The need for bone grafts to repair bone defects is rapidly accelerating as our population ages. Bone defects have been successfully treated using autografts and allografts. However, these are less than ideal approaches since autograft availability is limited and can result in donor-site morbidity while allografts can be immunologically rejected and have the potential to transmit disease. The use of putative human mesenchymal stem cells (hMSC) combined with biocompatible scaffolds has great potential for treating bone defects, which would otherwise be treated with autografts or allografts. Unfortunately, the expansion of hMSC in vitro, which could increase the therapeutic potential of hMSC, reduces their osteogenic potential. The ability to expand hMSC in vitro while maintaining, or even enhancing, their osteogenic potential, could greatly enhance their therapeutic potential. Therefore, the goal of this project is to identify specific biomaterial surface characteristics and biophysical signals that interact synergistically in optimizing hMSC differentiation toward the osteoblastic lineage. Our overall hypothesis is that biomaterial surface characteristics, specifically nanoscale topography, sensitize cells to fluid flow thus increasing the effect of fluid flow on expansion of hMSC while also enhancing the osteogenic potential of hMSC. Using unique fluid flow protocols, novel biomaterial surface characteristics, atomic force microscopy, genetic engineering and transgenic animal models we will identify an environment that optimizes differentiation of hMSC towards the osteoblastic lineage and the signal transduction pathways involved in this mechanism. We will then examine whether these pre-treated hMSC are more osteogenic in vivo than non pre-treated hMSC. We will accomplish this through the completion, over a 5 year period, of 4 aims: Aim 1, Examine the effect of surface topography on adhesion, proliferation and differentiation of hMSC, in the presence and absence of inhibitors of specific signaling pathways; Aim 2, Determine stiffness and mechanosensitivity of hMSC on surfaces with varying nanoscale topographies; Aim 3, Examine the effect of fluid flow, in the presence and absence of inhibitors of the PLC/calcineurin and PLC/ERK signaling pathways, on hMSC proliferation and differentiation; and Aim 4, Examine in vivo osteogenesis of hMSC seeded onto HA/TCP scaffolds. By completing these aims we will not only develop novel strategies for bone tissue engineering but also provide mechanistic insight into the regulation of hMSC proliferation and differentiation.