Immune-activating PTL/nanowire composite coating on Ti surface enables macrophage-driven bacterial clearance and osseointegration

Abstract

Implant-associated infections and loosening remain formidable clinical challenges. Conventional strategies such as incorporating antibiotics and metal ions can result in drug resistance and systemic toxicity, ultimately compromising tissue regeneration. Herein, we propose an immune-driven strategy for safe and effective anti-infection and pro-osseointegration, in which early immune activation promotes bacterial clearance, followed by timely resolution of inflammation to support angiogenesis and osteogenesis. As proof of the principle, gradient phase-transited lysozyme (PTL) on fixed size sodium titanate nanowires (PTL/nanowire composite coating) was constructed on Ti surface, creating a multifunctional platform for high-throughput screening (HTS) of optimal PTL dosages to support macrophage-driven bacterial clearance and osseointegration. The results revealed that PTL markedly enhanced bacterial clearance of macrophages (MΦs) through activating their Toll-like receptor 4 (TLR4) signaling pathway, while the nanowires beneath the PTL could promptly convert MΦs into pro-healing M2 phenotype, creating favorable macrophage-mediated immune microenvironment that promoted angiogenesis and osteogenesis. Among the tested formulations, moderate PTL dosage (PTL-3) achieved optimal balance of bacterial clearance and bone regeneration. In vivo experiments further corroborated the efficacy of PTL/nanowire composite coating in promoting infectious bone regeneration. This study underscores the potential of PTL/nanowire composite coatings for safely and effectively macrophage-driven bacterial clearance and osseointegration, paving the way for clinical translation in orthopedic and dental applications.

Graphical abstract

Highlights

• •PTL coatings activate macrophages via TLR4 to enhance bacterial clearance.
• •Moderate PTL dosage balances infection control with tissue regeneration.
• •Macrophage-driven antibacterial defense outperforms direct antibacterial activity.
• •PTL-3 coating creates optimal immune microenvironment for osseointegration.
• •Immune-activating strategy prevents implant infection without antibiotics.

1. Introduction

Implant-associated infections and loosen remain critical challenges in orthopedic and dental implantology [1]. Although surface modification strategies have advanced the osseointegration of titanium (Ti)-based implants, controlling chronic infections triggered by complex and unpredictable microenvironments remains difficult [2]. Conventional strategies, such as systemic antibiotics or antimicrobial ion-releasing coatings, provide short-term efficacy but frequently lead to antibiotic resistance, cytotoxicity, and impaired tissue repair [3]. Therefore, developing implant surfaces that seamlessly integrate infection control with tissue regeneration has become an urgent priority in materials science and regenerative medicine.

In this context, the concept of “immunoregulatory biomaterials” has emerged, aiming to achieve both infection control and tissue repair by modulating host immune responses [[4], [5], [6]]. Macrophages (MΦs), as central effector cells in osteoimmunology, play a pivotal role in infection recognition, inflammatory clearance, and subsequent tissue remodeling [[7], [8], [9], [10]]. Infection and oxidative stress activate the NF-κB pathway, driving MΦs toward a pro-inflammatory M1 phenotype with strong phagocytic capacity [11]. These M1 MΦs rapidly eliminate pathogens and necrotic tissue, laying the foundation for their transition to a tissue-repairing phenotype [12]. Thus, biomaterials that enable the timely activation and phenotypic transition of MΦs can harness host immunity to provide efficient antibacterial defense while minimizing the detrimental effects of prolonged inflammation on tissue regeneration. Such strategies hold great promise for the design of next-generation implant surfaces. For instance, layer-by-layer (LBL) assemblies and stimuli-responsive coatings have been designed to release bactericidal agents in response to the acidic microenvironment of early infection, followed by exposure of pro-osteogenic cues to promote bone healing [[13], [14], [15]]. Despite their efficacy, these systems often require complex drug-loading procedures and remain limited in precise spatiotemporal control. Therefore, a more robust and intrinsically bioactive platform is needed to autonomously direct macrophage-mediated responses without substantial reliance on exogenous therapeutic agents.

Protein-based bioactive molecules are particularly attractive for surface functionalization due to their unique immunorecognition properties [16,17]. Unlike metal ions or small-molecule drugs, proteins can directly interact with cell membrane receptors and immune-related signaling pathways, enabling precise modulation of the host immune microenvironment [18]. Lysozyme, a natural protein composed of 129 amino acids with four disulfide bonds, six tryptophan (Trp), three tyrosine (Tyr), and three phenylalanine (Phe) residues, is widely distributed in human tissues and secretions [19,20]. In addition to its well-documented antiviral, antibacterial, antihistamine, and antitumor activities, lysozyme possesses significant pharmacological potential [21]. Under physiological conditions, reduction of its disulfide bonds using tris (2-carboxyethyl) phosphine (TCEP) transforms lysozyme into phase-transited lysozyme (PTL). Prior studies have demonstrated that the.

Regulation of spatial conformation profoundly influences cellular recognition of biomaterials and subsequent immune responses [[22], [23], [24]], providing a strong foundation for PTL-based surface strategies. Although lysozyme and related antimicrobial proteins are increasingly used for implant surface modification, the dose-dependent balance between their antibacterial efficacy and tissue-repair potential remains poorly defined. Most studies examine either antimicrobial activity or isolated effects on host cells, leaving the optimal dosage window that can concurrently sustain immune regulation and support tissue regeneration unresolved. As a result, substantial variability in lysozyme loading strategies and biological outcomes persists, limiting reproducibility and hindering clinical translation. This issue is particularly pronounced in immunomodulatory biomaterials, where dose sensitivity and nonlinear immune responses mean that even small variations in coating thickness can shift outcomes from inflammatory to reparative. Establishing a quantitative and standardized dosing framework is therefore essential for the precise application of lysozyme coatings in immune modulation and tissue repair. Elucidating the relationships among PTL loading, MФs phenotypic dynamics, and downstream signaling pathways such as TLR4/NF-κB is crucial for designing implant surfaces that couple immune-driven antibacterial strategy. However, conventional fabrication approaches often rely on single-parameter control and discrete group comparisons, where discontinuous settings may obscure optimal conditions and structure–function correlations. This highlights the need for research paradigms that move beyond trial-and-error methods toward high-throughput screening (HTS) strategies capable of efficiently mapping material properties to biological performance.

HTS platforms offer a promising solution to these challenges by enabling the simultaneous construction of multiple gradient parameters within a single system [[25], [26], [27]]. These platforms facilitate the rapid assessment of how surface characteristics influence cellular functions through parallelized experiments. In recent years, HTS systems have been widely applied in studies of antibacterial coatings, immunoregulatory scaffolds, and bone repair materials, demonstrating clear advantages in elucidating material–cell interaction mechanisms [[28], [29], [30], [31]]. For instance, high-throughput preparation of gradient coatings has efficiently revealed dose-dependent patterns of cellular adhesion [32]. Similarly, gradients of RGD or antimicrobial peptide have been generated on Ti surfaces using titration and evaporation methods [28,33]. Collectively, these findings underscore the utility of HTS as both a mechanistic tool and a practical strategy for advancing the next-generation immunoregulatory implants with combined antibacterial and osseointegration functions.

Based on our previous findings, Na₂TiO₃ nanowires with diameters ranging from 5.63 to 14.25 nm exhibit optimal biological performance by promoting cell adhesion, proliferation, and osteogenic differentiation [30]. When combined with PTL, these nanowires are expected to exert synergistic effects, simultaneously enhancing antibacterial activity and osseointegration. Building on these advances and our prior optimization of nanowire dimensions, the present study introduces an immune-driven antibacterial strategy using high-throughput preparation methods. By employing a time-sequential microdroplet technique, we generated continuous-thickness PTL on nanowire structures, enabling systematic evaluation of coating morphology, chemical properties, and biological performance. Our results demonstrate that PTL significantly enhance immune-mediated bacterial clearance in the presence of MФs, primarily through activation of the TLR4 signaling pathway. Notably, a moderate PTL dosage (PTL-3) achieved an optimal balance, promoting bacterial clearance while simultaneously supporting angiogenesis and osteogenesis, thereby coupling infection control with osseointegration (Fig. 1). Furthermore, the therapeutic potential of PTL and nanowire composite coating was validated in a rat tibial defect model, where their anti-infective and pro-regenerative efficacy was confirmed. These findings provide both experimental and mechanistic evidence supporting the clinical translation of immunoregulatory biomaterials for bone repair applications.

Fig. 1.

Schematic of a dual-functional phase-transition lysozyme coating optimized by high-throughput screening for immuno-antibacterial activity and osteointegration.

