In a previous study, we showed that the combination of appropriately designed three-dimensional (3D) microcarrier scaffolds and fluid flow through and around the scaffolds during high aspect ratio vessel (HARV) rotation enhances the elaboration of mineralized bone matrix by osteoblast-like cells. In this study, we describe the ongoing characterization of our 3D culture system, including the investigation of interior fluid flow within the scaffolds and early stage integrin expression during hydrodynamic culture. Using theoretical and experimental methods, we have estimated that cells cultured on the interior of microcarrier scaffolds experience an interior nutrient flow velocity between 1 x 10(-3) and 1 x 10(-2) cm/s and maximum shear stress of 0.03 N/m(2). Under these conditions, osteoblast-like cells grew extensively in the interior regions of the scaffold and retained their osteoblastic phenotype as measured by alkaline phosphatase. In addition, flow cytometric analysis of the overall cell population showed that cells constitutively expressed integrin alpha3beta1 during 3D hydrodynamic culture.