to promote antibacterial properties according to claim 1, characterized in that: The immersion conditions are as follows: immersion temperature is 36-38°C, immersion time is 20-28 h, and the simulated body fluid is replaced every 4 hours; the simulated body fluid composition is: sodium chloride 9 g / L, calcium chloride 0.2 g / L, magnesium chloride 0.1 g / L, sodium dihydrogen phosphate 0.4 g / L, potassium chloride 0.4 g / L, and pH value is 7.

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8. The multifunctional coating that forms a long-term stable endogenous electric field to promote antibacterial properties obtained by the preparation method according to any one of claims 1 to 7.

9. The multifunctional coating for forming a long-term stable endogenous electric field to promote antibacterial properties according to claim 8, characterized in that: The multifunctional coating is a double-layer structure consisting of a tantalum bottom layer and a silver top layer. The tantalum bottom layer serves as the base layer of the coating and is directly sputtered on the surface of the medical metal matrix; the silver top layer is sputtered on the tantalum bottom layer and can release silver ions to form an endogenous electric field to inhibit the growth and reproduction of bacteria.

10. Use of the multifunctional coating for forming a long-term stable endogenous electric field to promote antibacterial properties as claimed in claim 8 or 9 in the preparation of dental implants, bone connection plates, porous scaffolds for bone reconstruction, intramedullary prostheses, internal fixation scaffolds, external fixation scaffolds or joint prostheses.

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