Topography enhances Runx2 expression in outgrowing cells from iPS cell-derived embryoid bodies

The effect of differing polystyrene substrate topographies on the osteogenic potential of the outgrowing cells (OGCs) formed from mouse-induced pluripotent stem (iPS) cells (miPSCs)-derived embryoid bodies (EBs) was investigated. Polystyrene substrates were sandblasted with 25, 50, and 150 μm aluminum oxide particles to obtain topographies with average Sa values of 0.6, 1.1, and 1.8 μm, respectively. 3D-SEM was used to evaluate substrate's topographies. Examination was done by scanning electron microscopy (SEM), by immunocytofluorescence (ICF) analysis for vinculin, Runx2 and collagen type I, and by quantitative RT-PCR (qRT-PCR) analysis for Runx2 and collagen type I. SEM and ICF analyses revealed that surface roughness caused cells elongation (2, 6, 8, 10 times for the NT, 0.6 μm, 1.1 μm, and 1.8 μm, respectively). Vinculin staining demonstrated how the Sa value affected cellular attachment to the substrate. FA points were randomly distributed on flat surfaces, but rough surfaces resulted in more concentrated FA points on the podia of the cells (11.7, 25.2, 26.7, 16.6 vinculin spots per 20 μm² for the NT, 0.6 μm, 1.1 μm, and 1.8 μm, respectively). qRT-PCR revealed that Runx2 expression was highest on day 16 on surfaces with Sa of 0.6 μm and 1.1 μm. Collagen type I expression increased from day 0 to day 16, no significance was found among the groups. In conclusion, surface topography affects cell shape and expression of early osteogenic potential in OGC, particularly surfaces with Sa values of 0.6 μm and 1.1 μm which showed the highest concentration of FA points on podia. These findings could be utilized in the development of inner surface topographies of scaffolds used with iPSCs. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2288-2296, 2019.