2. Materials and methods

2.1. Fabrication of gradient PTL and nanowire composite coatings

Commercially pure Ti foils (99.6%, dimensions: 32 × 10 × 0.2 mm) were used as substrates. The specimens were sequentially cleaned via ultrasonic treatment in acetone, ethanol, and deionized water for 20 min each, followed by air drying. To remove the native oxide layer, the foils were etched for 10 s in a mixed solution of hydrofluoric acid (HF, 40%), nitric acid (HNO₃, 65%), and deionized water in a 1:1:2 (v/v) ratio. The etched specimens were subsequently immersed in 2.5 M sodium hydroxide (NaOH) at room temperature for 30 min, ultrasonically cleaned for 5 min, and air-dried. These alkali-treated specimens were designated as PTL-0. To construct gradient PTL, PTL-0 specimens were vertically placed in 50 mL beakers. A mixed solution of lysozyme (Sigma-Aldrich, USA; concentrations of 0.1, 0.5, 1, and 2 mg/mL in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4) and 50 mM tris (2-carboxyethyl) phosphine (TCEP, Beyotime, China; prepared in 10 mM HEPES, pH adjusted with 5 M NaOH) was added dropwise at a rate of 0.9 mL/h at room temperature. After 24 h of reaction, the specimens were thoroughly rinsed with deionized water and air-dried. The stepwise increase in lysozyme concentration facilitated the formation of a gradient PTL coating on the Ti surface. To enable HTS of PTL dose-dependent responses, the gradient parameters were designed to cover the transition from partial PTL adsorption to near-complete nanowire encapsulation. Lysozyme concentrations of 0.1, 0.5, 1, and 2 mg/mL were therefore selected as a practical and controllable range to generate clearly distinguishable coating states within experimentally feasible times. The 0-24 h assembly window was chosen to capture the progression from the initial state to a near-saturated state, thereby yielding a broad thickness range.

2.2. Characterization of the gradient PTL and nanowire composite coatings

The surface and cross-sectional morphologies of the gradient PTL were examined using field-emission scanning electron microscopy (FE-SEM, JSM-IT800, JEOL, Japan). Thickness was quantified from cross-sectional SEM images by measuring 3 randomly selected positions per specimen (3 specimens per group) using Digimizer (perpendicular to the substrate). The roughness of the gradient PTL was detected using an atomic force microscope (AFM, Dimenson ICON, Bruker, USA). Surface chemical composition and elemental states were analyzed by X-ray photoelectron spectroscopy (XPS, K-alpha, Thermo Fisher, USA). Functional groups on the coatings were identified using diffuse reflectance Fourier-transform infrared spectroscopy (DR-FTIR, ALPHA II, Bruker, Germany). The Raman spectroscopy (Renishaw plc, Invia Reflex) was used to further determine the state of the lysozyme after the phase transition occurred. Surface wettability was evaluated using the sessile drop method with deionized water and measured with a contact angle analyzer (JC2000DF, Powereach, China). The nanowire/PTL composite coating was subjected to ultrasonic agitation at 50 W for 30 min at room temperature. After drying, the surface morphology of the coating was examined using SEM. The loading amount of PTL on the nanowires was evaluated using the Micro-BCA protein quantification kit. After washing with PBS, PTL-0, PTL-0.5, PTL-3 and PTL-24 were immersed in 1 ml 1% Sodium dodecyl sulfate (SDS, Sinopharm, China) and 8 M Urea (Sinopharm, China) prepared in 50 mM Tris-HCl. The specimen was eluted at 60 °C for 30 min. Subsequently, the total protein concentration in the obtained solution was detected using the Micro-BCA protein concentration determination kit.

2.3. Cell culture

Murine macrophage RAW 264.7 cells (MΦs; catalog number: TCM13) and human umbilical vein endothelial cells (ECs; EA.hy926, catalog number: GNHu39) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Bone marrow stem cells (BMSCs) were isolated from the femurs and tibias of 4-week-old Sprague-Dawley (SD) rats. For all experiments, passages 3 to 5 of BMSCs were used. All cells were cultured in a humidified incubator at 37 °C with 5% CO₂. MΦs and ECs were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; BI, China), 200 units/mL penicillin, 200 μg/mL streptomycin (Sangon Biotech, China), and 1.5 mg/mL sodium bicarbonate (NaHCO₃; Sinopharm, China). BMSCs were cultured in α-Minimum Essential Medium (α-MEM) supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2.2 mg/mL NaHCO₃. For osteogenic differentiation, BMSCs were pre-cultured in standard α-MEM medium for 3 days. The medium was then replaced with osteogenic induction medium, consisting of α-MEM supplemented with 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid, and 100 nM dexamethasone.

2.4. High-throughput screening of gradient PTL and nanowire composite coatings

2.4.1. Antibacterial assay of gradient PTL and nanowire composite coatings

The antibacterial activity of gradient composite coatings was evaluated using a Live/Dead Bacterial Staining Kit (DMAO & PI, Beyotime, China). Staphylococcus aureus (S. aureus, ATCC 25923) was cultured in sterile liquid medium containing 1 g peptone, 0.5 g NaCl, and 0.5 g beef extract per 100 mL for 12 h, and then diluted to a final concentration of 1 × 10⁶ CFU/mL. Gradient PTL-coated specimens were placed in 6-well plates, and 3 mL of bacterial suspension was added to each well after centrifugation at 3000 rpm for 5 min to remove the growth medium. Following incubation at 37 °C for 8 h, 150 μL of staining working solution was gently applied to the specimen surfaces and incubated in the dark for 15 min. Bacterial viability was observed and imaged using a confocal laser scanning microscope (CLSM, C2Plus, Nikon, Japan). For bacterial morphological analysis, S. aureus was centrifuged at 3000 rpm for 5 min and adjusted to a concentration of 5 × 10⁷ CFU/mL before inoculation onto gradient PTL coatings. After 24 h of incubation at 37 °C, the specimens were fixed in 3% glutaraldehyde for 1 h and dehydrated through a graded ethanol series (20%, 40%, 60%, 80%, 90%, and 100%, v/v) for 15 min at each step. The specimens were then gold sputter-coated, and bacterial morphology was examined using FE-SEM.

2.4.2. Immune response of macrophages cultured on gradient composite coatings

To evaluate MΦs morphology on gradient composite coatings, RAW 264.7 cells were seeded at a density of 2 × 10⁴ cells/cm² and cultured in standard medium for 24 h. For activation, lipopolysaccharide (LPS, 100 ng/mL, Beyotime, China) was added to the medium, followed by culture in serum-free medium for an additional 12 h. Specimens were rinsed three times with sterile phosphate-buffered saline (PBS), fixed with 2.5% glutaraldehyde at 4 °C for 40 min, and dehydrated through a graded ethanol series (50%, 60%, 70%, 80%, 90%, 95%, and 100%, v/v) for 10 min at each step. The cells were then air-dried, gold sputter-coated using an ion sputter coater, and observed via FE-SEM.

To access the ROS levels in MΦs cultured on the gradient composite coatings, the MΦs were seeded on gradient coatings at a density of 5 × 10⁴ cells/cm² for 24 h, activated by LPS (100 ng/mL) for 6 h and cultured for another 12 h. Afterwards, the cells were rinsed with sterile PBS three times and stained with the fluorescent probe 20,70-dichlorofluorescein diacetate (DCFH-DA, Beyotime, China) for 20 min at 37 °C. After rinsing with PBS again, the nuclei were counterstained with Hoechst 33342 (Beyotime, China) for 5 min. Then, the cells were visualized on a CLSM. The mean fluorescence intensity (MFI) of ROS per cell was determined by the NIH Image J 1.45 software.

To evaluate the apoptosis of MΦs on the gradient composite coatings, MΦs were seeded on the gradient composite coatings at a density of 5 × 10⁴ cells/cm² and cultured for 2 days. At the indicated time point, samples were gently rinsed with sterile PBS and stained with an Annexin V-FITC/PI kit (Yeasen, China) according to the manufacturer's protocol. Images were then randomly collected using CLSM.

To assess MΦs polarization on gradient composite coatings, immunofluorescence staining was performed. RAW 264.7 cells were seeded at a density of 5 × 10⁴ cells/cm² and cultured for 24 h, followed by LPS stimulation and further incubation for 12 h. The cells were fixed with 4% paraformaldehyde (PFA) for 30 min, washed three times with sterile PBS, permeabilized, and blocked with QuickBlock™ buffer (PBSTw, Beyotime, China) for 1 h. Specimens were incubated overnight at 4 °C with primary antibodies against inducible nitric oxide synthase (iNOS, Abcam, UK) and CD206 (Abcam, UK) to detect M1 and M2 phenotypes, respectively. After washing, the cells were incubated for 1.5 h at room temperature with secondary antibodies: goat anti-mouse IgG H&L (Alexa Fluor® 488, Abcam, UK) and goat anti-rabbit IgG H&L (Alexa Fluor® 555, Abcam, UK). Cell nuclei were counterstained with DAPI for 5 min, and the specimens were imaged using CLSM. The MFI of iNOS and CD206 were quantified using NIH ImageJ 1.45 software.

2.4.3. Biological response of ECs on gradient composite coatings

ECs were seeded onto gradient composite coatings at a density of 1 × 10⁴ cells/cm² and cultured under standard conditions for 24 h, following the procedures outlined in Section 2.4.2. Specimens were rinsed with sterile PBS, fixed in 2.5% glutaraldehyde, dehydrated through a graded ethanol series, and examined using FE-SEM. The area and perimeter of individual cells were quantified using Digimizer software.

Cell viability was assessed using the Live/Dead Viability/Cytotoxicity Kit (Invitrogen, USA). ECs were seeded at a density of 2 × 10⁴ cells/cm² and cultured for 1, 3, and 5 days. At each time point, specimens were rinsed three times with sterile PBS, stained with 150 μL of Live/Dead solution for 30 min in the dark, rinsed again with PBS, and imaged using CLSM. Viable and non-viable cells were quantified with NIH ImageJ 1.45 software.

