Simplified chemical deposition of Cu₂O/Ag nanoparticle on titanium alloy with robust early antibacterial activity and retained efficacy after mechanical abrasion

Abstract

Abstract

Implant-associated infections (IAIs) are severe complications following orthopedic procedures involving implanted materials. Previous researchers had created various antibacterial coatings to prevent early postoperative infections. Nevertheless, these coatings frequently lack the wear-resistant properties necessary for long-term effectiveness, and their production process is intricate. To overcome this challenge, we developed and employed a chemical technique, incorporating Ag or Cu₂O nanoparticles uniformly into the surface of titanium alloys to confer antibacterial properties. The microstructure and elemental composition of the coating were characterized using scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS). Our results demonstrated that the coating exhibited potent antibacterial activity, eliminating nearly all adhered bacteria within the first 6 hours. Prolonged friction test results revealed that the coating retained notable antibacterial activity and excellent biocompatibility. Notably, the straightforward fabrication process of this coating could allow for its application on implants of various shapes and materials, underscoring its potential for broad clinical adoption. In summary, this simple chemical method for surface modification of titanium alloys could provide long-lasting antibacterial properties, offering a cost-efficient and transformative strategy for preventing implant-associated infections.

Graphical Abstract

A. Preparation and characterization of the Ag or Cu2O nanoparticle coatings based on titanium alloy. This simple chemical deposition method for surface modification of titanium alloys provides long-lasting antibacterial properties and wear resistance. B. In-vitro antibacterial activity assay. The number of bacteria on the Ag and Cu-coated samples was significantly reduced compared to the control group. The bacterial surfaces in the samples showed significant damage, such as rupture, collapse, and distortion. C-D. In-vivo antibacterial assay of the acute osteomyelitis models and subcutaneous infection models. These nanoparticle coatings exhibit excellent in vivo antibacterial properties and maintain favorable antibacterial efficacy even after prolonged wear. Created in BioRender. Fang, J. (2025) https://BioRender.com/v64b128

Introduction

In the contemporary medical field, orthopedic implants are indispensable in fracture treatment and joint replacement surgeries. With the acceleration of population aging and advancements in medical technology, the demand for orthopedic implants has significantly increased. Due to their excellent mechanical properties, superior biocompatibility, low elastic modulus, and favorable chemical stability, titanium and its alloys have become one of the most used materials in orthopedic implants [1, 2]. However, titanium metal lacks antibacterial properties, making it prone to implant-related infection (IAIs). Once bacteria adhere to the implant surface during or after surgery, biofilm is highly likely to form. Biofilms are bacterial communities that secrete extracellular polymeric substances (EPS), protecting the bacteria within the biofilm.

Consequently, post-operative infections associated with biofilm formation are challenging to treat, hindering wound healing and bone integration and ultimately leading to surgical failure [3]. This situation not only imposes a substantial economic burden and resource waste on society and patients’ families but also significantly increases patient suffering [4, 5], thereby limiting the clinical performance of these materials. In the United States, 47 ± 2% of hip and 39 ± 2% of knee replacement surgeries require revision [6]. Various coatings have been designed on the implant surface to address this issue to prevent IAIs [7]. Some coatings covalently bind antibiotics to the implant surface [8, 9], while others impart nanoscale roughness to the surface through physical and chemical methods or develop antibacterial coatings by incorporating various metallic and non-metallic elements.

Among these, metallic nanoparticle coatings have garnered significant attention due to their unique antibacterial mechanisms. They can kill bacteria by utilizing antibacterial metallic nanoparticles and influencing them through nanostructure biomechanical mechanisms [10]. Specifically, silver (Ag) and copper (Cu) are notable for their strong antibacterial properties [11]. Several methods exist for preparing metallic nanoparticle coatings, including sputtering, anodic oxidation, acid etching, and micro-arc oxidation [12–16]. However, these preparation techniques often involve complex processes, imposing strict requirements on the substrate, such as the need for electrical conductivity or a regular flat surface. In clinical applications, customized medical implants or devices with specific shapes are often required to fit the anatomical structure of patients [17], which imposes limitations on these coating preparation techniques. Therefore, there is an urgent need for a convenient, efficient, and widely applicable antibacterial coating technology that allows for large-scale coating preparation.

Additionally, varying degrees of wear may occur during the implantation process and subsequent micro-movements of the implants in the body. The antibacterial properties of coatings after wear are critical for maintaining the long-term antibacterial efficacy of the implants, but limited studies have shown on this area.

Therefore, this study developed a facile fabrication method for Ag or Cu₂O nanoparticle coatings with dual functionality—antibacterial properties and wear resistance. Using a chemical reduction approach, metal nanoparticles were deposited onto titanium alloy plates. The coating was characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS), and its early-stage antibacterial efficacy was evaluated through a series of in vitro and in vivo experiments. Furthermore, in vitro wear testing was conducted to simulate long-term implant abrasion, allowing for additional assessment of the coating’s post-wear antibacterial performance and biocompatibility.

