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

Abstract

Human\nbone marrow mesenchymal stem cells (MSCs) cultured on three-dimensional\n(3D) nanofibrous scaffolds are known to undergo osteogenic differentiation\neven in the absence of soluble osteoinductive factors. Although this\nprocess of differentiation has been attributed to the shape that cells\nassume on the fibrous scaffolds, it is unclear how constriction of\ncell shape would contribute to the differentiation phenotype. Here,\nwe quantitatively compared cell and nuclear morphologies of cells\ncultured on 3D poly­(ε-caprolactone) (PCL) nanofibers (NF) and\ntwo-dimensional (2D) flat films using confocal fluorescence microscopy.\nWe discovered that while cells on the 2D films exhibited cellular\nand nuclear morphologies similar to those cultured on tissue culture\npolystyrene, cells cultured on the 3D NF showed distinct cell and\nnuclear morphologies, with lower areas and perimeters, but higher\naspect ratios. We next tested the effect of treatment of cells with\nactin-depolymerizing cytochalasin D and microtubule-depolymerizing\nnocodazole on these morphologies. In both 2D and 3D scaffolds, actin\ndepolymerization brought about gross changes in cell and nuclear morphologies.\nRemarkably, microtubule depolymerization resulted in a phenotype similar\nto actin depolymerization in cells cultured on 3D NF alone, indicating\na significant role for the microtubule cytoskeleton in the maintenance\nof cell shape and structure in 3D. The morphological changes of the\nnucleus that were apparent upon cytoskeletal perturbation were reflected\nin the organization of heterochromatin in the nucleus, with MSCs on\n3D alone exhibiting a differentiation phenotype. Finally, we tested\nthe effect of cytoskeletal depolymerization on mineralization of cells.\nAgain, we observed higher mineralization in cells cultured on 3D NF,\nwhich was lost in cells treated with either cytochalasin D or nocodazole.\nTaken together, our results suggest that both the actin and microtubule\ncytoskeletons contribute significantly toward maintenance of cell\nand nuclear shape in cells cultured on 3D scaffolds, and consequently\nto their osteogenic differentiation.