Titanium metal material with antibacterial and muscle-promoting functions as well as preparation method and application of titanium metal material

Abstract

The invention discloses a titanium metal material with antibacterial and muscle-promoting functions as well as a preparation method and application of the titanium metal material. The preparation method comprises the following steps: carrying out plasma pretreatment on a titanium substrate to improve the surface hydrophilicity; preparing a chitosan / HHC36 solution as a cation solution, and preparing a cellulose nano-whisker aqueous suspension as an anion solution; the two solutions are alternately spin-coated on the surface of a titanium substrate, and an LBL (layer-by-layer self-assembly) multilayer film formed by chitosan / HHC36 layers and cellulose nano-whisker layers alternately is constructed. The multi-layer structure can provide a continuous antibacterial effect after an operation, and the cellulose nanowhiskers form an orientation structure through spin coating, so that mouse skeletal muscle myoblasts can be induced to grow in the orientation direction, and ordered regeneration of soft tissues is effectively promoted. The titanium metal material with the antibacterial and muscle-promoting functions is suitable for anti-infection and tissue integration surface modification of implants in the orthopedics department and the stomatology department.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a titanium metal material with antibacterial and myogenic functions, and a preparation method and application thereof. Background Art

[0002] Medical implants (such as artificial joints, bone fixation plates, and dental implants) are widely used in clinical practice, but postoperative infection remains a major complication. Once bacteria colonize and form biofilms on implant surfaces, severe infection can necessitate implant removal and long-term antibiotic therapy. To reduce the risk of infection, researchers have explored various strategies for antimicrobial modification of implant surfaces, including antibiotic coatings, silver ion / nanosilver coatings, and antimicrobial peptide coatings. Among them, antimicrobial peptides (AMPs), a novel class of antimicrobial agents, have attracted considerable attention due to their broad spectrum, high efficacy, and resistance to drug resistance. For example, the AMP HHC36 (sequence: KRWWKWWRR), composed of nine amino acids, is a typical cationic antimicrobial peptide with potent antimicrobial activity against pathogens such as Staphylococcus aureus. HHC36 possesses unique advantages: broad antimicrobial activity (including against multidrug-resistant "superbugs"), efficacy at low concentrations of 0.3–11 μM, and minimal cytotoxicity to mammalian cells (even at concentrations as high as 251 μM, it causes little hemolysis). However, most current antimicrobial coatings focus solely on preventing infection, often suffering from issues such as excessively rapid drug release, short-lived antimicrobial duration, or insufficient support for surrounding tissue integration. Specifically, existing implant surfaces lack effective means to promote the regeneration of soft tissue / muscle tissue. This often hinders the orderly growth and integration of surrounding muscle or soft tissue after implantation, compromising long-term stability. Research has shown that constructing oriented nanostructures on a material surface can guide cell growth along specific directions. For example, spin-coated oriented cellulose nanowhiskers (CNWs) can induce myoblasts to adopt an elongated orientation and accelerate their fusion to form multinucleated myotubes. This phenomenon suggests that constructing oriented nanofiber structures could potentially promote the orderly regeneration of surrounding muscle or fibrous tissue, thereby improving the integration of the implant with the soft tissue interface. Chitosan, a natural polysaccharide polymer, has extensive research interest in implant coatings, as it possesses antimicrobial activity and promotes cell adhesion and growth. Therefore, if chitosan and oriented cellulose nanowhiskers can be combined and loaded with broad-spectrum antimicrobial peptides, it will be possible to simultaneously solve the two major problems of implant anti-infection and tissue regeneration. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, the present invention primarily aims to provide a method for preparing a titanium material with antibacterial and muscle-promoting properties. This method involves constructing a double-layer coating containing HHC36 chitosan / cellulose nanowhiskers on the titanium surface. This resulting titanium material possesses antibacterial and infection-fighting properties and promotes muscle tissue regeneration. This titanium material can be used for anti-infection and tissue regeneration repair on the surfaces of orthopedic or dental implants.

[0004] The second purpose of the present invention is to provide a titanium metal material with antibacterial and myopromoting functions prepared by the above preparation method.

[0005] The third purpose of the present invention is to provide an application of a titanium metal material with antibacterial and myogenic functions.

[0006] This application involves the fabrication of medical implants using titanium materials with antibacterial and myogenic properties. These implants can simultaneously achieve long-lasting antibacterial properties while guiding the orderly regeneration of surrounding soft tissues, such as muscle, thereby reducing infection rates and promoting implant soft tissue integration.

