Extracellular matrix (ECM) is a fibrous natural cell environment, possessing complicated micro-and nano- architectures, which provides signaling cues and influences cell behavior. Mimicking this three dimensional environment in vitro is a challenge in developmental and disease biology. Here, suspended multilayer hierarchical nanofiber assemblies fabricated using the non-electrospinning STEP (Spinneret based Tunable Engineered Parameter) fiber manufacturing technique with controlled fiber diameter (microns to less than 100 nm), orientation and spacing in single and multiple layers are demonstrated as biological scaffolds.

Hierarchical nanofiber assemblies were developed to control single cell shape (shape index from 0.15 to 0.57), nuclei shape (shape index 0.75 to 0.99) and focal adhesion cluster length (8-15 micrometer). To further investigate single cell-ECM biophysical interactions, nanofiber nets fused in crisscross patterns were manufactured to measure the "inside out" contractile forces of single mesenchymal stem cells (MSCs). The contractile forces (18-320 nano Newton) were found to scale with fiber structural stiffness (2 -100 nano Newton/micrometer). Cells were observed to shed debris on fibers, which were found to exert forces (15-20 nano Newton). Upon CO? deprivation, cells were observed to monotonically reduce cell spread area and contractile forces. During the apoptotic process, cells exerted both expansive and contractile forces. The platform developed in this study allows a wide parametric investigation of biophysical cues which influence cell behaviors with implications in tissue engineering, developmental biology, and disease biology.