Induction of osteoblast differentiation phenotype on poly(l-lactic acid) nanofibrous matrix

Thin sheets of nanofibrous (NF) poly(l-lactic acid) (PLLA) matrix were fabricated using a novel phase separation method, mimicking the structure of natural collagen fibers. In this study, the cell morphology, cytoskeleton and adhesion structure, proliferation and differentiation were investigated on NF PLLA matrix using an osteoblast cell line model. Scanning electron microscopy revealed that the MC3T3-E1 cells took a more rounded shape on NF matrix, with abundant interactions with nanofibers. There were no long dense stress fibers or typical focal adhesion structures formed on NF matrix. In consistence with the flat cell morphology and abundant focal adhesions, the cell proliferation was faster on flat PLLA films. With the addition of ascorbic acid (AA), cells were induced to differentiate both on NF matrix and flat films. Cells on NF matrix exhibited an enhanced osteoblast differentiation phenotype, with dramatically higher bone sialoprotein (BSP) gene expression (two orders of magnitude higher) and significantly higher alkaline phosphatase (ALP) activities. Strikingly, even without the addition of AA, thus no natural collagen fibers deposited into the matrix, the BSP gene expression was still highly up-regulated on NF matrix, showing a direct effect of PLLA nanofibers on BSP gene expression. Enhanced BSP gene expression was correlated with the down-regulation of the small GTPase RhoA activities. Inhibition of RhoA effector ROCK induced BSP gene expression of cells in AA-free medium on flat PLLA films. These results suggest that the nanofibers promote the differentiation of osteoblasts likely through RhoA-Rock signaling pathway.