For cytoskeletal observation, ECs were seeded at 1 × 10⁴ cells/cm² and cultured for 24 h. Cells were rinsed with PBS, fixed with 4% PFA for 30 min, and stained with 150 μL of FITC-phalloidin solution (Invitrogen, USA) for 40 min to label F-actin. Nuclei were counterstained with DAPI for 10 min, and specimens were imaged via CLSM. Intracellular nitric oxide (NO) production was detected using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA, Beyotime, China). ECs were seeded at 1 × 10⁴ cells/cm² and cultured for 24 h, then incubated with 150 μL of 5 μM DAF-FM DA solution for 20 min. Following three PBS rinses, specimens were observed using CLSM, and the MFI of NO was quantified using NIH ImageJ 1.45 software.

2.4.4. Biological BMSCs on gradient composite coatings

BMSCs were seeded on gradient composite coatings at a density of 1 × 10⁴ cells/cm² and cultured for 24 h. Cell morphology was examined using FE-SEM, following the procedure described in Section 2.4.3. Cell viability was evaluated using the Live/Dead staining protocol detailed in Section 2.4.3. BMSCs were seeded at a density of 2 × 10⁴ cells/cm² and cultured for 1, 3, and 5 days. For cytoskeletal organization analysis, BMSCs were seeded at a density of 1 × 10⁴ cells/cm² and cultured for 24 h. Cytoskeletal visualization was performed using the same protocol as described in Section 2.4.3.

2.5. In vitro antibacterial performance and mechanism of optimized coatings

2.5.1. Evaluation of antibacterial activity

Based on bacterial and cellular responses to the gradient coatings, representative parameters were selected to fabricate non-gradient composite coatings for further antibacterial evaluation. The antibacterial activity of these coatings was evaluated toward S. aureus. A 1 mL bacterial suspension (1 × 10⁶ CFU/mL) was added to specimens, with or without MΦs seeded at a density of 5 × 10⁴ cells/cm². After 12 and 24 h of incubation, bacterial suspensions were collected, serially diluted, and plated on agar plates for colony counting. The antibacterial rate (Ra) was calculated as: Ra (%) = [(A − B)/A] × 100%, where A and B represent the number of viable bacteria in the blank control and the specimen groups, respectively.

For the bacterial phagocytosis assay, MΦs were seeded on specimens surfaces at a density of 5 × 10⁴ cells/cm² and cultured for 24 h. Subsequently, 1 mL of bacterial suspension (1 × 10⁶ CFU/mL) was added to each well and co-incubated with the MΦs for 2, 4, 12, and 24 h. At each time point, the medium was replaced with fresh medium containing 100 μg/mL gentamicin and incubated for 1 h to eliminate extracellular bacteria. The infected MΦs were lysed with 1 mL of 1% Triton X-100, and the lysates were serially diluted and plated on agar for colony counting after 24 h of incubation.

To visualize bacterial phagocytosis, bacteria were fluorescently labeled using the Cell Proliferation and Tracing Kit (CFDA SE, Beyotime, China). Briefly, a bacterial suspension (1 × 10⁶ CFU/mL) was centrifuged to remove the supernatant, stained with CFDA SE for 30 min in the dark, and washed three times with PBS washes. The labeled bacteria were co-incubated with MΦs pre-seeded on different specimen surfaces for 2 and 4 h. After incubation, cells were washed with PBS to remove residual bacteria, counterstained with Hoechst (Beyotime, China) for 5 min and imaged using CLSM.

To further assess MΦ phagocytic capacity against S. aureus on different specimens, transmission electron microscopy (TEM, HT7800, Hitachi, Japan) was performed. MΦs were seeded onto each specimen at a density of 5 × 10⁴ cells/cm² and cultured for 24 h. Subsequently, 1 mL of S. aureus suspension was added for co-culture for 4 h and 24 h. At each time point, MΦs were harvested, centrifuged, and fixed with 2.5% glutaraldehyde at 4 °C for 12 h, followed by post-fixation with 1% osmium tetroxide for 2 h. The MΦs were then dehydrated through a graded ethanol series, embedded in resin, and sectioned into ultrathin slices (∼90 nm) for TEM observation of bacterial internalization.

2.5.2. Investigation of antibacterial mechanism

The role of the TLR4 in macrophage-driven antibacterial activity was examined using the TLR4 inhibitor TAK-242. To determine the optimal working concentration of TAK-242, MΦs were seeded in 24-well plates at a density of 5 × 10⁴ cells/cm² and cultured for 12 h. The medium was replaced with fresh medium containing TAK-242 at varying concentrations (0, 0.5, 1, 2, 4, 8, 16, 32, and 64 μM; Beyotime, China) for 4 h. Cells were subsequently washed with PBS and cultured in normal medium for another 4 h. Cell viability was assessed using the MTT assay, and absorbance was measured at 492 nm.

To evaluate the effect of TAK-242 on MΦs phagocytic activity, cells were seeded on specimen surfaces at a density of 5 × 10⁴ cells/cm² and cultured for 24 h. TAK-242 was added at the optimal concentration for 4 h, followed by the addition of 1 mL bacterial suspension (1 × 10⁶ CFU/mL). After 2 and 4 h of co-incubation, subsequent steps for bacterial phagocytosis and colony counting were performed as described in Section 2.5.1.

Immunofluorescence staining was conducted to evaluate TLR4 activation in MΦs cultured on PTL coatings. To assess the inhibitory effect of TAK-242 on LPS-induced TLR4 activation, cells were divided into three groups: (1) untreated, (2) LPS-stimulated (100 ng/mL), and (3) TAK-242-pretreated followed by LPS stimulation. After 24 h of culture, cells were fixed with 4% PFA for 30 min, washed three times with PBS, permeabilized, and blocked with QuickBlock™ buffer (PBSTw, Beyotime, China) for 1 h. Cells were incubated overnight at 4 °C with a primary antibody against TLR4 (ab22048, Abcam, UK), followed by incubation with Alexa Fluor® 488-conjugated goat anti-mouse IgG H&L secondary antibody (Abcam, UK) at room temperature for 1.5 h. Nuclei were counterstained with DAPI for 5 min. Stained cells were imaged using CLSM. The MFI of TLR4 expression was quantified using NIH ImageJ 1.45 software.

For further analysis, MΦs were seeded at 5 × 10⁴ cells/cm² on various PTL-coated surfaces and cultured for 24 h. Cells were fixed with 4% PFA for 30 min, followed by immunofluorescence staining for TLR4 as described above. Images were captured using CLSM, and the MFI of TLR4 expression was quantified with NIH ImageJ 1.45 software.

2.6. Immunomodulatory effects of MΦs on optimized composite coatings

2.6.1. Preparation of conditioned medium

MΦs were seeded at a density of 5 × 10⁴ cells/cm² on optimized PTL-coated surfaces in 6-well plates and cultured for 24 h. Following LPS stimulation, serum-free culture was performed as previously described in Section 2.5.2. The conditioned medium was collected, clarified by centrifugation at 4 °C and 1000 rpm for 20 min to remove cell debris, and the supernatant was retained for subsequent use. ECs were cultured in conditioned DMEM, prepared as a 1:1 mixture of fresh DMEM and MΦ-conditioned medium. Similarly, BMSCs were cultured in conditioned α-MEM, prepared as a 1:1 mixture of fresh α-MEM and MΦ-conditioned medium.

2.6.2. In vitro evaluation of angiogenic function of ECs in response to conditioned medium

The effects of MΦ-conditioned medium from PTL-coated surfaces on EC morphology, cytoskeletal organization, viability, and NO production were assessed as described in Section 2.4.3.

For CD31 expression, ECs were seeded at 1 × 10⁴ cells/cm² in 24-well plates and cultured for 24 h, followed by 48 h incubation in conditioned DMEM. Cells were fixed with 4% PFA for 30 min, washed three times with PBS, and incubated with a primary antibody against CD31 (Abcam, UK) at 4 °C for 12 h. After washing with PBS, cells were incubated with m-IgGκ BP-CFL 488 secondary antibody (Santa Cruz Biotechnology, USA) at room temperature for 1.5 h. Nuclei were counterstained with DAPI for 5 min. Images were obtained using CLSM, and CD31 MFI was quantified using NIH ImageJ 1.45 software.

For migration assays, ECs were seeded at 4 × 10⁴ cells/cm² in 24-well plates and cultured for 1 day. The medium was replaced with conditioned medium, and cells were further cultured for 2 days to reach confluence. A linear scratch was created on the bottom of each well with a 200 μL pipette tip. After three PBS washes to remove unattached cells, cultures were incubated in conditioned medium for 8, 16, and 24 h. Migration was observed using an inverted microscope (ECLIPSE Ts2R-FL, Nikon, Japan), and migration distances were quantified using Digimizer software.

For in vitro angiogenesis, ECs were seeded at 6 × 10⁴ cells/well on ECMatrix™ gel (ECM625, Millipore, USA) in 96-well plates and incubated with conditioned DMEM at 37 °C for 6 h and 12 h. At each time point, images were captured using an inverted microscope, and the number of branch points (nodes), mesh-like loops (circles), and tubular structures (tubes) was quantified using Photoshop CC software.

2.6.3. Evaluation of the osteogenic potential of BMSCs using conditioned medium

The morphology and osteogenic response of BMSCs cultured with macrophage-conditioned medium from optimized PTL-coated surfaces were assessed following the procedures described in Section 2.4.4.

For ALP Activity, BMSCs were seeded in 24-well plates at 1 × 10⁴ cells/cm² and cultured under standard conditions for 3 days, followed by incubation in conditioned osteogenic medium for 3 and 7 days. At each time point, cells were rinsed three times with sterile PBS, fixed with 4% PFA for 30 min, and stained with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) alkaline phosphatase (ALP) chromogenic reagent (Beyotime, China) for 12 h. ALP activity was qualitatively evaluated using a stereomicroscope (SMZ745T, Nikon, Japan).

