We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66. GPa). ⋙. dispersed (9.99. kPa). &gt
. central region (5.20. kPa). &gt
. peripheral regions (3.67. kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed. &gt
. central region. &gt
. peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25-40. kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site. © 2013 Elsevier Ltd.