This work aims to develop a simple yet wear-resistant antibacterial nanoparticle coating for orthopedic implants, providing a viable strategy to reduce production costs while enhancing the durability of antibacterial effects. The proposed coating holds significant potential for widespread clinical adoption due to its cost-effectiveness and functional advantages.

Materials and methods

Preparation of Ag or Cu₂O nanoparticle coatings and their post-friction surfaces

Medical-grade titanium alloy plates, Ti6Al4V, were selected as the substrate material for the coating. The critical reagents used in the experiment included CuSO4 solution (C128345-1L, Aladdin, China, 0.5 mol/L), AgNO₃ powder (209139-25 G, Merck KGaA, Germany, ≥99.0%), NaOH powder(S580606-500g, Aladdin, China, 97%), and aldehyde solution (F111936-500ml, Aladdin, China, 37 wt.% in H2O).A 0.5 mol/L AgNO₃ solution was prepared using sterile water, and a formaldehyde solution with a mass fraction of 35% was also formulated. Scheme 1 illustrates the schematic process for coating preparation. As depicted in Scheme 1A, Ti6Al4V plates were immersed in 150 mL of either 0.5 mol/L AgNO₃ solution or 0.5 mol/L CuSO₄ solution at 25 °C for 10 min. Then, the immersed Ti6Al4V plates were then thoroughly dried in an oven at 100 °C. Subsequently, as illustrated in Scheme 1B, to convert the adsorbed ions on the Ti6Al4V plates into metal oxide precipitates, the treated plates were immersed in 150 mL of 1 mol/L sodium hydroxide (NaOH) solution for 30 min. To reduce the precipitated oxides into nanoparticles, 10 mL of 35 wt.% formaldehyde solution was added to the system, and the mixture was heated to 100 °C under reflux using the setup shown in Scheme 1C for 30 min. The reflux heating apparatus consisted of a two-neck flask, a condenser, a beaker, and a supporting stand. According to the reaction device shown in Scheme 1C, the redox reactions were as follows [18]

$${\mathrm{Ag}}_2\mathrm{O}+{\mathrm{CH}}_2\mathrm{O}\to 2\mathrm{Ag}+\mathrm{HCOOH}$$

Scheme 1.

The simple preparation process of the Ag and Cu₂O nanoparticle coatings based on titanium alloy. Created in BioRender. Fang, J. (2025) https://BioRender.com/t23x126. A The Ti6Al4V plates were immersed in AgNO₃ solution or CuSO₄ solution for 10 min, followed by thorough drying in an oven to facilitate the adsorption of metal ions onto the metal surface. B The Ti6Al4V plates with adsorbed metal ions were immersed in a 1 mol/L sodium hydroxide (NaOH) solution for 30 min to convert the metal ions on the surface into metal oxide precipitates. C 10 ml of formaldehyde solution were introduced into the reaction system, followed by heating under reflux at 100 °C for 30 min, facilitating the reduction of metal oxides into nanoparticles. D The metal plates, after the completion of the reaction, were dried in an oven at 60 °C

And Cu (OH)₂ was reduced to nanoscale Cu₂O [19]. After 30 min of treatment, the titanium alloy plates were removed, as shown in Scheme 1D, thoroughly rinsed with deionised water, and dried in an oven at 60 °C. This process resulted in titanium alloy plates coated with Ag or Cu₂O nanoparticles, designated as Ag and Cu, respectively.

To further evaluate the changes in antibacterial properties and biocompatibility of the titanium alloy coatings after long-term in vivo wear, the samples were subjected to 100 cycles of wet abrasion testing using a scrubbing resistance tester for architectural coatings (PS2730, Pushen, China), according to the GB/T31410-2015 standard. The resultant materials were labeled as Ag100 and Cu100, respectively. Untreated medical-grade titanium alloy plates (Ti6Al4V) were designated as Ti. As illustrated in Fig. S1B of the Supplementary Materials, all samples were cut into 1 cm² squares for subsequent experimental evaluations. To better mimic clinical orthopedic applications, the antibacterial coating was additionally deposited onto conventional fracture fixation plates.

Characterization of the coatings

The microscopic surfaces of Ti, Ag, Cu, Ag100, and Cu100 samples were observed using Scanning Electron Microscopy in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University. The surface chemical composition of samples Ag and Cu was analyzed using energy-dispersive spectroscopy (EDS).

In-Vitro biocompatibility assay

MC3T3-e1 (CL-0710, Pricella, China) cells were used to assess the in vitro biocompatibility of different coating materials, with untreated titanium alloy (Ti) as the control group. The biocompatibility of each coating was evaluated by measuring cytotoxicity and its effects on cell adhesion and proliferation. Five groups of samples were prepared: Ti, Ag, Cu, Ag100, and Cu100. Each experiment was conducted three times for accuracy.

