AbstractLaminin-5 (Ln-5), an important ECM protein, plays a critical role in regulating the growth and differentiation of mesodermal tissues, including bone. Ln-5 can be secreted by the mesenchymal stem cells (MSCs), and Ln-5 promotes MSCs osteogenic differentiation. It has been demonstrated that substrate surface topography could regulate MSC secretion and differentiation. A better understanding of the mechanism of Ln-5 and surface roughness regulating MSC osteogenic differentiation, which would provide a guide way for the surface topography design and coating of orthopedic implants and cell culture substrates. However, few studies have investigated the relationship between surface roughness and the secretion of Ln-5 in the MSC osteogenic differentiation. Whether substrate surface topography regulates MSC differentiation via regulating Ln-5 secretion and how surface topography contributes to the secretion of Ln-5 are still not known. In this study, the influence of microscale roughness at different levels (R0, R1 and R2) on the secretion of Ln-5 of human bone marrow MSCs (hBMSCs) and subsequent osteogenic differentiation were examined. hBMSCs spreading, distribution and morphology were largely affected by different roughness levels. A significantly higher level of Ln-5 secretion was detected on R2, which correlated to the local cell density regulated by the rough surface. Ln-5 binding integrins (α2 and α3) were strongly activated on R2. In addition, the results from hBMSCs on R0 inserts with different cell densities further confirmed that local cell density regulated Ln-5 secretion and cell surface integrin activation. And the mineralization level of MSCs on R2 was remarkably higher than that on R0 and R1. These results suggested that hBMSC osteogenic differentiation level on R2 roughness was enhanced via increased Ln-5 secretion that was attributed to rough surface regulated local cell density. Thus, the microroughness could serve as effective topographical stimulus in cell culture devices and bone implant materials.