Optimized Biocompatibility of Anodic Tantalum Oxide Nanotube Arrays

Background

Tantalum (Ta) is a rare, hard, corrosion‑resistant, and bioinert metal whose ultrathin oxide film confers excellent biocompatibility. Its flexibility and low toxicity make it ideal for dental and orthopedic implants, bone reconstruction, and porous scaffolds that mimic native bone structure. Compared with titanium, tantalum supports greater extracellular matrix deposition, cellular adhesion, and proliferation. Recent studies have shown that nanostructured surfaces—especially self‑organized nanotube arrays—are pivotal in modulating cell behavior. Anodized Ta nanotubes have been reported to promote osseointegration more effectively than flat surfaces, and porous tantalum implants have achieved superior fixation in clinical trials. Building on prior work with TiO₂ nanotubes, this study focuses on TaO_x arrays of varying diameters, evaluating their influence on fibroblast adhesion and proliferation.

Methods

Preparation of TaO_x Nanotubes

Commercial Ta sheets (0.127 mm, 99.7 % purity) were ultrasonically cleaned in acetone, isopropanol, ethanol, and water. Anodization was performed at 20 °C in 4.9 wt % HF sulfuric acid using a two‑electrode cell (Ta anode, Pt counter). Applied voltages (10–40 V) yielded nanotube diameters from 20 to 90 nm. Samples were exposed to low‑intensity UV light (≈2 mW cm^–2) for 8 h before biocompatibility assays.

Material Characterization

Scanning electron microscopy (SEM) quantified nanotube dimensions; X‑ray diffraction (XRD) and transmission electron microscopy (TEM) confirmed amorphous structure; energy‑dispersive spectroscopy (EDS) verified composition. Contact angles were measured with the extension method using water and culture medium.

Human Fibroblast Cell Culture

MRC‑5 fibroblasts (BCRC No. 60023) were cultured in Eagle’s MEM with 10 % FBS, 2 mM L‑glutamine, 1.5 g L^–1 NaHCO₃, 0.1 mM non‑essential amino acids, and 1.0 mM pyruvate at 37 °C, 5 % CO₂. Cells were seeded onto autoclaved TaO_x sheets placed in 12‑well plates for subsequent assays.

Cell Adhesion Assay

Cells were seeded at 2.5 × 10³ cells cm^–2 and incubated for 3 days. After fixation, actin filaments were stained with rhodamine phalloidin and nuclei with DAPI. Fluorescence microscopy (AX80, Olympus) evaluated adhesion morphology. For SEM, cells were fixed in 2.5 % glutaraldehyde, dehydrated, critical‑point dried, and sputter‑coated with platinum.

Cell Proliferation Assay

Seeding density of 1 × 10⁴ cells cm^–2 was used for a 1‑week culture. Proliferation was quantified with the WST‑1 reagent (Roche); absorbance was read at 450 nm (Spectral Max250).

Statistical Analysis

Experiments were performed in triplicate with at least three independent repeats. Data are expressed as mean ± SD and analyzed by one‑way ANOVA (SPSS 12.0). A p‑value < 0.05 was considered significant.

Results and Discussion

SEM images (Fig. 1) confirm well‑defined nanotube arrays with diameters proportional to anodization voltage: 20, 35, 65, and 90 nm. The 20‑nm tubes exhibit slightly irregular walls, likely due to lower electric field strength. Cross‑sectional SEM (Fig. 2) shows consistent tube lengths across diameters. XRD and TEM (Fig. 3) confirm that all arrays remain amorphous, which is advantageous for biocompatibility.

SEM images of Ta foil and TaO_x nanotubes with diameters of 20, 35, 65, and 90 nm.

Cross‑sectional SEM of TaO_x nanotubes with diameters of 20, 35, 65, and 90 nm.

XRD spectra (a) and a TEM image of a 90‑nm TaO_x nanotube (b).

Wettability measurements (Fig. 4) reveal that all arrays are highly hydrophilic, with contact angles decreasing as diameter approaches 35 nm, then increasing at 20 nm. This trend aligns with Wenzel’s model, where increased geometric roughness lowers contact angle on hydrophilic surfaces. The calculated roughness factor (Fig. 5) confirms that 35‑nm tubes possess the highest roughness, explaining their superior hydrophilicity.

Water and culture medium droplets on Ta foil and TaO_x arrays (20–90 nm). Contact angles are indicated.

Schematic of nanotube geometry and calculated roughness factors for each diameter.

Fluorescence microscopy (Fig. 6) and SEM (Fig. 7) demonstrate that fibroblasts adhere and spread most effectively on 35‑nm tubes, forming robust actin networks and focal adhesions. The 20‑nm arrays, while offering more potential focal points, exhibit reduced hydrophilicity, impairing adhesion. Cell coverage area, quantified with ImageJ, mirrors the contact‑angle trend: maximal at 35 nm, lower at 20 nm and 90 nm.

Fluorescence images of fibroblast actin (red) and nuclei (blue) on Ta foil and TaO_x arrays (20–90 nm).

SEM images of fibroblast adhesion and proliferation on Ta foil and TaO_x arrays (20–90 nm). Coverage areas are marked.

The WST‑1 assay (Fig. 8) confirms that proliferation peaks on 35‑nm tubes, matching the wettability and adhesion data. No significant difference is observed between the flat Ta foil and TaO_x arrays overall, underscoring the importance of nanoscale geometry and surface energy in cell response.

Optical densities from WST‑1 assay for fibroblasts on Ta foil and TaO_x arrays (20–90 nm). Error bars represent SD.

Conclusions

Self‑organized TaO_x nanotube arrays with diameters from 20 to 90 nm were fabricated via anodization and characterized as predominantly amorphous. Wettability varied systematically with diameter, following Wenzel’s roughness model. In vitro fibroblast studies revealed a clear dependence on surface hydrophilicity and nanotube geometry; the 35‑nm arrays provided optimal focal adhesion density and cell coverage, resulting in the highest biocompatibility. These results demonstrate that tailoring TaO_x nanotube diameter and roughness can substantially improve the biological performance of tantalum‑based implants.