Co-culture of cells and materials

The cell concentration was adjusted to 5 × 10⁵ /ml. A 40 μL aliquot of the cell suspension was added to the surface of each metal plate, with a final count of 20,000 cells per group. The co-culture system was incubated for 4 h to allow the cells to adhere to the material surface fully. Then, 1 ml of culture medium was added to each well, fully submerging the materials.

Cytotoxicity assay

After 48 h of co-culture, the cytotoxicity of each sample (Ti, Ag, Cu, Ag100, Cu100) was assessed by measuring lactate dehydrogenase (LDH) release using an LDH cytotoxicity assay kit (C0016, Beyotime, China). The absorbance of each group was measured at 490 nm using a multifunctional microplate reader. Cytotoxicity was calculated based on the absorbance values using the following formula:

Cell viability (%) = [1 – (Absorbance of treated sample - Absorbance of control well)/(Absorbance of maximum enzyme activity - Absorbance of control well)] × 100%.

Live/dead cell staining assay and cell adhesion assay

The samples were co-cultured with cells for 72 h, with the culture medium replaced daily. After 72 h, the samples were removed. The adherent cells were stained using FITC-Phalloidin (40735ES75, Yeasen Biotechnology, Shanghai, China) and DAPI solution (C1005, Beyotime, China) for cell adhesion assay. While the sample were subjected to live/dead cell staining using the Calcein-AM/PI Double Staining Kit (C542, Dojindo, China). The samples were then observed and imaged using an upright fluorescence microscope (BX53). In the images, live cells were stained green, while dead cells were stained red.

Cell proliferation assay

For each group, metal plates (approximately 1 cm²) were immersed in a complete culture medium at a surface area-to-volume ratio of 1 cm²/mL and incubated for 24 h in an incubator (95% humidity, 5% CO₂, 37 °C). The extract supernatant was collected. MC3T3-e1 cells (5 × 10³ cells/well) were seeded into a 96-well plate with 100 μL of cell suspension per well. After 6 h of incubation to allow for cell adhesion, the culture medium was aspirated, and 100 μL of the corresponding extract (Ti, Ag, Cu, Ag100, Cu100) was added to each well. Cell proliferation was assessed at 24 h, 48 h, and 72 h using a CCK-8 kit (PF00004, Proteintech, China). The control group used an unsoaked titanium alloy culture medium.

In-Vitro antibacterial activity assay

Bacterial culture

The antibacterial performance of the samples was assessed using Gram-negative Escherichia coli (E. coli, ATCC 8099) and Gram-positive Staphylococcus aureus (S. aureus, ATCC 6538). The bacteria were cultured in broth medium at 37 °C and 200 rpm in a shaker for 12 h. The bacterial suspension was then diluted to a concentration of 10⁵–10⁶ CFU/mL for subsequent experiments. All media, samples, and containers used in the experiments were sterilized by autoclaving.

In-Vitro antibacterial assay

Antimicrobial zone testing was performed to evaluate the release kinetics of Cu²⁺ or Ag⁺ ions from the coating surfaces. Bacterial suspensions of E. coli were adjusted to 10⁵–10⁶ CFU/mL, and 250 μL aliquots were spread onto LB agar plates. Subsequently, titanium alloy orthopedic plates with different coatings were accordingly placed onto the inoculated agar surfaces. All samples were incubated at 37 °C for 24 h, after which the inhibition zones were measured. Direct contact antimicrobial activity was assessed by co-culturing bacterial suspensions with coated metal specimens. A 30 μL aliquot of the E. coli or the S. aureus suspension was evenly spread onto the surface of the metal plates. After co-culturing the bacteria and samples in an incubator at 37 °C for 6 h, the samples were carefully transferred to 15 mL centrifuge tubes, and 2 mL of sterile water was added. The samples were sonicated for 10 min to detach the bacteria from the surface. The bacterial suspension was collected, and 150 μL of the cleaning solution was spread on LB agar plates. The plates were incubated at 37 °C for 12 h, and the number of colony-forming units was photographed and counted according to GB/T4789.2 standards.

FE-SEM of bacteria

The bacterial suspension was diluted 10-fold, and 30 μL was added to the surface of the metal discs in 12-well plates. After co-culturing at 37 °C for 6 h, the bacteria were fixed overnight with 4% paraformaldehyde (PFA) and dehydrated. The samples were then observed using field-emission scanning electron microscopy (FE-SEM).

In-Vivo experiments

The in vivo experiments strictly adhered to the guidelines stipulated by the Chinese Law on Animal Experiments and received ethical clearance from the Ethics Committee of Zhejiang University School of Medicine (Ethics No. ZJU20240336).

