Titanium alloys, particularly TC4 (Ti6Al4V), are extensively employed as load-bearing biomedical implants due to their superior mechanical properties, excellent corrosion resistance, and favorable biocompatibility. However, their inherent bio-inertness often leads to inadequate osseointegration, while the lack of intrinsic antibacterial activity increases susceptibility to implant-associated infections, namely two major contributors to clinical failure. Conventional surface modification techniques frequently struggle to simultaneously enhance bioactivity and confer robust, long-lasting antibacterial efficacy. To address this critical challenge, the work aims to propose a one-step composite alkali-heat treatment (Cu-AHT) strategy, in which Cu2+ ions are incorporated into the conventional alkali-heat process to synergistically engineer a hierarchical micro-nano structure and introduce antibacterial Cu2O phases on TC4 surfaces.
The surface physicochemical properties of modified TC4 were systematically characterized. X-ray diffraction (XRD) confirmed the presence of α-Ti, β-Ti, and Cu2O phases on treated surfaces. The intensity of Cu2O diffraction peaks increased proportionally with the molar concentration of Cu(OH)2 (denoted as n(Cu(OH)2)) in the mixed alkaline solution, unequivocally verifying the successful incorporation of the antibacterial phase. Laser confocal microscopy revealed that Cu-AHT significantly increased surface roughness, with the arithmetic mean height (Sa) rising from (0.13±0.01) μm for untreated controls to (0.19±0.02) μm for treated specimens. Moreover, surface roughness exhibited a clear dependence on n(Cu(OH)2), demonstrating its tunability. Surface wettability was assessed via contact angle measurements with water, formamide, and diiodomethane as probe liquids. Contact angles for all three liquids were substantially lower on Cu-AHT-treated surfaces than on untreated TC4. Surface free energy (SFE), calculated with the van Oss-Chaudhury-Good (vOCG) method via MATLAB, significantly increased for all modified groups, confirming enhanced hydrophilicity-a key factor favoring initial protein adsorption and subsequent cell adhesion.
In vitro antibacterial performance was evaluated against Staphylococcus aureus, a predominant pathogen in orthopedic infections, with a direct contact assay. All Cu-AHT-treated specimens exhibited exceptional antibacterial efficacy, achieving bacterial reduction rates exceeding 99%. This potent antibacterial effect was attributed to the sustained release of Cu+ ions from the incorporated Cu2O phase, which disrupted bacterial membrane integrity and induced oxidative stress.
The in vitro biological response was systematically investigated with MC3T3-E1 osteoblast cells. CCK-8 assay revealed a distinct dose- and time-dependent pattern in cell metabolic activity. At day 5, the TC4-0.02 group exhibited the highest relative viability, while the TC4-0.03 group showed a sharp decline to approximately 10% of the control level, indicating that excessive copper loading (n(Cu(OH)2)=0.03) exceeded the biosafety threshold and induced significant long-term cytotoxicity. Calcein-AM staining corroborated these quantitative findings: TC4-0.02 surfaces displayed the densest and most uniformly distributed green fluorescence, whereas TC4-0.03 surfaces showed markedly reduced cell density and weaker fluorescence intensity. Phalloidin/DAPI co-staining further revealed that cells on TC4-0.02 surfaces exhibited the most extensive spreading, well-organized F-actin cytoskeletons, and abundant filopodia, indicating superior early-stage adhesion and migration capacity. In contrast, cells on TC4-0.03 surfaces displayed reduced spreading area and fewer pseudopodia, further confirming copper-induced cytotoxicity. Scanning electron microscopy (SEM) provided compelling morphological evidence that the unique micro/nano-porous network structure formed by Cu-AHT facilitated active cell embedding and tight mechanical interlocking between osteoblasts and the surface topography. Collectively, these assays demonstrate that Cu-AHT significantly enhances surface hydrophilicity, bioactivity, and osteogenic potential while concurrently imparting strong antibacterial properties. Critically, this work establishes a well-defined biosafety window for the Cu-AHT system: n(Cu(OH)2)=0.02 is identified as the optimal parameter that maximizes osteoblast adhesion, spreading, and long-term proliferation while maintaining potent antibacterial efficacy (>99% against S. aureus). In contrast, n(Cu(OH)2)=0.03 clearly exceeds the safety threshold, inducing progressive and dose-accumulative cytotoxicity characterized by metabolic collapse, reduced cell density, and impaired cytoskeletal development. These findings provide critical quantitative guidance for the rational design of antibacterial yet biocompatible titanium implants.
In summary, this one-step Cu-AHT strategy effectively addresses the dual challenges of bio-inertness and insufficient antibacterial performance on TC4 implants. The resulting surfaces exhibit a synergistic combination of enhanced hydrophilicity, robust antibacterial efficacy, significantly improved biocompatibility, and promising osteogenic activity. By elucidating the dose-dependent biological effects and establishing a clear safety boundary, this work provides not only a facile and scalable surface engineering approach but also essential design principles for the development of next-generation multifunctional titanium-based implants with optimized clinical performance and reduced failure rates.
Key words
titanium alloy /
surface modification /
composite alkali-heat treatment (Cu-AHT) /
micro-nano structure /
antibacterial activity /
bioactivity
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Funding
The National Key Research and Development of China (2024YFE0212600); National Training Program of Innovation and Entrepreneurship for Undergraduates（202510145035）
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