目的 打破传统表面改性技术难以协同优化生物活性与抗菌性能的局限。方法 本研究创新性地将Cu2+引入碱热处理工艺,在构建微纳结构的同时引入抗菌性离子,采用一步法复合碱热处理的方法协同调控TC4(Ti6Al4V)表面综合性能。采用X射线衍射仪(XRD)、激光共聚焦显微镜、接触角测量仪表征表面形貌与理化特性,采用直接接触法评估体外抗菌性能,并采用体外生物学研究方法对样品表面生物活性进行表征。结果 TC4经复合碱热处理后,表面可由XRD检测出α-Ti、β-Ti以及Cu2O的衍射峰,其中Cu2O衍射峰强度随混合碱液中n(Cu(OH)2)增加而增强,表明样品表面形成微米-纳米多级多孔粗糙结构并成功引入Cu2O相;表面粗糙度显著升高,并可由n(Cu(OH)2)定向调控;水、甲酰胺与二碘甲烷三组液体在处理后样品表面的接触角相比未处理样品皆有减小,并通过MATLAB计算得出处理后样品的相应表面自由能高于未处理样品,表面润湿性得到显著提升;在样品表面接种金黄色葡萄球菌,测得处理后样品抗菌率皆大于99%,呈强抗菌性;体外生物学实验也表明复合碱热处理赋予样品一定的生物活性和成骨活性。结论 本实验通过一步法复合碱热处理方法同时改善了TC4表面生物惰性与抗菌性,为优化钛基植入体综合性能提供了新的理论指导及技术支撑。
Abstract
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.
关键词
钛合金 /
表面改性 /
复合碱热处理 /
微纳结构 /
抗菌性 /
生物活性
Key words
titanium alloy /
surface modification /
composite alkali-heat treatment (Cu-AHT) /
micro-nano structure /
antibacterial activity /
bioactivity
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基金
国家重点研发项目（2024YFE0212600）; 国家级大学生创新创业训练计划资助项目（202510145035）
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