In-Vivo biocompatibility assay

BALB/c mice (male, ~4 weeks; ~20 ± 2 g) were randomly divided into five groups (Ti, Ag, Cu, Ag100, Cu100), with three mice per group. A 1.5 cm incision was made on the dorsal side of each mouse, and subcutaneous tissue was removed to create a pocket for the implantation of the corresponding metal plate. After 7 days, the implants were retrieved. The surrounding tissues were stained using a hematoxylin and eosin (HE) staining kit (C0105S, Beyotime, China). Inflammatory cells were counted manually three times for accuracy, and tissue inflammatory responses were evaluated.

Establishment of the implant-associated subcutaneous infection model and acute osteomyelitis model

To establish the implant-associated subcutaneous infection model, BALB/c mice (male, ~4 weeks; ~20 ± 2 g) were randomly divided into five groups, with three mice per group, labeled as the Ti group, Ag group, Cu group, Ag100 group, and Cu100 group. A 1.5 cm incision was made on the back of each mouse, and subcutaneous tissue was separated to create a pocket. Into this pocket, 30 µL of Escherichia coli suspension (10⁴ ~ 10⁵ CFU/mL) was injected, and a 1 cm² sample corresponding to each group was implanted.

To establish the acute osteomyelitis model, C57BL/6 mice (male, ~4 weeks; ~15 ± 2 g) were randomly divided into three groups, with three mice per group: Ti, Ag, and Cu. One of the knee joint cavities of each mouse was opened, and a customized handheld drill (approximately 1 mm in diameter) was used to create an opening in the distal femoral marrow cavity. A 10 µL suspension of E. coli (10⁵–10⁶ CFU/mL) was injected into each marrow cavity, and a sample strip (1 mm × 5 mm) was implanted into the marrow cavity. These procedures were performed under general anesthesia, induced by intraperitoneal injection of 0.15 mL of 1% sodium pentobarbital. After surgery, the mice were allowed to move freely in cages with sufficient water and food.

Evaluation of antibacterial activity in infection models

7 days after establishing the infection models, for the implant-associated subcutaneous infection model, the wounds of each mouse were photographed to assess healing progress. The subcutaneous implants were carefully retrieved, immersed in 1 mL of sterile water, and subjected to ultrasonic cleaning for 10 min. Afterwards, 150 µL of the cleaning solution was spread on LB agar plates. The surrounding skin and soft tissue were stained to assess tissue inflammation using a hematoxylin and eosin (HE) staining kit (C0105S, Beyotime, China). The inflammatory reaction around the implant was assessed by manually quantifying inflammatory cells within a defined size range. All counts were performed in triplicate to ensure accuracy.

For the acute osteomyelitis model, as described in Fig. 7A, the mice’s knee joint cavities were opened, and the implants were removed. The surrounding marrow tissue was imprinted onto LB agar plates. The implants were then placed in 1 mL of sterile water, sonicated for 10 min, and 150 µL of the cleaning solution was spread on LB agar plates. All plates were incubated at 37 °C for 12 h, and colony-forming units were photographed and counted according to the Chinese national standard GB/T4789.2.

Fig. 7.

In-vivo antibacterial assay of the acute osteomyelitis models. A The procedure to establish acute osteomyelitis models and test the antibacterial efficiency of implants in vivo. Created in BioRender. Fang, J. (2024) BioRender.com/q91r819. B The imprints of surrounding bone marrow tissue after implantation for 7 days. (Scare bar = 2 cm). C The spread plate images of E. coli seeding in the corresponding extraction solution from the acute osteomyelitis models. (Scare bar = 2 cm). D, E Quantitative tests of the number of CFU from the imprints of the surrounding bone marrow tissue and the spread plate images, respectively (Ns: non significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Statistical analysis

Data in this study were expressed as mean ± standard deviation (SD). Graphs and statistical analyses were performed using one-way ANOVA and t-test with GraphPad Prism software. The values of p < 0.05 were considered statistically significant.

Results

Characterization of Ag and Cu₂O nanoparticle coatings and post-wear coatings

The macroscopic appearance of the coatings is shown in Fig. S1B of the Supplementary Materials. The 1 cm² sample surfaces appear visually indistinguishable from traditional medical titanium alloy surfaces. The EDS spectra for Ag and Cu samples (Fig. 1A and B) revealed that the primary elements on the Ag coating were Ti, O, C, and Ag, with Ag only accounting for 0.32 wt%. The Cu coating consisted of Ti, O, C, and Cu, with Cu only accounting for 0.20 wt%. Furthermore, EDS analysis revealed an evenly distribution of Ag (silver) and Cu (copper) elements across the metal substrate surface. As the substrate material consists of titanium alloy (Ti6Al4V), EDS analysis of the untreated surface revealed a predominant titanium content of 90.55 wt%, as demonstrated in Supplementary Fig. S1A.