[0007] The primary purpose of the present invention can be achieved through the following technical solutions:

[0008] A method for preparing a titanium metal material with antibacterial and muscle-promoting functions comprises the following steps:

[0009] (1) Surface pretreatment of titanium substrate: A titanium substrate was selected, ground and polished, and then ultrasonically cleaned with acetone, anhydrous ethanol, and deionized water to remove surface oil and impurities. After cleaning, the titanium substrate was blown dry and plasma pretreated.

[0010] (2) preparing a chitosan / HHC36 layer by spin coating deposition: dissolving the antimicrobial polypeptide HHC36 in a chitosan solution to form a chitosan / HHC36 solution, dripping the chitosan / HHC36 solution onto the surface of the titanium substrate treated in step (1), spin coating, and drying to prepare a chitosan / HHC36 layer on the titanium substrate;

[0011] (3) Preparation of the second layer of cellulose nanowhisker layer by spin coating deposition: Cellulose nanowhiskers (CNWs) were ultrasonically dispersed in deionized water to prepare a stable cellulose nanowhisker suspension. The cellulose nanowhisker suspension was dropped onto the titanium substrate on which the chitosan / HHC36 layer had been formed, and the mixture was spin-coated and dried to prepare a cellulose nanowhisker layer on the titanium substrate.

[0012] (4) Layer-by-layer assembly (LBL structure): Repeat the spin coating process of steps (2) and (3) to alternately deposit chitosan / HHC36 layers and cellulose nanowhisker layers until a multilayer coating titanium material with the desired number of layers is obtained;

[0013] (5) Post-processing and sterilization: The assembled multi-layer coated titanium metal material is vacuum dried at room temperature to ensure that all layers are fully dried and the density of the film layer is improved, and disinfected and sterilized to prepare a titanium metal material with antibacterial and muscle-promoting functions.

[0014] Preferably, the titanium substrate in step (1) is a pure titanium substrate or a titanium alloy substrate.

[0015] Preferably, the plasma used in the plasma pretreatment in step (1) is oxygen plasma, the power of the plasma pretreatment is 50-100 W, and the treatment time of the plasma pretreatment is 5-10 min.

[0016] Preferably, the chitosan / HHC36 solution in step (2) uses chitosan with a deacetylation degree of ≥90%, which is dissolved in a 1 mol / L acetic acid solution at a mass volume ratio of 1 mg:5-10 mL to prepare a chitosan solution with an initial concentration of 5-10 mg / mL, which is then diluted with deionized water to a working concentration of 1-2 mg / mL, and HHC36 freeze-dried powder is added to the chitosan solution and stirred until completely dissolved, so that the final concentration of HHC36 reaches 1-2 mmol / L to prepare a chitosan / HHC36 solution.

[0017] Preferably, the rotation speed of the high-speed spin coating in step (2) is 2000-4000 rpm, and the time is 20-60 s; the rotation speed of the drying is 5000-8000 rpm, and the time is 40-60 s.

[0018] Preferably, the spin coating speed in step (2) is 3000 rpm, and the spin coating time is 30 s.

[0019] Preferably, the drying speed in step (2) is 5000 rpm and the drying time is 50 s.

[0020] Preferably, the preparation method of the cellulose nano whisker suspension in step (3) is as follows:

[0021] Cellulose and sulfuric acid are mixed in a mass-to-volume ratio of 1 g:87.5 ml, and after acid treatment, a cellulose / sulfuric acid solution is prepared; deionized water is added to the cellulose / sulfuric acid solution, residues are removed, centrifugation is performed, the supernatant is discarded, the solution is washed with deionized water, dialyzed until the pH value is close to neutral, and ultrasonic treatment is performed to prepare a cellulose nanowhisker suspension; the mass fraction of the sulfuric acid is 50-70%; the temperature of the acid treatment is 45-55°C, and the time of the acid treatment is 20-30 minutes; the cellulose is any one of Tunisian ascidian cellulose, microcrystalline cellulose, or plant cotton pulp; the centrifugal speed is 6000-8000 rpm, and the centrifugal time is 5 minutes; the ultrasonic treatment frequency is 40 kHz, and the ultrasonic treatment time is 2 minutes; and the concentration of the cellulose nanowhisker suspension is 0.45% w / v.

[0022] Preferably, the spin coating speed in step (3) is 2000-4000 rpm, and the spin coating time is 20-60 s; the drying speed is 5000-8000 rpm, and the drying time is 40-60 s.

[0023] Preferably, 10 double layers are alternately deposited in step (4), i.e. a total of 20 layers, to construct a functional coating of sufficient thickness.

[0024] Preferably, the vacuum drying time in step (5) is 2 hours.

[0025] Preferably, the disinfection and sterilization in step (5) is carried out by using ethylene oxide sterilization or ultraviolet irradiation to disinfect and sterilize the titanium metal material.

