Anodically oxidized titanium surfaces, prepared by spark discharge, have micro-submicron surface topography and nano-scale surface chemistry, such as hydrophilic functional groups or hydroxyl radicals in parallel. The complexity of the surface characteristics makes it difficult to draw a clear conclusion as to which surface characteristic, of anodically oxidized titanium, is critical in each biological event. This study examined thein vitrobiological changes, induced by various surface characteristics of anodically oxidized titanium with, or without, release of hydroxyl radicals onto the surface. Anodically oxidized titanium enhanced the expression of genes associated with differentiating osteoblasts and increased the degree of matrix mineralization by these cellsin vitro. The phenotypes of cells on the anodically oxidized titanium were the same with, or without, release of hydroxyl radicals. However, the nanomechanical properties of thisin vitromineralized tissue were significantly enhanced on surfaces, with release of hydroxyl radicals by oxidation effects. In addition, the mineralized tissue, produced in the presence of bone morphogenetic protein-2 on bare titanium, had significantly weaker nanomechanical properties, despite there being higher osteogenic gene expression levels. We show that enhanced osteogenic cell differentiation on modified titanium is not a sufficient indicator of enhancedin vitromineralization. This is based on the inferior mechanical properties of mineralized tissues, without either being cultured on a titanium surface with release of hydroxyl radicals, or being supplemented with lysyl oxidase family members.