For type I collagen secretion, Sirius Red staining was performed. BMSCs were seeded at 1 × 10⁴ cells/cm² in 24-well plates, cultured for 3 days under standard conditions, and then incubated with conditioned medium for 7 and 14 days. At each time point, cells were washed three times with PBS, fixed with 4% PFA for 30 min, and stained with 0.1% Direct Red 80 (Sigma-Aldrich, USA) for 18 h. Excess dye was removed with 0.1 M acetic acid, and samples were air-dried for qualitative evaluation under a stereomicroscope. For quantification, the bound dye was eluted using 1 mL NaOH/methanol solution for 30 min with vigorous pipetting, and absorbance was measured at 570 nm using a microplate reader.

For extracellular matrix mineralization, Alizarin Red S staining was performed. BMSCs were seeded in 24-well plates, cultured for 3 days under standard conditions, and then incubated with conditioned medium for 7 and 14 days. At each time point, cells were washed three times with PBS, fixed with 75% ethanol for 1 h, and stained with 40 mM Alizarin Red S solution (pH 4.2) for 30 min. Specimens were rinsed with deionized water, air-dried, and qualitatively evaluated under a stereomicroscope. For quantification, bound dye was eluted using 500 μL 10% cetylpyridinium chloride solution (MREDA, China) in 10 mM Na₃PO₄ (Xiya, China) for 2 h, and absorbance was measured at 570 nm using a microplate reader.2.7. In vivo Experiments.

2.6.4. Surgical procedure

The Animal Ethics Committee of Taiyuan University of Technology (TYUT2025120101) authorized all animal experiment processes and surgery protocols, which were carried out following international animal welfare standards. Cylindrical Ti specimens (Φ1.5 × 3 mm) were fabricated based on optimized parameters for in vivo implantation. Male Sprague-Dawley rats (∼350 g) were used as experimental subjects. Anesthesia was induced by intraperitoneal injection of 1% pentobarbital sodium at a dosage of 30 mg/kg. A 1.5 mm-diameter defect was drilled into the tibia plateau using a surgical electric drill under continuous saline irrigation to prevent thermal damage. Implants pre-inoculated with S. aureus were inserted into the tibia to establish an implant-associated bone infection model. To standardize bacterial inoculation across groups, all implants were exposed to the same bacterial strain under identical inoculation conditions. Briefly, the specimens were immersed in 5 mL of an S. aureus suspension (1 × 10⁸ CFU/mL) and incubated for 24 h to allow biofilm formation on the implant surfaces. Each group was randomly assigned 15 rats. Following implantation, muscle and skin were sutured in layers. Implant placement was immediately confirmed using X-ray imaging (SD5.0A, Wanke, China), with rats positioned supine on the imaging platform. All procedures were performed under sterile conditions. Rats were euthanized at 1, 3, and 10 days, and 8 weeks post-implantation. Tibia containing implants were harvested for further analysis.

2.6.5. Antibacterial activity

At 1st and 3rd days post-implantation, three rats from each group were euthanized, and implants were carefully retrieved from the tibia. Each implant was placed into a 1.5 mL centrifuge tube containing sterile PBS and subjected to ultrasonic agitation for 30 s to dislodge bacteria. The resulting suspensions were serially diluted, plated on agar, and incubated for 24 h for bacterial colony counting. Simultaneously, femoral specimens were processed for histological analysis. Specimens were fixed in 4% PFA, followed by decalcification, dehydration, paraffin embedding, and sectioning. Sections were ground and polished, then stained with Giemsa and hematoxylin & eosin (H&E) for microscopic evaluation.

2.6.6. Inflammatory response

To evaluate the inflammatory response at the bone-implant interface, Western blot analysis was performed on 3rd and 10th day post-implantation. Three rats per group were euthanized, and macroscopic images of the implantation site were recorded. Implants and surrounding bone tissue were harvested, homogenized on ice for 30 min in RIPA lysis buffer (Beyotime, China) containing protease and phosphatase inhibitors (Thermo Fisher Scientific, USA), and centrifuged at 12,000 rpm for 20 min at 4 °C. Supernatants were collected, and total protein concentrations were measured using a BCA Protein Assay Kit (Beyotime, China). Protein samples (3 μg/μL) were mixed with 5 × SDS loading buffer (4:1, v/v), denatured at 100 °C, and 15 μL of each sample was subjected to SDS-PAGE, followed by transfer to PVDF membranes. Membranes were blocked with Block™ Western Buffer (P0252, Beyotime, China) for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibodies against the following proteins: iNOS (ab178945), IL-1β (ab254360), CD206 (ab64693), Arg1 (ab239731), Ang1 (ab183701), CD31 (ab9498), OPN (sc-21742), ALP (sc-271431), and GAPDH (sc-365062). Antibodies were obtained from Abcam (UK) or Santa Cruz Biotechnology (UK) as indicated. After incubation with HRP-conjugated secondary antibodies, protein bands were visualized using enhanced chemiluminescence (ECL) reagents (BeyoECL Star, Beyotime, China) and imaged using a chemiluminescence system (Tanon-5200, China).

2.6.7. Osseointegration analysis

At 8 weeks post-implantation, femoral specimens were harvested for high-resolution micro-computed tomography (micro-CT) analysis (nanoVoxel-1000, San Ying). A 100 μm zone extending from the implant surface was defined as the region of interest (ROI). Three-dimensional reconstruction and quantitative bone analysis were conducted using DragonFly software to determine bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). For histological analysis, specimens were fixed, decalcified, dehydrated, embedded, and sectioned. After grinding and polishing, sections were stained with H&E and Masson's trichrome for morphological evaluation.

2.7. Statistical analysis

All experiments were conducted in triplicate, with three specimens per group. Representative images were selected for qualitative analysis. Quantitative data are presented as mean ± standard deviation (SD). Statistical comparisons were performed using one-way analysis of variance (ANOVA) in SPSS 14.0 software (IBM, USA). Statistical significance was defined as p < 0.05 (significant), p < 0.01 (highly significant), and p < 0.001 (very highly significant).

3. Results

3.1. Construction and characterization of the gradient PTL platform

As illustrated in Fig. 2a, a gradient PTL platform was fabricated using a high-throughput preparation strategy. Morphological analysis (Fig. 2b, c and e) revealed that with increasing immersion time, nanowires became progressively coated, forming a layer with thickness ranging from approximately 3.34 nm to 38.91 nm. After 24 h, the surface exhibited a dense granular morphology, with the underlying nanostructure no longer visible. At a low lysozyme concentration (0.1 mg/mL), PTL coverage was incomplete (Fig. S1a). In contrast, complete coverage was achieved within 6 h and 1.5 h for the 1 mg/mL and 2 mg/mL groups, respectively (Fig. S1b and c). The surface roughness (Ra) results were presented in Fig. 2d. The original nanowire surface showed an Ra of 34.7 nm. With increasing PTL deposition time, the Ra value first decreased to 18.1 nm and then increased to 37.7 nm. DR-FTIR analysis (Fig. 2f) confirmed successful PTL deposition. All groups, except for the 0 h control, displayed characteristic absorption peaks at 1515 cm⁻¹ (C-O-H), 1649 cm⁻¹ (C=O), 3300 cm⁻¹ (-NH₂), and 3744 cm⁻¹ (-OH), with increasing peak intensities correlating with increasing coating thickness. Contact angle measurements (Fig. 2g and h) showed a gradual increase in contact angle with thicker coatings, indicating a reduction in surface.

Fig. 2.

Physicochemical properties of the PTL gradient platform: (a) High-throughput fabrication of the phase-transition lysozyme (PTL) gradient platform; (b) Surface SEM image of the gradient platform prepared with 0.5 mg/mL PTL solution; (c) Cross-sectional morphology of the gradient platform; (d) Surface AFM image of the gradient platform prepared with 0.5 mg/mL PTL solution; (e) Quantification of PTL coating thickness across the gradient; (f) FTIR spectra of the platform surface; (g) Quantification of surface water contact angles; (h) Representative images of water contact angles; (i) XPS survey spectra of the platform surface; (j) High-resolution XPS spectra of Ti 2p; (k) High-resolution XPS spectra of N 1s.

Hydrophilicity. Compared with the lysozyme, the specimens treated with TCEP exhibited significant spectral changes in disulfide bonds, C skeleton, and amide I (particularly around 505, 927 and 1665-1670 cm⁻¹), indicating that the conformational environment of the reduced protein has changed (Fig. S2). XPS analysis (Fig. 2i–k) further validated PTL deposition, showing enhanced N 1s and S 2p signals alongside progressive attenuation of the Ti 2p signal, which is consistent with increasing surface coverage. Furthermore, changes in the actual protein loading were quantitatively assessed using the Micro BCA protein assay to determine the amount of immobilized PTL (Fig. S3). As soaking time increased, the protein loading density rose from 0 to 56.85 μg/cm², further supporting the change in coating thickness observed previously. Notably, the composite coating remained structurally intact and well-adhered even after 30 min of high-intensity ultrasonic cleaning, as confirmed by SEM characterization (Fig. S4). Such robust adhesion ensures that the functional interface can withstand the mechanical challenges during surgical insertion and long-term physiological exposure.