[0026] The method for preparing the titanium metal material with antibacterial and myopromoting functions of the present invention activates the surface of the titanium substrate and improves its hydrophilicity through pretreatment. The contact angle of the titanium substrate surface treated with plasma can be reduced from about 90° when it is not treated to close to 0°, showing super hydrophilicity. This hydrophilic surface is conducive to the uniform deposition and firm adhesion of the subsequent coating. In the process of multi-layer layer-by-layer assembly, each layer is briefly dried after deposition to ensure that the interlayer bonding is firm before spin coating of the next layer. Through this layer-by-layer self-assembly method, the present invention constructs a two-component multilayer coating with alternating arrangements of chitosan / HHC36 layers and cellulose nano whisker layers on the titanium surface. Since chitosan is a cationic polysaccharide and the surface of the sulfated cellulose nano whisker carries a negative charge, the electrostatic interaction between the two can enhance the bonding force between the layers, so that the formed multilayer film structure is stably attached to the substrate surface and is not easy to peel off.

[0027] The titanium coating structure, which exhibits antibacterial and myogenic properties, exhibits ordered nanoscale layers: the chitosan / HHC36 layer provides the antibacterial component and an adhesion interface, while the cellulose nanowhisker layer forms an oriented nanofiber network. Under optimized spin-coating parameters, the cellulose nanowhiskers are oriented along the shear direction within the coating, forming a regular, striped nanofiber texture on the coating surface.

[0028] This oriented nanofiber structure can provide contact guidance signals for cells, inducing cells to grow and arrange along the direction of the nanofibers: the preliminary cell culture results of the present invention show that mouse skeletal muscle myoblasts (C2C12) are arranged radially and orderly on the surface of the coating (radially extending with the implant as the center) (see Figure 2 ), while on the unmodified titanium surface they grew randomly without a specific orientation; mouse skeletal muscle myoblasts also showed an elongated orientation and fused into myotubes on the oriented cellulose nanowhisker coating surface, suggesting that the coating can effectively promote the orderly regeneration of muscle tissue. (See Figure 3 ) Meanwhile, the sustained release of HHC36 from the chitosan matrix provides continuous antibacterial protection while cells grow.

[0029] The in vitro antibacterial experiment verified that the coating of the present invention has a significant inhibitory effect on Staphylococcus aureus: for example, after the coating sample was incubated with Staphylococcus aureus for 24 hours, the colony forming unit (CFU) was reduced by more than 99% (see Figure 4 ). Thus, the coating of the present invention imparts excellent antibacterial properties to titanium materials, and is expected to play a role in the early prevention of infection in implants.

[0030] The second object of the present invention can be achieved through the following technical solutions:

[0031] A titanium metal material with antibacterial and muscle-promoting functions is prepared by the above preparation method.

[0032] The third object of the present invention can be achieved through the following technical solutions:

[0033] Application of a titanium metal material with antibacterial and myopromoting functions in medical implants.

[0034] Preferably, the medical implant is an orthopedic or dental implant.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention's method for preparing a titanium material with antibacterial and myopromoting properties utilizes plasma pretreatment followed by spin coating for layer-by-layer self-assembly, successfully constructing a dual-layer functional coating on a titanium surface, effectively combining antibacterial and muscle tissue regeneration functions. The antimicrobial peptide HHC36 in the surface coating of the titanium material exhibits broad-spectrum, high efficacy, and a low risk of drug resistance. Implants made with this titanium material can continuously kill surface bacteria early after implantation, preventing infection. Furthermore, the cellulose nanowhiskers, formed through spin coating, form an oriented nanofiber topology that provides structural guidance to surrounding cells, similar to the myoblast fiber bundles found in the natural extracellular matrix. This promotes myoblast growth and integration into the implant surface, thereby facilitating wound healing and long-term stable fixation. This titanium material preparation method is gentle and simple, requiring no harsh conditions such as high temperature and high pressure. The materials used are all biocompatible polymers or natural nanomaterials, with no toxic side effects on tissues. By adjusting parameters such as the number of spin-coated layers and solution concentration, the coating thickness, drug content, and release rate can be flexibly controlled, offering excellent adjustability and versatility. In summary, the present invention provides a simple and feasible implant surface functionalization scheme, which significantly improves the anti-infection performance and soft tissue regeneration and integration ability of titanium-based implant materials, and has broad application prospects in the fields of orthopedic implants and dental implants. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the process flow of the titanium metal material with antibacterial and muscle-promoting functions described in Example 1;

