Osteoblast response to nanostructured and phosphorus-enhanced titanium anodization surfaces

Abstract

Titanium surface modification by anodization has been shown to promote bone formation. However, interactions between anodized layers and bone cells depend upon the anodized layer surface composition and morphology. The present study evaluated the effect of anodized layer phosphorus content, crystallinity, surface porosity, and roughness on the MC3T3-E1 osteoblast response. Commercially pure titanium specimens were anodized in two mixed-acid electrolytes to final forming voltages of 108 V and 180 V. The oxide crystallinity, surface porosity, surface roughness, and elemental composition of each specimen type were determined by X-ray diffraction, scanning electron microscopy, atomic force microscopy, and energy dispersive spectroscopy analyses. Osteoblast-like MC3T3-E1 cell attachment, proliferation, differentiation, and mineralization were assessed using optical and fluorescence microscopy, total protein content, alkaline phosphatase activity, osteocalcin production, and alizarin red assays. Oxide crystallinity and surface roughness increased with anodization forming voltage in each electrolyte. Phosphorus (P) uptake increased with forming voltage in the phosphoric acid containing electrolyte. The total protein assay showed P-incorporated, low surface pore density oxides to exhibit initially delayed proliferation compared to non-P containing high surface pore density oxides. Alkaline phosphatase and osteocalcin assays showed trends of early differentiation and maturation for P-incorporated, low surface pore density oxides compared to non-P containing high surface pore density oxides. Highly crystalline anodized layers were shown to induce enhanced mineralization compared to non-anodized titanium specimens. Overall, the biochemical influence of P-incorporation into the oxide layers showed trends of earlier osteoblast differentiation and maturation. The combination of P-incorporation, an anatase phase oxide, a low surface pore density, and a high surface roughness showed the highest mineralization levels.