# Mesenchymal Stem Cell Differentiation Driven by Osteoinductive Bioactive Nanoscale Topographies
Molecules of varied sizes, ranging from full-length ECM proteins to short linear peptides have been investigated as possible ways of assigning bioactivity to a material surface [4; 19; 20; 21; 22; 23; 24; 25]. Although the use of full-length ECM proteins has proven to be a successful way of controlling cell behavior on bioactive materials, their use is hindered due to intrinsic limitations (e.g., poor stability, safety concerns) [19]. To overcome these shortcomings, synthetic peptides encompassing only the amino acids (aa) necessary to support a particular biological activity have been investigated [20; 26]. Mimetic peptides can be synthesized with high purity, lower costs, and specific active sites can be engineered in a controlled way [19]. Moreover, contrary to proteins, conformation and density of short molecules can be controlled when bound to a material [19]. The most representative motif used for the improvement of cell adhesion is the sequence of a arginine-glycine-asparatic (RGD), which in vivo mediates the binding of ECM proteins (e.g., fibronectin) to transmembrane integrin receptors [27; 28; 29]. The growth factors most commonly used for the enhancement of osteogenic differentiation of hMSCs are bone morphogenetic proteins (BMPs), particularly BMP-2 [30; 31]. Most studies take advantage of only the sequence responsible for the osteogenic activity of this molecule to functionalize biomaterials for bone tissue engineering applications [19; 32; 33]. The combination of a peptide promoting cell adhesion with one promoting cell differentiation for the co-functionalization of a biomaterial has been reported to further enhance differentiation when compared with the grafting of only one peptide sequence, such as a BMP-2 mimetic peptide [34; 35; 36; 37; 38]. Several studies can be found reporting also synergistic effects of combining nanodopographies with chemical cues on osteogenic differentiation of hMSCs or osteoblast progenitors [39; 40; 41]. Guided by these considerations, this study aims at investigating osteogenic differentiation of hMSCs cultured on Si nanopillar arrays functionalized with RGD peptide and/or a BMP-2 mimetic peptide and comparing it with the hMSC response on bare un-functionalized counterparts. The peptide immobilization strategy involves the grafting of an amino functional organosilane to the silicon surface and changing the terminal amine to a maleimide-functional surface by coupling a heterobifunctional crosslinker. The maleimideterminated surfaces then are coupled with a peptide through the thiol group on the terminal cysteine of the peptide. Geometry of the arrays was defined according to [PERSON] et al. [18]. The prepared surfaces were carefully characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). To evaluate to which extent hMSC differentiation was promoted, expression of early (runt-related transcription factor 2, Runx2, and collagen type I, Col1A1) and late (osteopontin, OPN, and osteoclast, OCN) osteogenic differentiation markers [34, 36] was investigated by immunofluorescence and quantitative polymerase chain reaction (RT-qPCR).