Rotating bioreactors have been used to overcome the limitations of passive nutrient diffusion in three-dimensional (3D) constructs for tissue engineering of bone. It is hypothesized that conventional scaffolds undergo repeated wall collisions in rotating bioreactors, which may disrupt bone tissue formation. In this study, we investigated the effects of wall collision on osteoblastic cells cultured on a microsphere based scaffold of varying densities in comparison to water. The conventional heavier than water (HTW; density > 1 g/cm3) scaffolds were fabricated by sintering HTW microspheres of 85:15 poly (lactide-co-glycolide) (PLAGA), and mixed scaffolds were designed by mixing lighter than water (LTW; density < 1 g/cm3) and HTW microspheres of PLAGA. We quantified average velocities of the two types of scaffolds using a particle tracking system, and no significant difference in average velocities was observed between the two types of scaffolds. However, HTW scaffolds have frequent wall collision and mixed scaffolds can avoid wall collision in bioreactors. When human Saos-2 osteoblast like cells were cultured on the scaffolds in bioreactors for 16 days, bone cell proliferation and cell differentiation on HTW scaffolds were significantly inhibited as compared to those cultured on mixed scaffolds in rotating bioreactors. These results indicate that collision between scaffolds and bioreactor wall is a confounding factor in osteoblastic cell proliferation and differentiation. These studies provide a foundation for development of 3D scaffolds for tissue engineering of bone in rotating bioreactors.