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Titanium implants that have been grit-blasted and acid-etched to produce a rough microtopography support more bone integration than do smooth-surfaced implants. In vitro studies have suggested that this is due to a stimulatory effect on osteoblasts. It is not known if grit-blasted and acid-etched Ti6Al4V implants also stimulate osteoblasts and increase bone formation clinically. In this study, we examined the effects of micrometer-scale-structured Ti6Al4V surfaces on cell responses in vitro and on tissue responses in vivo.Ti6Al4V disks were either machined to produce smooth surfaces with an average roughness (Ra) of 0.2 μm or grit-blasted, resulting in an Ra of 2.0, 3.0, or 3.3 μm. Human osteoblast-like cells were cultured on the disks and on tissue culture polystyrene. The cell number, markers of osteoblast differentiation, and levels of local factors in the conditioned media were determined at confluence. In addition, Ti6Al4V pedicle screws with smooth or rough surfaces were implanted into the L4 and L5 vertebrae of fifteen two-year-old sheep. Osteointegration was evaluated at twelve weeks with histomorphometry and on the basis of removal torque.The cell numbers on the Ti6Al4V surfaces were lower than those on the tissue culture polystyrene; the effect was greatest on the roughest surface. The alkaline-phosphatase-specific activity of cell lysates was decreased in a surface-dependent manner, whereas osteocalcin, prostaglandin E2, transforming growth factor-β1, and osteoprotegerin levels were higher on the rough surfaces. Bone-implant contact was greater around the rough-surfaced Ti6Al4V screws, and the torque needed to remove the rough screws from the bone was more than twice that required to remove the smooth screws.Increased micrometer-scale surface roughness increases osteoblast differentiation and local factor production in vitro, which may contribute to increased bone formation and osteointegration in vivo. There was a correlation between in vitro and in vivo observations, indicating that the use of screws with rough surfaces will result in better bone-implant contact and implant stability.The osteointegration of screws with rough microtopographies is likely to be better than that of screws with smoother surfaces.