[0038] Figure 2 This is an atomic force microscope (AFM) image of the surface morphology and orientation characteristics of the cellulose nanowhisker coating of the titanium metal material coating with antibacterial and muscle-promoting functions prepared in Example 1. Figure 2 (a) Surface morphology of a cellulose nanowhisker coating formed by spin coating, showing that the nanowhiskers are highly oriented in a single direction. Figure 2 (b) is the orientation distribution diagram of cellulose nanowhiskers corresponding to Figure 2(a), showing good directional consistency; Figure 2 (c) is the surface morphology of the cellulose nanowhisker coating formed by the drop-addition method, in which the nanowhiskers are distributed in a disorderly manner and in random directions. Figure 2 (d) is the orientation distribution diagram of cellulose nanowhiskers corresponding to Figure 2(c), with a wide angle distribution and no obvious orientation trend; Figure 2 (e) is a box plot of the orientation angles of cellulose nanowhiskers under different substrate treatments. The results show that spin coating treatment improves the orientation of cellulose nanowhiskers and enhances their directional consistency.

[0039] Figure 3This is a confocal fluorescence microscopic image of C2C12 cell myotube formation on the surface of the titanium metal material coating with antibacterial and myogenic function prepared in Example 1, in which myosin heavy chain (MHC) staining is green and cell nuclei staining is blue, which is used to evaluate the effects of different surface treatments on myogenic differentiation, among which: Figure 3 (a) Fluorescence image of C2C12 cells cultured on an untreated titanium sheet for 4 days; Figure 3 (b) Fluorescence image of C2C12 cells cultured on a titanium sheet treated with LBL spin coating for 4 days; Figure 3 (c) Fluorescence image of C2C12 cells cultured on an untreated titanium sheet for 7 days; Figure 3 (d) Fluorescence image of C2C12 cells cultured on a titanium sheet treated with LBL spin coating for 7 days;

[0040] Figure 4 This is a confocal fluorescence microscopic image of C2C12 cell myotube formation on the surface of the titanium metal material coating with antibacterial and myogenic function prepared in Example 2, in which myosin heavy chain (MHC) staining is green and cell nuclei staining is blue, which is used to evaluate the effects of different surface treatments on myogenic differentiation, among which: Figure 4 (a) with Figure 4 (b) Fluorescence images after culturing for 4 and 7 days, respectively, according to Example 2;

[0041] Figure 5 This is the antibacterial rate result of the titanium metal material with antibacterial and muscle-promoting functions prepared in Example 2 against Staphylococcus aureus. DETAILED DESCRIPTION

[0042] Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but the present invention is not limited to these embodiments.

[0043] Example 1: Preparation of coated titanium sheet

[0044] like Figure 1 The figure shows a process flow diagram of the titanium metal material with antibacterial and muscle-promoting functions described in Example 1; the titanium substrate surface is plasma pretreated and spin-coated to construct a chitosan / HHC36 layer and a cellulose nano whisker layer;

[0045] In this embodiment, 1×1 cm 2 , a 1mm thick pure titanium sheet was used as a substrate to prepare a titanium material with antibacterial and muscle-promoting functions. The specific steps are as follows:

[0046] (1) Surface pretreatment of titanium substrate: A pure titanium disc was selected as the titanium substrate, and the disc was ground and polished. The pure titanium disc was placed in acetone, anhydrous ethanol and ultrapure water for ultrasonic cleaning for 5 minutes each to remove surface oil and impurities. The disc was rinsed with ultrapure water and blown dry. Then, the surface of the titanium disc was treated with oxygen plasma (power 100W) for 5 minutes. After treatment, the surface of the titanium disc changed from hydrophobic to hydrophilic, and the water droplet contact angle decreased from about 85° to below 5°, showing superhydrophilicity, indicating that the surface was clean and activated successfully.

[0047] (2) The first layer of spin coating deposition is used to prepare a chitosan / HHC36 layer: the antimicrobial polypeptide HHC36 is dissolved in a chitosan solution to form a chitosan / HHC36 solution, the chitosan / HHC36 solution is added dropwise to the surface of the titanium substrate treated in step (1), spin-coated and dried to prepare a chitosan / HHC36 layer on the titanium substrate; the chitosan / HHC36 solution uses chitosan with a deacetylation degree of ≥90% (viscosity-average molecular weight of about 100 kDa), the chitosan is dissolved in 1 mol / L acetic acid to obtain a chitosan solution with a concentration of 10 mg / mL, and the chitosan solution is diluted to 1 mg / mL with deionized water, and then HHC36 freeze-dried powder is added and stirred to dissolve, and the HHC36 concentration is adjusted to 1 mmol / L. The viscosity of the solution is moderate, and the solution is allowed to stand to eliminate bubbles before use; the spin coating speed is 3000 rpm and the spin coating time is 30 s; the drying speed is 5000 rpm and the drying time is 50 s;

[0048] (3) Preparation of the second layer of cellulose nanowhisker layer by spin coating deposition: Cellulose nanowhiskers (CNWs) were ultrasonically dispersed in deionized water to prepare a stable cellulose nanowhisker suspension. The cellulose nanowhisker suspension was dropped onto the titanium substrate on which the chitosan / HHC36 layer had been formed, and the mixture was spin-coated and dried to prepare a cellulose nanowhisker layer on the titanium substrate.

