Hydrodynamic control of titania nanotube formation on Ti-6Al-4V alloys enhances osteogenic differentiation of human mesenchymal stromal cells.

In order to obtain bioactive bone-implant interfaces with enhanced osteogenic capacity, various approaches have been developed to modify surface physicochemical properties of bio-inert titanium and titanium alloys. One promising strategy involves fabricating highly ordered nanotubes (NT) on implant surfaces via electrochemical anodization. However, few studies have applied this technique to Ti-6Al-4V alloys most commonly adopted for the fabrication of osteo-integrated surfaces on orthopedic implants. In this study, we investigated the influence of electrolyte hydrodynamics to NT fabrication on Ti-6Al-4V in ethylene glycol based electrolyte and evaluated the osteogenic differentiation capacity of human mesenchymal stromal cells (hMSCs) on different diameter NT surfaces. Computational Fluid Dynamics (CFD) analysis was used to simulate electrolyte flow profiles under various stirring conditions (e.g. stirrer bar location and flow direction) and their correlation to NT formation. Polished Ti-6Al-4V disks (240 grit) were anodized at 20 and 40 V under optimal electrolyte flow conditions for comparison of NT diameter-controlled osteogenic differentiation and mineralization potential of hMSCs over 21 days culture in osteogenic media. Ti-6Al-4V surfaces anodized with 20 and 40 V resulted with NTs diameter approx. 39 and 83 nm, respectively. Electrolyte hydrodynamics (flow profile) significantly influenced the uniformity of NT formation. Here, a uniform velocity and shear stress profile at the surface promoted homogeneous NT growth, whereas large variation in either flow velocity or shear stress to the surface impaired mature NT formation. After 21 days of culture, fluorescence staining demonstrated significantly greater osteocalcin and osteopontin expression, and increased mineralized deposits (xylenol orange staining) on fluctuating NT surfaces anodized under 20 V (Ø 39 nm) relative to flat NT layer anodized with 40 V (Ø 83 nm) and polished controls. This study provides a systematic investigation of NT formation with respect to the electrolyte hydrodynamic effects to NT growth on Ti-6Al-4V alloys, demonstrating the feasibility of a one-step anodization process for generating uniform NT under optimal hydrodynamics. Optimized wavy micro-/nano-topography with Ø 39 nm NT stimulated osteogenic differentiation capacity of hMSCs on Ti-6Al-4V alloys and confirmed the potential application of anodization to improve osteo-integrative surfaces in orthopedic implants.