Fig. 1.

Material characterization. A, B EDS spectra of the Ag and Cu₂O coatings, respectively (Scare bar = 10 µm). C Surface morphologies characterization of different samples under SEM (Scare bar = 10µm (up row) and 1 µm (down row))

The SEM images (Fig. 1C) show that the NaOH-treated coatings displayed a significantly rougher surface compared to untreated titanium plates, with the untreated Ti showing a relatively smooth surface. As shown in Fig. 1C, due to their high relative atomic masses, silver (Ag) and copper (Cu) exhibit strong electron backscattering capability, consequently appearing brighter in electron microscopy images. Meanwhile, some dispersed nanoparticles formed amorphous structures that subsequently aggregated. In contrast, the Ag100 and Cu100 groups displayed significantly rougher surface morphology due to frictional treatment. Usually chemically deposited Ag nanoparticles typically have diameters of 15–20 nm [18], and the Cu₂O nanoparticles are approximately 10 nm in size [20].

The adsorption mechanism in this study is primarily based on the chemical characteristics of the titanium alloy surface oxide layer (TiO₂). In copper or silver ion solution, ions are adsorbed onto the surface via electrostatic interactions with hydroxyl groups [21]. Treatment with sodium hydroxide further increases the hydroxyl density and may lead to the formation of an intermediate phase. Under heating conditions, formaldehyde reduces Cu²⁺ to form Cu₂O nanoparticles and Ag⁺ to form Ag nanoparticles, and the bonding with the substrate is governed by both chemical bonding and mechanical interlocking. This process is consistent with the reduction kinetics of formaldehyde in chemical copper plating and the passivation behavior of the titanium surface [22]. Further surface characterization will be conducted to confirm the presence of interfacial chemical bonds.

In-Vitro biocompatibility assay

Biocompatibility is crucial as orthopedic implants will directly interact with host cells and tissues [1]. Figure 2A shows the live/dead staining results, where most cells in all groups were viable, with only a few dead cells indicating outstanding in vitro biocompatibility of the material. Additionally, live cell counts were conducted in the same size field of view three times, and the results are shown in Fig. 2B. The data show that the number of viable cells in the Ag and Cu was significantly higher than in the Ti, with statistical significance. The number of viable cells in the Cu100 and Ag100 was lower than that in the Ti, but the differences between these two groups and the control were not statistically significant (P > 0.05).

Fig. 2.

In-vitro biocompatibility assay. A Live/dead cell staining assay. (Scare bar = 50 µm). B Relative quantification of live/dead cell staining. C Cellular morphology of MC3T3-E1 cells co-cultured with the samples (scale bar = 50μm, blue for nuclei, green for actin). D Relative quantification of cell adhesion assay (ns: non significance, *i < 0.05, **p < 0.01, ***p < 0.001)

Excellent cell adhesion indicates that the material has premium biocompatibility at the bone-contacting interface, which is beneficial for bone integration and the biological fixation of the implant [23, 24]. Figure 2C shows that MEC3T3-e1 cells were well-distributed in a reticular pattern across the surfaces of all samples, with extensive adhesion and prominent filopodia extension, consistent with the typical morphology of MC3T3-e1 cells. The number of cells was also sufficient, with very few spherical cells or cells lacking filopodia. This suggests that the coatings, even after long-term wear, did not disrupt the cytoskeletal organization and supported initial cell adhesion.

Cell adhesion was further quantified by counting DAPI-stained cells in different fields of view, and the results are shown in Fig. 2D. There were no statistically significant differences in cell adhesion numbers between the groups and the Ti control. According to previous studies, materials with cell viability rates above 95% are considered to have good biocompatibility [25]. LDH assays were used to evaluate the in vitro cytotoxicity of the materials, and the results are shown in Fig. 3A. The Ti, Ag, and Ag100 groups’ cell viability was above 95%. Although the cell viability of the Cu and Cu100 groups was slightly lower, at 92.9% and 94.7%, respectively, it was still above 92%, and no significant differences were found between the groups (P > 0.05). The results of the CCK-8 assay, displayed in Fig. 3B, indicate a steady increase in absorbance in all groups at 24, 48, and 72 h, suggesting that the cells were increasing in all groups, with statistically significant differences observed (P < 0.001). These findings indicate that the coating treatment and long-term wear did not negatively affect cell proliferation.

Fig. 3.

Biocompatibility assay. A LDH cytotoxicity assay of different samples. B Cell proliferation experiments of different samples on days 1, 2, and 3. C Relative quantification of inflammatory cells of tissue contact with the samples for 7 days. D H&E staining of subcutaneous tissue contact with the samples for 7 days (Scare bar = 100 µm) (ns: non significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

In-Vivo biocompatibility assay

Figure 3D shows the HE staining results of the subcutaneous tissue above the implants in each group. The images indicate that the epidermal structure in all groups remained intact, with no signs of ulceration or detachment. The dermal collagen fibers were dense, and skin appendages such as hair follicles and sebaceous glands were visible. All groups exhibited only mild inflammatory responses.