3.2. High-throughput screening of the gradient PTL and nanowire composite coating

As shown in Fig. 3a and Fig. S5a, immunomodulatory and antibacterial properties were evaluated by independent culturing MФs and S. aureus on the gradient PTL platform. SEM images (Fig. S5b) showed a gradual transition in bacterial morphology from intact to wrinkled and ruptured with increasing coating dosage, a trend more evident in the higher concentration groups. Live/Dead bacterial staining further confirmed this observation, red fluorescence (indicating dead bacteria) intensified with increasing PTL loading, demonstrating a positive correlation between antibacterial activity and both concentration and dosage (Fig. S5c). In contrast, the 0.1 mg/mL group exhibited only mild membrane disruption after 24 h, suggesting limited antibacterial efficacy. Furthermore, the antibacterial efficacy of the PTL gradient was quantified. As shown in Fig. S5d, the antibacterial rate exhibited a clear dose-response relationship. While the low-concentration group (0.1 mg/mL) showed limited inhibitory effects, the antibacterial rate significantly increased in the higher-concentration groups, reaching approximately 70% at the highest dosage (2 mg/mL). The morphology of MΦs on PTL-coated surfaces is shown in Fig. 3b. At low magnification, MΦs were evenly distributed across all specimens, confirming that the coatings supported cellular adhesion. High-magnification images revealed coating dose-dependent morphological changes. From 0 to 3 h, MΦs displayed spindle-like shapes with abundant lamellipodia, indicative of good spreading. However, from 6 h onward, lamellipodia became filamentous and substrate adhesion decreased. Cells adopted more spherical or star-like morphologies, suggestive of impaired spreading. At 0.1 mg/mL, MΦs remained uniformly adherent with well-developed lamellipodia (Fig. S6a), whereas higher concentrations induced earlier onset of morphological impairment. Specifically, spherical cells were observed as early as 3 h in the 1 mg/mL group and at 1.5 h in the 2 mg/mL group. To further investigate MФs polarization, immunofluorescence staining for iNOS (M1 marker) and CD206 (M2 marker) was performed (Fig. 3c). At 0-3 h, iNOS expression was weak across all groups with no significant differences. However, with prolonged PTL loading, iNOS expression increased while CD206 expression decreased. Quantitative analysis (Fig. 3d and e) indicated that low-dose PTL favored M2 (repair-oriented) polarization, whereas high-dose PTL promoted M1 (pro-inflammatory) polarization. ROS staining showed that MΦs cultured on the PTL/nanowire composite coating exhibited higher levels than those in the control group (Fig. 3f), with ROS levels increasing with PTL dosage. Quantitative analysis further confirmed this trend (Fig. 3g). Immunofluorescence observations at 48 and 72 h further confirmed this trend. The polarization pattern at 48 h remained similar to that at 24 h (Fig. S7). At 72 h, decreased iNOS and increased CD206 expression were observed in the PTL-0, PTL-0.5, and PTL-3 groups, indicating a tendency toward inflammation resolution, whereas PTL-24 showed no evident reversal of the pro-inflammatory phenotype. The Annexin V/PI staining results of MΦs on the PTL-coated surface were shown in Fig. S8. No red dead cells and green apoptotic cells were observed at any location, indicating that the PTL-24 phenotypic change was not driven by cell apoptosis.

Fig. 3.

Antibacterial performance and MΦ immune response on the PTL gradient platform: (a) Schematic of high-throughput screening on the PTL gradient platform; (b) SEM morphology of MΦs on the platform surface; (c) Immunofluorescence staining of MΦs on the platform surface; (d) Quantification of iNOS fluorescence intensity; (e) Quantification of CD206 fluorescence intensity; (f) ROS of MΦs on the platform surface; (g) Quantification of ROS fluorescence intensity. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using one-way ANOVA with a Tukey post hoc test.

Angiogenesis is a critical prerequisite for osseointegration [34], while the osteogenic activity of BMSCs on implant surfaces plays a pivotal role in determining long-term implant success [35]. To this end, the behavior of ECs and BMSCs on gradient PTL-coated surfaces was systematically evaluated (Fig. S9a). As shown in Fig. S9b, EC morphology varied notably along the gradient. In the 0 to 6 h region, ECs exhibited larger spreading areas and longer perimeters (Fig. S9c and d). However, from 12 h to 24 h, their spreading capacity declined, and cell adhesion became restricted. High-magnification images revealed that ECs at early positions (0 to 6 h) maintained a flattened polygonal morphology with tight substrate attachment and prominent lamellipodia, whereas those in later regions (12 h to 24 h) displayed a raised, rounded morphology with limited surface contact. Cytoskeletal staining (Fig. S10) further supported these observations: ECs in the 0 h to 3 h region showed abundant and well-organized F-actin filaments, while cells in regions with higher dose PTL (over 12 h) exhibited weakened and disorganized actin structures. Live/Dead staining (Fig. S11) indicated no cytotoxic effects across the gradient, as no red fluorescence (dead cells) was detected. Strong green fluorescence, denoting high viability, was observed in the 0 to 12 h range, whereas higher dose PTL (over 18 h) reduced both green fluorescence intensity and overall cell density. Collectively, these findings suggest that low-dose PTL coatings promote EC adhesion, spreading, and proliferation. Given the central role of NO in angiogenesis [36], intracellular NO production was further evaluated. As shown in Fig. S12a and b, NO fluorescence intensity peaked in the 0.5 to 3 h region, significantly higher than at 0 h or later gradient positions, indicating enhanced angiogenic potential in thinner PTL coatings.

BMSCs morphology on the gradient PTL surfaces is shown in Fig. S13. In the 0.5 to 3 h region, BMSCs exhibited a flattened, polygonal morphology with increased spreading area, while cells in the 6 h and beyond regions displayed an elongated spindle-like shape with reduced spreading. Quantitative analysis (Fig. S13b and c) confirmed a gradual decline in both spreading area and perimeter as PTL dosage increased. High-magnification images revealed that high-dose PTL impaired intimate contact between cells and the substrate. Cytoskeletal staining (Fig. S14) corroborated these findings. BMSCs on thinner coatings formed well-organized actin networks and retained intact cytoskeletal architecture, while high-dose PTL disrupted actin filament organization and decreased expression. Live/Dead staining (Fig. S15) showed no evidence of cytotoxicity in any group, as no red fluorescence was detected. However, from 3 h onward, green fluorescence intensity and total cell number progressively declined, indicating reduced cell proliferation. Taken together, these results demonstrate that optimally thin PTL coatings support EC and BMSC adhesion, spreading, and proliferation. In contrast, excessive PTL thickness and the resulting loss of nanowire surface topography negatively affect cellular behavior and bioactivity.

3.3. Effect of PTL and nanowire composite coating on antibacterial properties

Achieving a balance between antibacterial efficacy and cellular compatibility remains a major challenge in implant material design [37]. While enhanced antibacterial performance is desirable, it is often accompanied by adverse effects on cell viability and function. Based on preliminary HTS of bacterial inhibition and cell compatibility, a PTL concentration of 0.5 mg/mL was selected as optimal for the fabrication of non-gradient coatings. Four types of specimens were prepared for further evaluation: a nanowire substrate without PTL (PTL-0), and PTL-loaded coatings with immersion durations of 0.5 h (PTL-0.5), 3 h (PTL-3), and 24 h (PTL-24). These specimens were systematically assessed in terms of antibacterial performance, macrophage-mediated immune response, angiogenesis, and osteogenesis, with the aim of identifying the optimal coating parameters for both infection control and tissue integration.

Effective infection control is essential for the treatment of infectious bone defects, as it establishes the foundation for subsequent osseointegration and bone healing. The antibacterial activity of the four PTL-coated groups was first evaluated. As shown in Fig. S16a, after 12 h of incubation with S. aureus, the PTL-0 group showed no significant reduction in bacterial count compared with the uncoated Control group. PTL-24 exhibited a slight reduction. After 24 h, all PTL-coated groups demonstrated measurable antibacterial effects, with bacterial inhibition rates of 15.2% (PTL-0.5).

22.4% (PTL-3), and 46.6% (PTL-24). Despite the moderate antibacterial effect observed in the PTL-24 group, the results were still below the threshold typically required for clinical infection control requirements. MΦs, as key innate immune cells, serve as the first line of defense by recognizing pathogens, migrating to infection sites, and polarizing toward a pro-inflammatory M1 phenotype to facilitate bacterial clearance [38]. Therefore, the contribution of MΦs to the antibacterial response was further investigated. Following 1 day of MΦs culture on PTL-coated surfaces, S. aureus was introduced. As shown in Fig. S16b, the presence of MΦs significantly enhanced antibacterial activity, with inhibition rates increasing to 84.9% (PTL-0.5), 90.7% (PTL-3), and 97.8% (PTL-24). These values were significantly higher than those observed for the coatings alone, underscoring both the crucial role of MΦs in antibacterial defense and the ability of PTL coatings to potentiate their function. To further evaluate MΦs phagocytic activity, MΦs were co-cultured with the PTL-coated specimens and S. aureus. At designated time points, extracellular bacteria were eliminated with gentamicin, and intracellular bacterial was quantified after MФs lysis with Triton X-100. As shown in Fig. 4b, no significant differences in phagocytosis were observed among groups at 2 h. However, by 4 h, phagocytic efficiency increased significantly and correlated positively with PTL dosage. Fluorescence staining results (Fig. 4c and d) corroborated these findings. With prolonged incubation (12 h and 24 h), intracellular bacterial counts declined progressively across all groups (Fig. S17), indicating sustained macrophage-driven bacterial clearance facilitated by the immunostimulatory effects of PTL. TEM analysis further verified that the PTL coating enhances MФ phagocytic activity (Fig. 4e). After 4 h, MФs in the PTL-3 group contained numerous bacteria enclosed within well-defined phagosomes and phagolysosomes, maintaining intact morphology and dense cytoplasm indicative of active phagocytosis. In contrast, the PTL-0 group showed few internalized bacteria, pronounced vacuolization, and disrupted membranes, reflecting weakened phagocytic function. After 24 h, the PTL-3 group exhibited increased phagosome formation and bacterial degradation while preserving cellular integrity, whereas the PTL-0 group displayed aggravated vacuolization, membrane injury, and residual extracellular bacteria. These results confirm that the PTL coating sustains MФ phagocytic activity, promoting efficient bacterial clearance.

