Role of Microtubules in Osteogenic Differentiation of Mesenchymal Stem Cells on 3D Nanofibrous Scaffolds.

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 tested the effect of treatment of cells with actin-depolymerizing cytochalasin D and microtubule-depolymerizing nocodazole on these morphologies. In both 2D and 3D scaffolds, actin depolymerization brought about gross changes in cell and nuclear morphologies. Remarkably, microtubule depolymerization resulted in a phenotype similar to actin depolymerization in cells cultured on 3D NF alone, indicating a significant role for the microtubule cytoskeleton in the maintenance of cell shape and structure in 3D. The morphological changes of the nucleus that were apparent upon cytoskeletal perturbation were reflected in the organization of heterochromatin in the nucleus, with MSCs on 3D alone exhibiting a differentiation phenotype. Finally, we tested the effect of cytoskeletal depolymerization on mineralization of cells. Again, we observed higher mineralization in cells cultured on 3D NF, which was lost in cells treated with either cytochalasin D or nocodazole. Taken together, our results suggest that both the actin and microtubule cytoskeletons contribute significantly toward maintenance of cell and nuclear shape in cells cultured on 3D scaffolds, and consequently to their osteogenic differentiation.