Based on the inflammatory cell counts in the subcutaneous tissue under the same magnification (Fig. 3C), the Ag, Cu, and Ag100 groups had significantly fewer inflammatory cells than the Ti group, with statistical significance. The Cu100 showed no significant difference in the number of inflammatory cells compared to the Ti.

In-Vitro antibacterial activity assay

As shown in Fig. S1C, the antibacterial zone test revealed no detectable inhibition zones in any experimental groups. The results of direct contact antibacterial assays against both Escherichia coli and Staphylococcus aureus are presented in Figs. 4 and 5, respectively. The Escherichia coli colonies are shown in Fig. 4A. Compared to the control group, the number of bacteria of the Ag or Cu group was significantly reduced. The bacterial count on the Cu100 and Ag100 samples was comparable to that of the Ti group. To further evaluate the antibacterial performance of the groups, bacterial colony counts from three repeated experiments were recorded. As shown in Fig. 4B, both the Ag and Cu groups exhibited significant antibacterial activity (P < 0.001). Notably, there was no significant difference between the Ag100 and Ti groups (P > 0.05), indicating a decline in antibacterial performance for Ag after 100 wet abrasion cycles. In contrast, the Cu100 group still showed a statistically significant difference compared to the Ti group (P < 0.05). The bacterial colonies of Staphylococcus aureus are presented in Fig. 5A. Compared with the control group, all experimental groups exhibited significant reduction in bacterial colonization. Notably, the Ag100 and Cu100 groups maintained marked antibacterial efficacy against S. aureus even after abrasion treatment. To systematically evaluate the antimicrobial performance, colony-forming units (CFU) were quantified through triplicate independent experiments. As demonstrated in Fig. 5B, statistically significant differences were observed in bacterial colony counts between Ti and other groups, confirming the antimicrobial superiority of the experimental treatments.

Fig. 4.

In-vitro antibacterial activity assay. A The spread plate images of E. coli seeding in the corresponding extraction solution. (Scare bar = 1 cm). B Relative quantification of the number of CFU on the plates from different samples. C SEM images of E. coli morphology on the surfaces of various samples (Scare bar = 10 µm (up row) and 1 µm (down row)). D Relative quantification of the number of E. coli on the different samples at 2000 magnification (ns: non significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 5.

In-vitro antibacterial activity assay. A The spread plate images of S. aureus seeding in the corresponding extraction solution. B Relative quantification of the number of CFU on the plates from different samples. C SEM images of S. aureus morphology on the surfaces of various samples (Scare bar = 10 µm (up row) and 1 µm (down row)). D Relative quantification of the number of S. aureus on the different samples at 2000 magnification (ns: non significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

The FE-SEM images illustrating the adhesion of Escherichia coli and Staphylococcus aureus on the sample surfaces are presented in Figs. 4C and 5C, respectively. At a magnification of ×2000, the Ti group exhibited the highest bacterial adhesion density, while all other groups demonstrated significantly lower bacterial adhesion compared to the Ti group. To further evaluate the antimicrobial efficacy of each group, quantitative bacterial counting was performed under lower magnification, as depicted in Figs. 4D and 5D. All experimental groups demonstrated significantly enhanced antibacterial activity relative to the control group (P < 0.0001). Notably, the Ag100 and Cu100 groups maintained comparable antibacterial performance to their respective Ag and Cu counterparts, with no statistically significant decline observed in antimicrobial efficacy, indicating that the coatings maintained good in vitro antibacterial performance even after prolonged wear.

Moreover, the research showed that nanoparticles can disrupt bacterial cell membranes, forming pores [26]. As illustrated in Figs. 4C and 5C, scanning electron microscopy at a magnification of 30,000× revealed structurally intact cellular membranes in both Escherichia coli and Staphylococcus aureus specimens from the Ti group. In contrast, bacterial surfaces from other experimental groups exhibited conspicuous morphological alterations including membrane rupture, cellular collapse, surface wrinkling, and structural distortion. These observed ultrastructural modifications suggest that the coatings induced considerable structural compromise to bacterial cell walls, thereby disrupting their normal physiological processes and demonstrating significant bactericidal efficacy.