Fig. 4.

PTL coating enhances MΦ phagocytosis of bacteria: (a) Schematic of the experimental procedure for bacterial phagocytosis by MΦs on PTL-coated surfaces; (b) Phagocytic capacity of MΦs on PTL-coated surfaces; (c) Fluorescence images of bacterial phagocytosis by MΦs; (d) Quantification of fluorescence intensity of phagocytosis at different time points; (e) TEM images of phagocytosis at different time points. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using one-way ANOVA with a Tukey post hoc test.

3.4. Mechanism of macrophage-mediated antibacterial activity

Building on the above findings, we further investigated the mechanism by which PTL enhance MΦs phagocytosis. TLR4, a pattern recognition receptor abundantly expressed on immune cells, plays a central role in mediating inflammatory responses [39]. Upon activation by LPS, TLR4 initiates the NF-κB signaling cascade through p65 phosphorylation and nuclear translocation, triggering the release of pro-inflammatory mediators [40]. To assess whether MФs phagocytosis was mediated via TLR4 signaling, the specific inhibitor TAK-242 was employed. MTT assay results (Fig. S18) revealed a significant reduction in MФs viability at TAK-242 concentrations above 8 μM. Thus, 8 μM was selected for subsequent experiments. Immunofluorescence staining (Fig. S19a) confirmed a marked increase in TLR4 expression following LPS stimulation, whereas TAK-242 treatment significantly reduced fluorescence intensity (Fig. S19b), validating effective inhibition of TLR4 signaling. Following TLR4 blockade, MФs cultured on PTL-coated surfaces exhibited significantly reduced phagocytic efficiency. As shown in Fig. 5a, bacterial uptake remained consistently low across all groups after 2 h, and did not improve with prolonged incubation, indicating that TLR4 inhibition abolished the PTL-induced enhancement of phagocytosis. Further evaluation of TLR4 expression in MΦs cultured on various PTL-coated surfaces (Fig. 5b) demonstrated significant upregulation in a coating dose-dependent manner relative to the control group. These results suggest that PTL function as immunoregulatory cues by activating TLR4 on MΦs, thereby enhancing their phagocytic activity for more effective clearance of S. aureus (Fig. 5c). Mechanistically, we propose that PTL proteins are enriched in hydrophobic amino acid. This hydrophobic amino acid is preferentially recognized by TLR4, activating downstream inflammatory pathways, promoting cytokine release, and enhancing MФs phagocytic capacity to facilitate bacterial elimination.

Fig. 5.

Mechanistic investigation of MΦ phagocytosis on different PTL-coated surfaces: (a) Phagocytic capacity of MΦs on PTL coatings after TAK-242 treatment; (b) Qualitative and quantitative analysis of TLR4 activation in MΦs on PTL coatings; (c) Schematic illustration of the PTL-mediated antibacterial mechanism in MΦs. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using one-way ANOVA with a Tukey post hoc test.

3.5. Effects of the PTL-induced MΦ-mediated immune microenvironment on angiogenesis and osteogenesis

Persistent induction of pro-inflammatory MФs at bone defect sites, especially under infectious conditions can exacerbate inflammation and inhibit new bone formation [41,42]. Conversely, a well-modulated immune microenvironment facilitates the recruitment and functional activation of ECs and BMSCs, thereby promoting angiogenesis and osteogenesis essential for successful osseointegration. To evaluate the impact of the PTL-induced immune milieu on regenerative processes, conditioned media were collected from MΦs cultured on different PTL-coated specimens and applied to ECs (Fig. 6a). As shown in Fig. S20, conditioned media influenced EC morphology. Low-magnification images indicated uniform adhesion across all groups. However, at medium magnification, ECs in the PTL-3 group exhibited the largest spreading areas over time, while those in the PTL-24 group exhibited reduced spreading. High-magnification images revealed impaired lamellipodia in PTL-24, suggesting compromised adhesion under an excessively pro-inflammatory environment. Cytoskeletal staining further corroborated these findings (Fig. S21). Next, NO production was assessed as a key marker of endothelial function. As shown in Fig. 6b and c, NO levels were slightly lower in the PTL-0.5, PTL-3, and PTL-24 groups compared to PTL-0. Among these, PTL-3 exhibited the highest NO expression, indicating that moderate immune activation best supports endothelial function. Immunofluorescence staining of CD31 (Fig. 6d and e) revealed the highest expression in the PTL-0 and PTL-0.5 groups, with slightly reduced but still substantial expression in PTL-3. In contrast, CD31 expression was significantly diminished in PTL-24, underscoring the inhibitory effect of an excessively pro-inflammatory environment on angiogenic signaling. In vitro angiogenesis assays (Fig. 6f) demonstrated that all groups were capable of forming capillary-like structures, although their number declined over time. Quantitative analysis (Fig. 6g and h) confirmed that PTL-0, PTL-0.5, and PTL-3 maintained strong angiogenic potential, whereas PTL-24 exhibited significantly impaired performance. Similarly, scratch wound assays (Fig. 6i and.

Fig. 6.

Effects of the MΦ-mediated immune microenvironment on ECs: (a) Schematic of the experimental procedure for investigating the MΦ-mediated immune microenvironment; (b, c) Qualitative and quantitative analysis of NO production; (d, e) Qualitative and quantitative analysis of CD31 expression; (f-h) Qualitative and quantitative analysis of in vitro tube formation; (i, j) Qualitative and quantitative analysis of ECs migration. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using one-way ANOVA with a Tukey post hoc test.

6j) confirmed that all conditioned media supported EC migration, but the migratory capacity was markedly reduced in the PTL-24 group. These results collectively highlight the importance of immune modulation, while moderate immune activation (PTL-3) enhances EC function and angiogenesis, excessive stimulation (PTL-24) disrupts vascular performance, emphasizing the need for optimized immuno-regulatory coatings.

The impact of macrophage-conditioned media on BMSC osteogenic differentiation is shown in Fig. 7. Morphological observations (Fig. S22) revealed that BMSCs in the PTL-0, PTL-0.5, and PTL-3 groups exhibited larger spreading areas, flattened polygonal morphologies, and abundant lamellipodia that features indicative of favorable adhesion and activation. In contrast, BMSCs exposed to PTL-24 displayed smaller, spindle-like morphologies, suggesting impaired adhesion and cellular activation. The experimental procedure is shown in Fig. 7a. These morphological trends were corroborated by osteogenesis-related staining. ALP activity staining (Fig. 7b) showed a progressive increase in deep blue NBT/BCIP staining across groups, with PTL-3 exhibiting the highest ALP activity and PTL-24 the lowest. Similar trends were observed in COL-I secretion (Fig. 7c) and ECM mineralization (Fig. 7d). Quantitative analysis (Fig. 7e and f) confirmed statistically significant differences among groups. Collectively, these results demonstrate that PTL coatings distinctly modulate the MΦ-mediated immune microenvironment, which in turn governs angiogenesis and osteogenesis. High PTL loading (PTL-24) drives MΦs toward an excessively pro-inflammatory M1 phenotype, leading to the release of inflammatory mediators that inhibit BMSCs osteogenic differentiation. In contrast, moderate PTL loading (PTL-3) fosters a regenerative MΦ-mediated immune microenvironment, thereby enhancing both angiogenesis and osteogenesis.

Fig. 7.

Effects of the MΦ-mediated immune microenvironment on BMSCs: (a) Schematic of the experimental procedure for investigating the MΦ-mediated immune microenvironment; (b) Optical images of ALP activity of BMSCs after 3 and 7 days of osteogenic induction; (c) Optical images of type I collagen production after 7 and 14 days; (d) Optical images of ECM mineralization after 14 and 21 days; (e, f) Quantitative analysis of type I collagen expression and ECM mineralization. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using one-way ANOVA with a Tukey post hoc test.

3.6. In vivo response to PTL and nanowire composite coatings

In vitro findings demonstrated that PTL-0, PTL-0.5, and PTL-3 effectively modulated the MΦ-mediated immune microenvironment and promoted angiogenesis and osteogenesis, while PTL-0 and PTL-0.5 offered limited antibacterial activity. Although PTL-24 exhibited stronger antibacterial effects, it simultaneously triggered excessive inflammation that compromised osseointegration. To validate these results and determine the optimal coating for clinical application, an in vivo infectious bone defect model was established to evaluate antibacterial performance, inflammatory response, and tissue regeneration (Fig. 8a). X-ray imaging confirmed accurate implant placement within the defect, without displacement or loosening (Fig. S23). For early-stage antibacterial evaluation, implants were retrieved on postoperative days 1 and 3 for bacterial quantification. On the 1st day, PTL-0 implants exhibited extensive.