[0049] The preparation method of the cellulose nano whisker suspension is as follows:

[0050] Cellulose and sulfuric acid are mixed in a mass-to-volume ratio of 1 g:87.5 ml, and after acid treatment, a cellulose / sulfuric acid solution is prepared; deionized water is added to the cellulose / sulfuric acid solution, residues are removed, centrifuged, the supernatant is discarded, the solution is washed with deionized water, dialyzed until the pH is close to neutral, and ultrasonically treated to prepare a cellulose nanowhisker suspension with a concentration of 0.45% w / v; the mass fraction of the sulfuric acid is 64%; the temperature of the acid treatment is 45°C, and the time of the acid treatment is 30 minutes; the cellulose is microcrystalline cellulose; the speed of the centrifugation is 8000 rpm, and the time of the centrifugation is 5 minutes; the frequency of the ultrasonic treatment is 40 kHz, and the time of the ultrasonic treatment is 2 minutes; the speed of the spin coating in step (3) is 3000 rpm, and the time of the spin coating is 30 seconds; the speed of the drying is 5000 rpm, and the time of the drying is 50 seconds.

[0051] The specific method of the dialysis is as follows:

[0052] The supernatant was discarded, and the cellulose / sulfuric acid solution, which had been washed seven times with deionized water, was placed in a dialysis bag and dialyzed in deionized water for seven days, with the dialysis water regularly replaced until the pH of the solution approached neutral. The resulting suspension after dialysis was ultrasonically treated for two minutes to obtain a uniformly dispersed cellulose nanowhisker suspension. Transmission electron microscopy revealed that the cellulose nanowhiskers were nanorod-like particles with a length of 200 nm and a diameter of 10 nm.

[0053] (4) Multilayer layer-by-layer assembly (LBL structure): Repeat the spin coating process of steps (2) and (3), alternately depositing chitosan / HHC36 layers and cellulose nanowhisker layers until a multilayer coated titanium metal material with the desired number of layers is obtained; in this embodiment, a total of 10 double layers are repeatedly deposited (the cycle of chitosan / HHC36 layers and cellulose nanowhisker layers is repeated 10 times, ultimately forming a 20-layer stacked structure); finally, the titanium sheet coated with the multilayer film is placed in a vacuum drying oven and dried at room temperature for 2 hours to obtain the target coated titanium sheet sample, i.e., the titanium metal material with antibacterial and myogenic functions;

[0054] (5) Post-processing and sterilization: The assembled multi-layer coated titanium metal material is vacuum dried at room temperature to ensure that all layers are fully dried and the film density is improved, and disinfected and sterilized to prepare a titanium metal material with antibacterial and muscle-promoting functions; the vacuum drying time is 2 hours; the disinfection and sterilization uses ethylene oxide sterilization or ultraviolet irradiation to disinfect and sterilize the titanium metal material.

[0055] After 10 double-layer assemblies of the titanium metal material with antibacterial and muscle-promoting functions described in this embodiment, the total coating thickness is about 3.5 μm. After drying, the coating is uniform and transparent with no cracks or peeling when observed with the naked eye. The adhesion test shows that the coating is firmly attached to the titanium surface without obvious peeling. Figure 2As shown in FIG, the atomic force microscope (AFM) image of the surface morphology and orientation characteristics of the cellulose nanowhisker coating of the titanium metal material coating with antibacterial and muscle-promoting function prepared in this embodiment is characterized, wherein: Figure 2 (a) Surface morphology of a cellulose nanowhisker coating formed by spin coating, showing that the nanowhiskers are highly oriented in a single direction. Figure 2 (b) is the orientation distribution diagram of cellulose nanowhiskers corresponding to Figure 2(a), showing good directional consistency; Figure 2 (c) Surface morphology of the cellulose nanowhisker coating formed by dropwise addition, showing that the nanowhiskers are randomly distributed and oriented. Figure 2 (d) is the orientation distribution diagram of cellulose nanowhiskers corresponding to Figure 2(c), with a wide angle distribution and no obvious orientation trend; Figure 2 (e) is a box plot of the orientation angles of cellulose nanowhiskers under different substrate treatment methods. The results show that spin coating treatment improves the orientation of cellulose nanowhiskers and enhances their directional consistency.

