The present report is an extension of our preceding publication in Biomaterials (2013) entitled "Effect of RGD nanospacing on differentiation of stem cells." Cell-adhesive peptide arginine-glycine-aspartate (RGD) was nanopatterned on a non-fouling poly(ethylene glycol) (PEG) hydrogel, and mesenchymal stem cells (MSCs) derived from rat bone marrow were cultured on the patterned surfaces at nanospacings from 37 to 124 nm. Cell adhesion parameters such as spreading areas varied with RGD nanospacings significantly. The differences were well observed at both the first and eighth days, which confirmed the persistence of this nanospacing effect on our nanopatterns. The proliferation rate also varied with the nanospacings. Osteogenic and adipogenic inductions were undertaken, and a significant influence of RGD nanospacing on stem cell differentiation was found. The effect on differentiation cannot be simply interpreted by differences in cell adhesion and proliferation. We further calculated the fractions of single, coupled, and multiple cells on those nanopatterns, and ruled out the possibility that the extent of cell-cell contact determined the different differentiation fractions. Accordingly, we reinforced the idea that RGD nanospacing might directly influence stem cell differentiation.
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