Chitosan is a naturally occurring polysaccharide, which has proven to be an attractive candidate for bone tissue engineering, due to its ability to promote osteoblast mineralization. Electrospinning has become a well-established cell scaffold processing technique, as it produces a high surface area to volume fibrous material that can mimic the three dimensionality of the extracellular matrix of a cell. In this study, we have investigated the osteoblast response to two different chemically crosslinked (hexamethylene-1,6-diaminocarboxysulfonate (HDACS) and genipin) electrospun chitosan scaffolds and their film counterparts in order to determine how material chemistry influences cellular behavior in conjunction with material topology. In addition, material properties of each fiber scaffold such as porosity and tensile strength were considered. MLO-A5 osteoblast cells grown on chitosan-HDACS scaffolds were found to display a more organized cellular network, along with significantly more filopodia extensions, compared to those grown on chitosan-genipin scaffolds. After 2 days of growth on chitosan-HDACS fibers, a higher level of alkaline phosphatase expression in MLO-A5 cells was reported compared to that of either chitosan-genipin fibers or films. These results indicate that not only chemistry, but also surface topology is an important effecter of cellular behavior. Ultimately, chitosan-HDACS fiber scaffolds provided an adequate substrate for osteoblast attachment and proliferation. (C) 2015 Wiley Periodicals, Inc.