[0056] like Figure 3 As shown, the confocal fluorescence microscopy images of C2C12 cell myotube formation on the surface of the titanium metal material coating with antibacterial and myogenic function prepared in this example, wherein the myosin heavy chain (MHC) staining is green and the cell nucleus staining is blue, are used to evaluate the effects of different surface treatments on myogenic differentiation, wherein: Figure 3 (a) Fluorescence image of C2C12 cells cultured on an untreated titanium sheet for 4 days; Figure 3 (b) Fluorescence image of C2C12 cells cultured on a titanium sheet treated with LBL spin coating for 4 days; Figure 3 (c) Fluorescence image of C2C12 cells cultured on an untreated titanium sheet for 7 days; Figure 3 (d) Fluorescence image of C2C12 cells cultured on the titanium sheet surface treated with LBL spin coating after 7 days.

[0057] Depend on Figure 3 As can be seen, C2C12 cells seeded on the treated titanium surface (the antibacterial, myogenic titanium surface prepared in this example) exhibited significant muscle fusion and myosin heavy chain (MHC) expression. However, the degree of muscle fusion and MHC expression observed on the control surface (untreated titanium) was lower, and myotube staining was also less than that on the treated titanium surface.

[0058] Example 2: Preparation of coated titanium sheet

[0059] like Figure 1 The figure shows a process flow diagram of the titanium metal material with antibacterial and muscle-promoting functions described in Example 2; the titanium substrate surface is plasma pretreated and spin-coated to construct a chitosan / HHC36 layer and a cellulose nano whisker layer;

[0060] In this embodiment, 1×1 cm 2 , a 1mm thick pure titanium sheet was used as a substrate to prepare a titanium material with antibacterial and muscle-promoting functions. The specific steps are as follows:

[0061] (1) Surface pretreatment of titanium substrate: A pure titanium disc was selected as the titanium substrate, and the disc was ground and polished. The pure titanium disc was placed in acetone, anhydrous ethanol and ultrapure water for ultrasonic cleaning for 5 minutes each to remove surface oil and impurities. The disc was rinsed with ultrapure water and blown dry. Then, the surface of the titanium disc was treated with oxygen plasma (power 100W) for 5 minutes. After treatment, the surface of the titanium disc changed from hydrophobic to hydrophilic, and the water droplet contact angle decreased from about 85° to below 5°, showing superhydrophilicity, indicating that the surface was clean and activated successfully.

[0062] (2) The first layer of spin coating deposition is to prepare a chitosan / HHC36 layer: the antimicrobial polypeptide HHC36 is dissolved in a chitosan solution to form a chitosan / HHC36 solution, the chitosan / HHC36 solution is added dropwise to the surface of the titanium substrate treated in step (1), spin-coated at high speed, and dried at a higher speed to prepare a chitosan / HHC36 layer on the titanium substrate; the chitosan / HHC36 solution uses chitosan with a deacetylation degree of ≥90% (viscosity-average molecular weight of about 100 kDa), the chitosan is dissolved in 1 mol / L acetic acid to obtain a chitosan solution with a concentration of 10 mg / mL, and the chitosan solution is diluted to 1 mg / mL with deionized water, and then HHC36 freeze-dried powder is added and stirred to dissolve, and the HHC36 concentration is adjusted to 1 mmol / L. The viscosity of the solution is moderate, and the solution is allowed to stand to eliminate bubbles before use; the spin coating speed is 3000 rpm and the spin coating time is 30 s; the drying speed is 5000 rpm and the drying time is 50 s;

[0063] (3) Preparation of the second layer of cellulose nanowhisker layer by spin coating deposition: Cellulose nanowhiskers (CNWs) were ultrasonically dispersed in deionized water to prepare a stable cellulose nanowhisker suspension. The cellulose nanowhisker suspension was dropped onto the titanium substrate on which the chitosan / HHC36 layer had been formed, and the mixture was spin-coated and dried to prepare a cellulose nanowhisker layer on the titanium substrate.

[0064] The preparation method of the cellulose nano whisker suspension is as follows:

[0065] Cellulose and sulfuric acid are mixed in a mass-to-volume ratio of 1 g:87.5 ml, and after acid treatment, a cellulose / sulfuric acid solution is prepared; deionized water is added to the cellulose / sulfuric acid solution, residues are removed, centrifuged, the supernatant is discarded, the solution is washed with deionized water, dialyzed until the pH is close to neutral, and ultrasonically treated to prepare a cellulose nanowhisker suspension with a concentration of 0.45% w / v; the mass fraction of the sulfuric acid is 64%; the temperature of the acid treatment is 45°C, and the time of the acid treatment is 30 minutes; the cellulose is microcrystalline cellulose; the speed of the centrifugation is 8000 rpm, and the time of the centrifugation is 5 minutes; the frequency of the ultrasonic treatment is 40 kHz, and the time of the ultrasonic treatment is 2 minutes; the speed of the spin coating in step (3) is 2000 rpm, and the time of the spin coating is 40 seconds; the speed of the drying is 5000 rpm, and the time of the drying is 50 seconds.