Fig. 8.

In vivo antibacterial performance and histological evaluation: (a) Schematic of the in vivo experimental procedure; (b) In vivo antibacterial effects; (c) Quantitative analysis of antibacterial efficacy in vivo; (d) H&E and Giemsa staining of bone tissue on days 1 and 3 post-bone grafting. Post-implantation. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using one-way ANOVA with a Tukey post hoc test.

Bacterial colonization. In contrast, PTL-0.5, PTL-3, and PTL-24 achieved bacterial clearance rates of approximately 25.5%, 33.9%, and 62.1%, respectively (Fig. 8b). On the 3rd day, natural host immune responses reduced bacterial loads in the PTL-0 group to a 50.1% clearance rate. However, PTL-0.5, PTL-3, and PTL-24 exhibited significantly enhanced antibacterial efficacy, with clearance rates of 73.4%, 92.2%, and 98.9%, respectively (Fig. 8c). These findings suggest that PTL coatings synergize with host immunity to enhance bacterial eradication. Histological analysis provided further insight into bacterial persistence and inflammatory status. Giemsa staining (Fig. 8d) showed bacterial presence surrounding all implants on 1st day, with bacterial loads decreasing proportionally with increasing coating thickness. On the 3rd day, bacterial presence was substantially reduced, consistent with quantitative plate-count results. H&E staining revealed severe inflammation across all groups on the 1st day, most pronounced in PTL-0 and PTL-24. On the 3rd day, persistent inflammatory infiltration was still evident in PTL-24, whereas PTL-3 showed clear signs of inflammation resolution and tissue regeneration. These findings underscore the unique ability of PTL-3 to balance effective antibacterial activity with controlled immune regulation, thereby establishing a local microenvironment favorable for bone repair and osseointegration.

To further elucidate the mechanisms by which PTL coatings regulate the MΦ-mediated immune microenvironment and facilitate tissue regeneration, Western blot analysis was performed on postoperative 3 and 10 days (Fig. 9a). As shown in Fig. 9b, on the 3rd day, pro-inflammatory markers iNOS and IL-1β were elevated in all groups, with PTL-24 exhibiting the highest expression, indicating a strong inflammatory response. At this early stage, anti-inflammatory proteins CD206 and Arg-1 remained low across all groups. On the 10th day, pro-inflammatory markers had significantly declined in the PTL-0, PTL-0.5, and PTL-3 groups, while anti-inflammatory markers were markedly upregulated, with PTL-3 showing the most pronounced effect (Fig. 9c). In contrast, the PTL-24 group maintained high levels of pro-inflammatory proteins and failed to restore anti-inflammatory responses, indicating a risk of chronic inflammation. Angiogenesis-related proteins (Ang1 and CD31) and osteogenesis-related markers (OPN and ALP) were also evaluated. PTL-3 showed the highest expression levels of these markers, significantly outperforming other groups, suggesting robust vascularization and osteogenic induction. Macroscopic observations (Fig. 9d) further corroborated these findings.

Fig. 9.

In vivo osseointegration outcomes: (a-c) Western blot analysis of peri-implant bone tissue collected 3 and 10 days after implantation; (d) Macroscopic images of the implantation site at 3 and 10 days; (e) Micro-CT 3D reconstruction of newly formed bone surrounding the implant; (f) H&E staining of peri-implant tissue after 8 weeks; (g) Masson staining of peri-implant tissue after 8 weeks; (h-k) Quantitative analysis of bone structural parameters, including BV/TV, Tb.N, Tb.Th, and Tb.S. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 using one-way ANOVA with a Tukey post hoc test.

On the 3rd day, all groups exhibited signs of wound bleeding and edema, most pronounced in PTL-24. On 10th day, wounds in the PTL-0, PTL-0.5, and PTL-3 groups showed substantial healing and were covered by visible membranes. In contrast, PTL-24 presented with thick, opaque fibrous tissue obscuring the implant, indicating excessive fibrosis and delayed remodeling.

At week 8, Micro-CT imaging and 3D reconstruction revealed abundant new bone formation in the PTL-3 group, far exceeding that of other groups (Fig. 9e). Quantitative analysis (Fig. 9h–k) showed that BV/TV (49%), Tb.Th (0.12 mm), and Tb.N (7.51 mm⁻¹) were significantly higher in PTL-3, while Tb.Sp (0.036 mm) was the lowest, indicating the dense, mature bone regeneration. Histological analysis via H&E and Masson staining (Fig. 9f and g) confirmed these results. PTL-0 and PTL-0.5 exhibited limited bone regeneration due to persistent infection, with poor integration between implant and host bone. In contrast, PTL-3 demonstrated abundant new bone tightly integrated with the implant, indicating superior osseointegration. The PTL-24 group exhibited ongoing inflammation, minimal bone formation, and disrupted repair. Masson staining also revealed extensive collagen deposition in the PTL-3 group, confirming its enhanced bone matrix formation capacity. The semi-quantitative results of collagen deposition further confirmed the above findings (Fig. S24). To assess biosafety, major organs (heart, liver, spleen, lung, and kidney) were examined histologically at week 8 (Fig. S25). No pathological abnormalities were observed, confirming the systemic biocompatibility and safety of PTL coatings.

4. Discussion

In this study, we developed a gradient PTL coating that enhances macrophage-driven immunomodulatory and antibacterial functions. Through systematic HTS of the gradient platform, a coating formed at 0.5 mg/mL for 3 h (PTL-3), with an approximate thickness of 10.85 nm, was identified as optimal. Given the dose-dependent immunomodulatory effects of PTL coatings, precise and reproducible PTL gradient deposition is essential. Here, PTL dose was controlled by a small set of defined parameters, including lysozyme concentration, droplet volume, deposition speed, and total deposition time, enabling a deterministic relationship between programmed deposition time and effective PTL thickness. Cross-sectional imaging and blinded quantitative analysis at multiple random sites further verified the uniformity of coating thickness within samples and its reproducibility across batches. This formulation achieved a balance between effective antibacterial activity and robust osseointegration. The findings establish a new paradigm wherein immune-activating biomaterials mobilize host MΦs to combat infection, which distinct from conventional strategies reliant on antibiotics or metal ions [6,43,44]. The immunomodulation-based approach presented here thus provides a unique direction for implant surface functionalization, differing fundamentally from traditional release-dependent or passive anti-adhesive strategies.

The high-throughput fabrication strategy was instrumental in enabling comprehensive evaluation of surface properties and biological responses under varying PTL loading conditions. Based on our previous work, Na₂TiO₃ nanowires with optimal dimensions were firstly synthesized on Ti surface via alkaline etching [30], and gradient coatings were then deposited on the nanowires by precisely modulating PTL concentration and immersion time. This approach preserved substrate nanostructure while generating a spectrum of coating parameters on a single platform, thereby enhancing screening throughput and data consistency. As immersion time increased, nanowires became progressively encapsulated by PTL, ultimately forming a dense granular layer that eventually obscured the underlying nanostructure. Notably, although PTL initially promoted macrophage-mediated bacterial clearance, the highest concentration (2 mg/mL) significantly impaired its pro-healing activity. This reduction in bioactivity was closely linked to the particle aggregation shown in Fig. S1. Large PTL aggregates may physically shield the bioactive sodium Na₂TiO₃ nanowires, thereby reducing exposure to the nano-topographical cues necessary for M2 polarization [30]. Moreover, excessive PTL aggregation may act as a damage-associated signal, leading to excessive immune activation. The presence of hydrophobic amino acid residues in PTL reduced surface hydrophilicity, which significantly impacted cell behavior. Notably, cell spreading area decreased, pseudopodia transitioned from lamellipodia to filopodia, and cells adopted a more spherical morphology, indicating the reduced adhesion and functional activity. These surface modifications were confirmed through DR-FTIR and XPS analyses. PTL loading intensified N and S signals while attenuating the Ti 2p peak, consistent with complete PTL coverage. These physical and chemical alterations directly influenced cell-bacteria interactions, as evidenced by MΦ morphological changes, polarization states, and antibacterial responses across the gradient. This underscores the critical role of surface property modulation in directing biological performance. Compared to conventional surface engineering strategies, the integration of gradient fabrication and HTS in this study provided superior control over experimental parameters and facilitated the identification of optimal functional outcomes. Traditional methods typically apply a single concentration or produce uniform coatings, limiting the exploration of dose-response effects [45,46]. Although techniques such as spraying or plasma treatment can enhance surface features, they lack the capacity for efficient multi-parameter screening [47,48]. The gradient system developed here represents a transition from empirical to data-driven biomaterial design, offering a robust platform for the rational development of tailored, multifunctional implant coatings. Given the potential for paracrine cross-talk between adjacent regions on a continuous gradient substrate, the gradient platform in this study was primarily used for trend discovery, while the main conclusions were further confirmed using independently prepared discrete PTL-coated specimens. Although the current HTS platform was developed on planar surfaces, it could be further adapted to complex 3D structures, including porous scaffolds, by integrating microfluidic-assisted infiltration, gradient additive manufacturing, or rotation-compensated dip-coating. This would enable precise compositional gradient construction within internal pores and offer a robust strategy for optimizing the spatiotemporal biological performance of complex 3D implants.

