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Human bone marrow mesenchymal stem cells (MSCs) cultured on three-dimensional (3D) nanofibrous scaffolds are known to undergo osteogenic differentiation even in the absence of soluble osteoinductive factors. Although this process of differentiation has been attributed to the shape that cells assume on the fibrous scaffolds, it is unclear how constriction of cell shape would contribute to the differentiation phenotype. Here, we quantitatively compared cell and nuclear morphologies of cells cultured on 3D poly(ε-caprolactone) (PCL) nanofibers (NF) and two-dimensional (2D) flat films using confocal fluorescence microscopy. We discovered that while cells on the 2D films exhibited cellular and nuclear morphologies similar to those cultured on tissue culture polystyrene, cells cultured on the 3D NF showed distinct cell and nuclear morphologies, with lower areas and perimeters, but higher aspect ratios. We next …