Stem cells transit along a variety of lineage-specific routes towards differentiated phenotypes. including both passive (topography order and substrate stiffness) and dynamic mechanical inputs can further TR-701 regulate these phenotypic shifts [4 5 These microenvironmental cues are particularly important for tissue engineering where stem cells must often interface with a biomaterial substrate that TR-701 can instruct tissue formation or serve as a vehicle for targeted delivery of cells in vivo. Design of the material microenvironment for example through engineering specific receptor-ligand interactions onto the material surface can modulate the extent to which differentiation occurs [6 7 Similarly the topography of the interacting surface can be altered to impact stem cell fate whether these cells are delivered with or invade into the biomaterial post-implantation [8 9 While the mechanism by which these passive topographical stimuli elicit changes in stem cell activity is not yet clear their influence occurs over a range of length scales and appears to influence the differentiation process directly. Nanofibrous scaffolds formed by the process of electrospinning are TR-701 commonly employed for tissue engineering with stem cells [10]. These scaffolds provide a biomimetic fibrous microenvironment with polymeric fibers that recreate the length scale encountered by cells within their normal extracellular milieu. Adult MSCs seeded onto these scaffolds can differentiate along multiple lineages [11] Nanofibrous scaffolds by virtue of their nano-scale features also influence cell shape and therefore biologic responses directly. For example primary chondrocytes on nanofibrous scaffolds produce higher levels of cartilage-specific matrix compared to the same cells seeded on micron-scale fibers of the same composition [12]. Nanofibrillar surfaces also control mouse embryonic fibroblast morphology and cytoskeletal organization [13] enhance proliferation and self-renewal of mouse embryonic stem cells [14] and activate cytoskeletal remodeling through the small GTPase Rac [15]. We have recently shown that alignment of this nanofibrous microenvironment can direct actin stress fiber organization in adult human mesenchymal stem cells [16]. This in turn directs the ordered deposition of matrix which in the long term translates to improved construct mechanical properties [17 18 Of particular note and in comparison to traditional pellet culture the aligned topography provided by these organized nanofibrous patterns can foster fibrous over cartilaginous differentiation of MSCs [19]. In addition to these passive cues provided by material microenvironments active mechanical cues likewise exert control over stem cell differentiation. Physical forces applied to MSCs in vitro often carried out via deformation of scaffolds with custom mechanical devices (e.g. [20 21 can increase collagen gene expression by MSCs after one day [22] and improve osteogenesis and mineral deposition MEN2B over several days [23]. On dynamically loaded unpatterned surfaces most cells reorient such that their long axis is perpendicular to the prevailing stain direction [24 25 To force cells to adopt a specific morphology with respect to the applied strain constraints have been applied via aligned microgrooves on elastomeric subtracts produced with soft lithography [24 26 Using such methods Kurpinski and co-workers showed that with dynamic tensile deformation applied in the microgroove direction MSCs increased both proliferation and smooth muscle marker gene expression while decreasing chondrogenic matrix marker expression [27]. Interestingly when strain was applied perpendicular to the cell axis a different set of genes was activated and proliferation rates were no longer altered suggesting that mechanosensing by MSCs is anisotropic (direction dependent). Cells are inextricably linked to their extracellular environment via complex interpenetrating cytoskeletal networks [28 29 These networks provide a rapid and efficient means by which extracellular and intracellular perturbations can be transmitted to cell structures such TR-701 as the nucleus [30-32]. Nuclear shape and deformation in turn correlate with gene expression changes. For example when pre-osteoblastic cells are confined to specific micropatterned geometries an ideal ratio of nuclear area to height promotes collagen gene expression [33]. In tissues and tissue-like engineered constructs nuclear deformation is associated with changes in cellular biosynthetic activities [34 35 In.