In this review, the appropriate use of cell culture to evaluate substrate effects on osteoblast behavior during the process of osseointegration has been considered in the context of existing reports. The interactions of osteoblasts with different substrates can be measured in terms of cytotoxicity, attachment, proliferation, and differentiation. The osteoblast culture systems that produce an osteoblast matrix opposing implant material substrates provide one model for evaluating the implant-bone interface. Alterations in osteoblast behavior at different culture substrates may reflect clinical determinants of bone formation at these substrates in vivo; however, cell responses in vitro have not been compared or correlated with in vivo outcomes. Legitimate interpretations of in vitro experiments are discussed in terms of practical, technical, and biologic limitations presented by the cell culture approach. Cell culture provides access to molecular and cellular information that fosters nanostructural engineering approaches to implant design and significant hypotheses to be tested in vivo. In this way, cell culture offers unique insights into the process and phenomenon of osseointegration. Keywords: biomaterials, cell culture, interface, osteoblast, review, surface topography