[0066] The specific method of the dialysis is as follows:

[0067] The supernatant was discarded, and the cellulose / sulfuric acid solution, which had been washed seven times with deionized water, was placed in a dialysis bag and dialyzed in deionized water for seven days, with the dialysis water regularly replaced until the pH of the solution approached neutral. The resulting suspension after dialysis was ultrasonically treated for two minutes to obtain a uniformly dispersed cellulose nanowhisker suspension. Transmission electron microscopy revealed that the cellulose nanowhiskers were nanorod-like particles with a length of 200 nm and a diameter of 10 nm.

[0068] (4) Multilayer layer-by-layer assembly (LBL structure): Repeat the spin coating process of steps (2) and (3), alternately depositing chitosan / HHC36 layers and cellulose nanowhisker layers until a multilayer coated titanium metal material with the desired number of layers is obtained; in this embodiment, a total of 10 double layers are repeatedly deposited (the cycle of chitosan / HHC36 layers and cellulose nanowhisker layers is repeated 10 times, ultimately forming a 20-layer stacked structure); finally, the titanium sheet coated with the multilayer film is placed in a vacuum drying oven and dried at room temperature for 2 hours to obtain the target coated titanium sheet sample, i.e., the titanium metal material with antibacterial and myogenic functions;

[0069] (5) Post-processing and sterilization: The assembled multi-layer coated titanium metal material is vacuum dried at room temperature to ensure that all layers are fully dried and the film density is improved, and disinfected and sterilized to prepare a titanium metal material with antibacterial and muscle-promoting functions; the vacuum drying time is 2 hours; the disinfection and sterilization uses ethylene oxide sterilization or ultraviolet irradiation to disinfect and sterilize the titanium metal material.

[0070] like Figure 4 As shown, the confocal fluorescence microscopy images of C2C12 cell myotube formation on the surface of the titanium metal material coating with antibacterial and myogenic function prepared in this example, wherein the myosin heavy chain (MHC) staining is green and the cell nucleus staining is blue, are used to evaluate the effects of different surface treatments on myogenic differentiation, wherein: Figure 4 (a) with Figure 4 (b) Fluorescence images of C2C12 cells cultured on a titanium sheet treated in this example after 4 and 7 days. While the C2C12 cells exhibited clear muscle fusion and myosin heavy chain (MHC) expression, the high-speed spin coating rate of the CNW layer in Example 2 was lower than in Example 1, preventing the formation of highly oriented CNWs. Consequently, the distribution of myotubes was inferior to that of the highly directional CNW film surface in Example 1.

[0071] Performance testing:

[0072] The coated titanium sheet (a titanium material with antibacterial and myogenic properties) prepared in this experiment was used for in vitro antibacterial and cell culture testing. The antibacterial experiments used Staphylococcus aureus as the indicator bacteria, and tested the in vitro antibacterial properties of four materials: the titanium sheet treated with LBL spin coating, a titanium sheet, a coating coated with 20 layers of CNWS by LBL spin coating, and a coating coated with 20 layers of chitosan / HHC36 solution by LBL spin coating.

[0073] Cell compatibility and tissue growth-promoting properties were tested using mouse skeletal myoblast L929 cells. The cells were seeded onto the surfaces of the LBL-spin-coated titanium sheets of the present invention and control titanium sheets, respectively. After 4 and 7 days of culture, cell morphology was observed using fluorescent staining. The results showed that C2C12 cells seeded onto the LBL-spin-coated titanium sheets exhibited significant muscle cell fusion and MHC protein expression.

[0074] like Figure 5 As shown, the coating of the present invention not only imparts excellent antibacterial properties to titanium materials but also guides orderly cell growth, promoting soft tissue healing and regeneration. This dual-functional titanium coating has potential applications in medical implants where both infection prevention and muscle / soft tissue integration are required (e.g., where bone fixation devices penetrate the skin or dental implants penetrate the gums).