In-Vivo antibacterial assay

Subcutaneous infection and acute osteomyelitis models were established in mice to evaluate the in vivo antibacterial performance of the coated titanium plates. Figure 6A shows the condition of the wounds in the subcutaneous infection model 7 days post-implantation. The Ti group exhibited significant infection around the wound, with visible redness, swelling, and poor healing. In contrast, the Cu and Ag groups showed smaller areas of inflammation, with the scars gradually healing. The Ag100 and Cu100 groups still had small areas of inflammation. The results of the bacterial spread plates are shown in Fig. 6B, which shows the Ti group had the highest number of bacteria. In contrast, the bacterial count in the other groups was significantly reduced, with the Ag and Cu groups showing the most notable reductions. Bacterial colony counts from the bacteria spread plates are shown in Fig. 6D. All groups had significantly fewer bacteria than the Ti group, although Ag100 and Cu100 had more bacteria than the Ag and Cu groups. However, Ag100 and Cu100 still showed significant differences from the Ti group (P < 0.0001).

Fig. 6.

In-vivo antibacterial assay of the subcutaneous infection models. A Surgical incision healing at 7 days after operation. (Scare bar =1 cm). B Each group’s spread plate images of E. coli of the subcutaneous infection models. (Scare bar =2 cm). C H&E staining of subcutaneous tissue contact with the samples and E. coli for 7 days. (Scare bar = 100 µm). D Quantitative tests of the number of CFU from spread plate images in the antibacterial assay in vivo. E Relative quantification of inflammatory cells of the tissue in subcutaneous infection models. (Ns non significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Figure 6C shows the HE staining results of the soft tissue surrounding the implants. Inflammatory cell infiltration was extensive in the Ti, Ag100, and Cu100 groups, while the Ag and Cu groups had fewer inflammatory cells. Inflammatory cell counts, shown in Fig. 6E, revealed that the Ag, Ag100, and Cu groups had fewer inflammatory cells than the Ti group. The difference between Ti and these groups was still statistically significant (P < 0.0001). While the Cu100 group showed a decrease in inflammatory cells compared to the Ti group, the difference was not statistically significant (P > 0.05).

The establishment of the acute osteomyelitis model is depicted in Fig. 7A. 7 days after model induction, the implants were sonicated, and the cleaning solution was plated onto LB agar while the surrounding bone marrow tissue was imprinted onto agar plates. Figure 7B shows the imprinting results, where the Ti group exhibited significant bacterial growth, while the Ag and Cu groups showed little to no bacterial growth. Bacterial colony counts from the imprinted plates are shown in Fig. 7D. The Ag and Cu groups had significantly fewer bacteria than the Ti group (P < 0.0001). Figure 7C shows the results of the bacteria spread plates, where the Ti group still exhibited considerable bacterial growth. In contrast, the bacterial count in the Ag and Cu groups was significantly reduced. Figure 7E shows bacterial colony counts from the bacteria spread plates, demonstrating that the Ag and Cu groups had significantly fewer bacteria than the Ti group (P < 0.0001). These results indicate that the coatings retain antibacterial solid performance within the bone marrow cavity.

Discussion

In this study, we designed a simple and durable antibacterial nanoparticle coating with wear-resistant properties. The coating exhibited strong antibacterial properties in the early stage and still had excellent antibacterial properties in vivo and in vitro after 100 cycles of friction. Meanwhile, this coating has premium biocompatibility without compromising its antibacterial effect. Besides, the procedure of this coating is simple, which is convenient for large-scale manufacturing, proving a promising option for IAIs in orthopedic surgery.

IAIs remain a significant challenge in the field of orthopedics. The severe consequences of these infections stem from several factors: (1) Unlike infections in the absence of implants, once an infection occurs, the implant material serves as a substrate for bacterial adhesion, leading to biofilm formation on its surface [27]. (2) In the in-vivo environment, bacteria irreversibly and specifically adhere to the material surface, binding tightly, which differs from bacterial adhesion in vitro [28]. (3) In implant-associated infections, bacterial adhesion triggers chronic inflammation, forming granulation tissue and fibrous encapsulation, creating an immune-escape microenvironment [29, 30]. Therefore, developing implant materials with long-term antibacterial properties is critical to reducing the risk of postoperative infection.

Metal nanoparticles, as a novel class of antimicrobial agents, exhibit excellent antibacterial properties. Their antibacterial mechanisms can be categorized into direct contact-killing effects and ion-mediated killing effects [31, 32]. The EDS analysis of the coating in this study revealed that Ag accounted for only 0.32 wt% and Cu for 0.20 wt% in the coating composition. Furthermore, the inhibition zone assay demonstrated no significant bactericidal regions, indicating that the surface-released metal ion concentrations from the coating were insufficient to exert significant bactericidal effects. The antimicrobial activity of this coating is therefore primarily attributed to the direct contact-killing mechanism.