The treatment of infectious bone defects critically depends on effective infection control, as thorough debridement and systemic antibacterial therapy are prerequisites for establishing a microenvironment conducive to osseointegration and bone regeneration [49]. In this context, we comprehensively evaluated the antibacterial performance of optimized PTL coatings. A key finding is that, PTL coatings exhibit only modest direct antibacterial effects under our conditions, whereas their principal benefit lies in potentiating host antibacterial immunity by modulating MΦ function. Notably, thicker PTL layers substantially promoted MФ phagocytosis of S. aureus, achieving up to 99% antibacterial efficacy within 24 h, whereas thinner layers failed to control infection promptly. Mechanistic investigations identified TLR4 as a central mediator of this immune-enhancing effect. TLR4, a pattern recognition receptor broadly expressed on MФs, plays a pivotal role in sensing pathogen-associated molecular patterns (PAMPs), triggering NF-κB signaling, and initiating pro-inflammatory responses. Our findings demonstrate that the gradient PTL coating serves as an effective TLR4 agonist, significantly enhancing MΦ phagocytic efficiency in a dose-dependent manner. This immunostimulatory effect stems from the amyloid-like physicochemical properties of PTL, which enable preferential binding to TLR4 and subsequent activation of downstream signaling pathways [50,51]. The immune-activating capacity of the PTL coating is fundamentally associated with its amyloid-like structure. Unlike native lysozyme, phase transition exposes dense hydrophobic amino acid residues that are normally buried within the protein interior. Mechanistically, these exposed hydrophobic domains may function analogously to the lipid A moiety of LPS. Whereas LPS engages the MD-2 hydrophobic pocket through its fatty acid chains [52], PTL may use its organized hydrophobic patches to achieve similar interfacial recognition, thereby promoting TLR4 dimerization and subsequent NF-κB activation [53]. This hydrophobic pattern-recognition mechanism is consistent with the established role of TLR4 as a sensor of misfolded protein aggregates and diverse PAMPs, and provides a plausible theoretical basis for the macrophage-mediated bacterial clearance observed in this study. Previous studies have shown that bacterial recognition activates small GTPases such as Cdc42 and Rac1, which drive cytoskeletal rearrangement and promote phagosome formation [54,55]. Concurrently, Rab7 regulates phagosome trafficking to lysosomes, while VAMP7 and Syntaxins mediate phagosome-lysosome membrane fusion to generate bactericidal phagolysosomes [56]. In addition, LC3-II recruitment further enhances lysosomal degradative capacity [57]. Therefore, as a pivotal innate immune receptor, TLR4 engagement directly augments MΦs phagocytosis, providing a mechanistic basis for the observed PTL-induced antibacterial effects. Moreover, the increased surface hydrophobicity of PTL was found to reduce MΦs adhesion, inducing a rounded morphology with elevated membrane tension, features that are known to promote pseudopodia extension and phagocytic cup formation [58]. This biophysical adaptation further amplifies the phagocytic response, underscoring the dual contribution of biochemical signaling and mechanical modulation in PTL-mediated immune activation. It was further validated using TAK-242, a selective TLR4 inhibitor. TAK-242 treatment significantly suppressed MΦ phagocytic capacity and attenuated PTL-induced pro-inflammatory signaling, confirming the central role of TLR4 in mediating the immunostimulatory effects PTL. These findings are consistent with the recognized role of TLR4 in detecting LPS and other pathogen-derived molecules [40], supporting a novel immunomodulatory strategy for biomaterial design. Hydrophobicity may influence complement activation by modulating plasma protein adsorption, suggesting that PTL could alter the early protein corona and opsonization cues. However, complement responses are governed not only by wettability, but also by the identity and conformation of adsorbed proteins and nanoscale topography. Therefore, complement endpoints will be quantified in future plasma-exposure studies. Unlike conventional strategies that rely on prolonged antibiotic administration or metal ion-based coatings which pose risks of cytotoxicity and antimicrobial resistance, PTL coatings enhance innate immune clearance by activating host immunity while maintaining biocompatibility. Metal-ion systems (e.g., Ag/Cu/Zn) often require high local release to remain bactericidal, which may impair peri-implant cells or tissues when dosing is difficult to control [13,44]. Although antibiotic-loaded coatings provide strong early killing, biofilm tolerance and sustained sub-inhibitory exposure may increase selection pressure for resistant phenotypes [[59], [60], [61]]. In contrast, the PTL/nanowire interface promotes macrophage-mediated antibacterial activity while supporting subsequent inflammation resolution and osseointegration through persistent topographical cues. This study therefore provides both mechanistic insights and design principles for the development of next-generation antibacterial and regenerative biomaterials. Although S. aureus was chosen as a clinically dominant orthopedic pathogen, future studies will validate the PTL/nanowire interface against Gram-negative bacteria and antibiotic-resistant strains (e.g., MRSA) to further establish broad-spectrum translational relevance.

Osseointegration is a highly coordinated process involving the interplay of multiple cell types, mainly including MΦs, ECs, and BMSCs [47,62]. The emerging field of osteoimmunology has garnered increasing attention, as immune regulation during the early stages of osseointegration is critical for long-term success. Among these cell types, MΦs serve as pivotal regulators that orchestrate the balance between inflammation and regeneration [63]. In this study, the medium-loading PTL coating (PTL-3) was identified as the optimal formulation, striking a balance between antibacterial efficacy and immunomodulation. Moderate early-phase pro-inflammatory activation facilitated effective debridement and vascular matrix remodeling, which subsequently supported vascularization and extracellular matrix mineralization. In contrast, the low-dose PTL (PTL-0.5) elicited only mild inflammation and insufficient M2 polarization, while the high-dose PTL (PTL-24) triggered excessive inflammation and prolonged M1 dominance, thereby impairing endothelial angiogenesis and BMSC-mediated osteogenesis. These observations align with previous reports demonstrating the detrimental effects of chronic inflammation on tissue regeneration [64,65]. The dynamic transition from M1 to M2 MΦ phenotypes is essential for establishing a pro-regenerative MΦ-mediated immune microenvironment. In the early phase, M1 phenotype secrete pro-inflammatory cytokines that aid in bacterial clearance and basement membrane degradation [66]. This is followed by a shift toward M2 polarization, characterized by the release of anti-inflammatory cytokines and growth factors that stabilize nascent vasculature and promote matrix remodeling. Together, the current in vitro and in vivo data support a time-dependent trend toward early immune activation followed by later inflammation resolution in the PTL-3 group, thereby exhibiting superior angiogenic and osteogenic performance. Compared with other immunomodulatory biomaterials, such as IL-4-loaded bio-ceramics, which promote M2 polarization but lack sufficient early inflammatory activation [67]. The PTL coating offers stage-specific immune modulation within a single, unified formulation. This simplifies design complexity while enhancing therapeutic efficiency. However, research on the immune microenvironment has primarily focused on the immune state mediated by MΦ adhesion, future studies should delve deeper into their early recruitment mechanisms. In vivo data further validated these findings, with PTL-3 significantly improving bone microarchitectural parameters compared to other strategies, highlighting its potential to enhance both early implant stability and long-term osseointegration. A limitation of this study is that only male rats were used for the in vivo evaluation. Therefore, the impact of gender differences should be considered in future research.

Overall, this study demonstrates that the PTL-3 coating modulates MΦ phenotypes to couple rapid bacterial clearance with efficient tissue regeneration. It introduces an integrated immune-driven antibacterial strategy for orthopedic implant surface design. By concurrently enhancing immune clearance, mitigating infection risk, and fostering a regenerative microenvironment, the PTL-3 group exhibits strong bioactivity and translational potential within the tested range. This strategy provides a robust framework for developing next-generation bone repair materials, although further clinical validation will be critical for widespread clinical adoption.

5. Conclusion

This study demonstrates that immune-activating PTL/nanowire composite coatings constructed on Ti surfaces offer a safe and effective strategy for preventing implant-associated infections while enhancing osseointegration. Utilizing a high-throughput gradient platform, we identified that PTL significantly enhance host defense by activating MΦs via a TLR4-dependent pathway, achieving bacterial clearance rates of up to 99%. The immunomodulatory effects of the coating were found to be dose-dependent, with moderate loading (PTL-3) achieving an optimal balance between immune activation and tissue regeneration. This formulation effectively promoted endothelial angiogenesis and BMSC-mediated osteogenic differentiation. In vivo evaluation further validated these findings. PTL-3 coatings achieved rapid infection control (96% bacterial clearance by day 3) and robust bone regeneration (BV/TV of 49% by week 8), without inducing systemic toxicity or organ pathology. Collectively, these findings establish PTL/nanowire-based immune-activating coatings is a clinically promising strategy to safely and effectively address implant-associated infections and loosening. This strategy holds strong translational potential for orthopedic and dental implants, particularly in clinical scenarios where infection remains a significant challenge.

Ethics approval and consent to participate

The Animal Ethics Committee of Taiyuan University of Technology (TYUT2025120101) authorized all animal experiment processes and surgery protocols, which were carried out following international animal welfare standards.

CRediT authorship contribution statement

Ruiyue Hang: Methodology, Investigation, Data curation, Conceptualization. Huanming Chen: Software, Formal analysis. Liwei Yang: Software, Formal analysis. Ruoyu Di: Software, Formal analysis. Yuyu Zhao: Conceptualization. Runhua Yao: Investigation. Xiaohong Yao: Supervision, Resources, Project administration, Funding acquisition. Huaiyu Wang: Writing – review & editing, Supervision. Yin Xiao: Writing – review & editing, Supervision, Resources, Conceptualization. Ruiqiang Hang: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: