# Mesenchymal Stem Cell Differentiation Driven by Osteoinductive Bioactive Nanoscale Topographies
## 4 Discussion  
Reverse micelles of initial block copolymer (BCPs) granted the possibility of creating ordered polymeric arrays with uniformity over large areas (full wafers) which could be used as masks for the patterning of the underlying substrate with high processing reproducibility. The ability of creating organized nanostructure arrays with high throughput is essential for the subsequent utilization of these samples for cell culture. Such characteristics are essential for the use of silicon nanopillars on studies of hMSC differentiation. A more detailed discussion on this point has been previously reported [18]. XPS characterization showed that surface modification process was successful on the topographies tested. Moreover, since identical atomic % were observed on all surfaces after peptide grafting, it could be concluded that the surface chemistry on all patterns was similar. It was essential to confirm that surface chemistries were homogeneous across a sample area and identical between topographical conditions due to the high sensitivity of hMSCs to small alterations of surface chemistry of a material [34,48]. A uniform distribution of the molecules on the surface was expected on both nanopillar arrays. The theoretical size of unfolded BMP-2 mimetic peptide bound to the surface was \(\sim\)6 nm, which is less than a fifth of the lowest separation for the array with reduced periodicity (A, 36 nm separation between features). Hence, separation between nanopillars should not result in a preferential distribution of molecules on the nanopatterns. Previous studies demonstrated that cells adhere only to the top part of the pillars when cultured on nanopillar arrays, thus, making molecular distribution on pillar tops to be much more important compared to the inter-feature concavities [49,50]. The top parts of the pillars are  
Figure 5: Normalized gene expression from hMSCs cultured for 2 weeks in basal medium, taking non-modified flat silicon as control. Tables summarize significant differences between conditions (grey: non-significant, green: significant differences. \({}^{\star}\) represents \(p<0.05\), \({}^{\star\ast}\)\(p<0.01\), \({}^{\star\ast\ast}\)\(p<0.001\)). even more accessible for surface-functionalized as compared to a flat surface due to their three-dimensional profiles. Additionally, curvature on the pillar tops is not prominent, with typical rounding radii of 45 nm, which is approximately 10 times larger than the thickness of the molecular layer thickness. Such curvature should not adversely impact the molecular binding to the surface. hMSCs were cultured for two weeks in basal medium independently of the assay (immunofluorescence or RT-qPCR). Flat and nanopatterned samples were tested right after fabrication or functionalized with RGD or/and BMP-2 mimetic peptide to investigate which could be the best surface for the promotion of osteogenic differentiation of hMSCs. The nanostructure arrays were selected according to results we previously reported [18]. In that study, modulation of osteogenic differentiation of hMSCs by silicon nanopillar arrays was investigated in vitro. A set of six nanopillar arrays of different dimensions was used as substrates for hMSC culture in basal medium. It was observed that osteogenic differentiation was enhanced for cells cultured on nanopillars of large diameter (100 nm) and height (80 nm). Moreover, it was verified that spacing between features needed to be tuned according to the age of the cell donor to further increase the rate of osteodifferentiation. In agreement with our previous study, after two weeks, non-modified Nanoarray A appeared to be the best surface for the control of hMSC commitment and differentiation towards the osteoblastic lineage, as shown by immunofluorescence and RT-qPCR results [18]. When comparing the expression of the different markers from cells cultured on this pattern with the remaining samples (flat or B), significantly higher levels were observed on Nanotopography A.