In terms of short-term antibacterial effects, compared to studies where Ag nanoparticles were loaded into TiO₂ nanotubes [12], the nanoparticles in this study were directly exposed to the material surface. This design not only enables allows for direct nanoparticle contact to kill bacteria but also inhibits bacterial adhesion. Studies have shown that orthopedic implants are most susceptible to bacterial adhesion within the first six hours after implantation [33]. The in vitro antibacterial results of this study demonstrate that the coating exhibited a strong early bactericidal effect after 6 h of co-incubation with bacteria. In the in vivo antibacterial tests, results from the Ag and Cu groups in the subcutaneous infection model demonstrated that implants with this coating exhibited a strong antibacterial effect in the early stages. Furthermore, the osteomyelitis model confirmed this early-stage efficacy. However, it should be noted that the acute osteomyelitis model, established to probe the early-phase antibacterial activity, did not include the Ag100 and Cu100 groups. While this marks a limitation of the present work, it paves the way for future investigations into the medium- and long-term antibacterial properties of these coatings in a chronic osteomyelitis setting. The bacterial challenge in this experiment was harsher than typical clinical scenarios, confirming that the coating meets early antibacterial requirements.

Regarding long-term antibacterial performance, since implants remain in the body for extended periods, friction with tissues and bones is unavoidable. Therefore, the coating must retain antibacterial properties after long-term wear. This study simulated long-term in vivo wear through 100 wet abrasion cycles, and the Ag100 and Cu100 groups still exhibited strong antibacterial effects. Although Ag100’s antibacterial performance decreased in vitro, in actual clinical scenarios, the bacterial concentration the material faces after long-term wear is already reduced due to early antibacterial action, and the dynamic nature of the in-vivo environment further dilutes bacterial concentrations via body fluids. Thus, Ag100 operates in a less stringent environment in clinical applications than in experimental conditions. SEM observations revealed significant damage to the bacterial cell walls in the Ag, Cu, Ag100, and Cu100 groups. In the in-vivo subcutaneous implantation infection model, the Ag100 and Cu100 coatings demonstrated excellent antibacterial performance. Overall, this study has shown that the Ag and Cu nanoparticle coatings exhibit long-term antibacterial activity.

The cytotoxicity and antibacterial effects of metal nanoparticles are often dose-dependent [34]. Previous studies have shown that eukaryotic cells can internalize metal nanoparticles, which release metal ions inside the cell, disrupting cellular functions and damaging genetic material [35]. It has been established that there is a concentration range of nanoparticles that own lower cytotoxicity with significant antibacterial properties. Compared to coatings that combine nanoparticles with PEI, BG, or PLGA [36–38], the coating in this study contains no organic components, simplifying the structure while maintaining excellent biocompatibility without sacrificing antibacterial efficacy. Moreover, this study observed that although the cell viability of Cu and Cu100 in the LDH assay was slightly below 95%, it remained above 92%, and Cu²⁺ is readily diluted in the in vivo environment [39], further reducing toxicity. Additionally, the coating exhibited good cell adhesion, with well-spread cytoskeletons in all groups. In vivo experiments showed that the coating did not induce significant tissue inflammation compared to Ti. Besides, it could further optimize the Ag and Cu content and nanoparticle size by controlling the soaking time of titanium alloy plates, the concentration of metal ions in the solution, and the reducing agents through more research in the future to achieve coatings with enhanced antibacterial efficacy and biocompatibility. Therefore, this coating demonstrates good biocompatibility.

These antibacterial nanoparticle coatings are suitable for titanium implants and can also be applied to implants of various shapes and materials, offering broad clinical application potential. Compared to traditional coating methods, the chemical method used in this study is not limited by material shape or conductivity, making it simple, eco-friendly, and widely applicable, including in daily life applications such as hospital elevator buttons, stair railings, etc.

Conclusion

In summary, a simple redox reaction successfully incorporated Ag or Cu₂O nanoparticles onto titanium alloy plates, uniformly adhering to the surface. The coating exhibited significant antibacterial activity during the early stages, killing nearly all bacteria in the culture medium within the first 6 hours. Even after 100 wet abrasion cycles, the coating maintained excellent antibacterial performance. This relatively durable antibacterial effect provides an effective solution for preventing both early and late-stage infections in orthopedic implant surgeries. More importantly, the coating demonstrated outstanding biocompatibility. In the future, further optimization of nanoparticle concentration by adjusting solution concentrations and soaking times could enhance antibacterial performance and biocompatibility. Additionally, the coating’s simple preparation process and broad applicability suggest it holds tremendous potential for clinical translation and application. Given the urgent need to prevent implant-associated infections in orthopedic surgery, this coating offers a promising strategy for surface modification of medical implants.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Ethical approval

The in vivo experiments strictly adhered to the guidelines stipulated by the Chinese Law on Animal Experiments and received ethical clearance from the Ethics Committee of Zhejiang University School of Medicine (Ethics No. ZJU20240336).

Supplementary information

The online version contains supplementary material available at 10.1007/s10856-025-06991-3.