[0075] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a titanium metal material with antibacterial and muscle-promoting functions, characterized in that: The following steps are included: (1) Surface pretreatment of titanium substrate: A titanium substrate was selected, ground and polished, and then ultrasonically cleaned with acetone, anhydrous ethanol, and deionized water to remove surface oil and impurities. After cleaning, the titanium substrate was blown dry and plasma pretreated. (2) preparing a chitosan / HHC36 layer by spin coating deposition: dissolving the antimicrobial polypeptide HHC36 in a chitosan solution to form a chitosan / HHC36 solution, dripping the chitosan / HHC36 solution onto the surface of the titanium substrate treated in step (1), spin coating, and drying to prepare a chitosan / HHC36 layer on the titanium substrate; (3) Preparation of a second layer of cellulose nanowhisker layer by spin coating deposition: cellulose nanowhiskers were ultrasonically dispersed in deionized water to prepare a stable cellulose nanowhisker suspension, and the cellulose nanowhisker suspension was dropped onto the titanium substrate on which the chitosan / HHC36 layer had been formed, and the mixture was spin-coated and dried to prepare a cellulose nanowhisker layer on the titanium substrate; (4) Multilayer layer-by-layer assembly: Repeat the spin coating process of steps (2) and (3) to alternately deposit chitosan / HHC36 layers and cellulose nanowhisker layers until a multilayer coated titanium material with the desired number of layers is obtained; (5) Post-processing and sterilization: The assembled multi-layer coated titanium metal material is vacuum dried at room temperature to ensure that all layers are fully dried and the density of the film layer is improved, and disinfected and sterilized to prepare a titanium metal material with antibacterial and muscle-promoting functions.

2. The method for preparing the titanium metal material with antibacterial and muscle-promoting functions according to claim 1, characterized in that: The titanium substrate in step (1) is a pure titanium substrate or a titanium alloy substrate.

3. The method for preparing the titanium metal material with antibacterial and muscle-promoting functions according to claim 1, characterized in that: The plasma used in the plasma pretreatment in step (1) is oxygen plasma, the power of the plasma pretreatment is 50-100W, and the processing time of the plasma pretreatment is 5-10min.

4. The method for preparing the titanium metal material with antibacterial and muscle-promoting functions according to claim 1, characterized in that: The chitosan / HHC36 solution in step (2) uses chitosan with a deacetylation degree of ≥90%, which is dissolved in a 1 mol / L acetic acid solution at a mass volume ratio of 1 mg:5-10 mL to prepare a chitosan solution with an initial concentration of 5-10 mg / mL, which is then diluted with deionized water to a working concentration of 1-2 mg / mL. HHC36 freeze-dried powder is added to the chitosan solution and stirred until completely dissolved to make a final concentration of HHC36 reach 1-2 mmol / L to prepare a chitosan / HHC36 solution.

5. The method for preparing the titanium metal material with antibacterial and muscle-promoting functions according to claim 1, characterized in that: The rotation speed of the high-speed spin coating in step (2) is 2000-4000 rpm, and the time is 20-60 s; the rotation speed of the drying is 5000-8000 rpm, and the time is 40-60 s.

6. The method for preparing the titanium metal material with antibacterial and muscle-promoting functions according to claim 1, characterized in that: The preparation method of the cellulose nano whisker suspension in step (3) is as follows: Cellulose and sulfuric acid are mixed in a mass-to-volume ratio of 1 g:87.5 ml, and after acid treatment, a cellulose / sulfuric acid solution is prepared; deionized water is added to the cellulose / sulfuric acid solution, residues are removed, centrifugation is performed, the supernatant is discarded, the solution is washed with deionized water, dialyzed until the pH value is close to neutral, and ultrasonic treatment is performed to prepare a cellulose nanowhisker suspension; the mass fraction of the sulfuric acid is 50-70%; the temperature of the acid treatment is 45-55°C, and the time of the acid treatment is 20-30 minutes; the cellulose is any one of Tunisian ascidian cellulose, microcrystalline cellulose, or plant cotton pulp; the centrifugal speed is 6000-8000 rpm, and the centrifugal time is 5 minutes; the ultrasonic treatment frequency is 40 kHz, and the ultrasonic treatment time is 2 minutes; and the concentration of the cellulose nanowhisker suspension is 0.45% w / v.

7. The method for preparing the titanium metal material with antibacterial and muscle-promoting functions according to claim 1, characterized in that: The spin coating speed in step (3) is 2000-4000 rpm, and the spin coating time is 20-60 s; the drying speed is 5000-8000 rpm, and the drying time is 40-60 s.

8. The method for preparing the titanium metal material with antibacterial and muscle-promoting functions according to claim 1, characterized in that: The disinfection and sterilization in step (5) is carried out by using ethylene oxide sterilization or ultraviolet irradiation to disinfect and sterilize the titanium metal material.

9. A titanium material with antibacterial and muscle-promoting functions, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the titanium metal material with antibacterial and myopromoting functions according to claim 9 in medical implants.

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