# Mesenchymal Stem Cell Differentiation Driven by Osteoinductive Bioactive Nanoscale Topographies
## 3 Results
### Immunofluorescence Assays  
After two weeks of culture on the selected nanostructures either plain or functionalized with RGD or/and BMP-2 mimetic peptide, hMSCs were fixed and immunostained to investigate the expression of Runx2 and OPN by immunofluorescence (Figure 4). It was observed that, independent of the surface chemistry (RGD or/and BMP-2 functionalization), Runx2 expression was higher on Nanotopography A (pillars with reduced spacing). Regarding flat silicon surfaces, it was observed that expression of Runx2 could be enhanced if the substrate was functionalized with RGD peptide or, to a lesser extent, co-functionalized with both peptides. Still, for all cases the fluorescence signal detected on \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline  & \multirow{2}{*}{**Bond**} & \multirow{2}{*}{**F**} & \multicolumn{4}{c}{**Atomic \%**} & \multirow{2}{*}{**B**} \\  & & & **Clean** & & **BMP-2** & **Clean** & **BMP-2** & **Clean** & **BMP-2** \\ \hline \multirow{3}{*}{Si} & Si\({}^{0}\) & 50.3 & 39.1 & 41.6 & 33.5 & 45.5 & 32.9 \\  & Si\({}^{0}\)C\({}_{3}\) & 2.1 & 2.3 & 3.4 & 4.1 & 1.8 & 4.0 \\  & SiO\({}_{2}\)C\({}_{2}\), SiO\({}_{3}\)C & 5.6 & 5.2 & 7.7 & 5.2 & 8.3 & 4.6 \\ \hline \multirow{3}{*}{C} & C-C & 10.3 & 16.8 & 14.4 & 19.1 & 12.0 & 19.6 \\  & C-O & 3.2 & 5.3 & 3.3 & 5.1 & 3.0 & 6.1 \\  & COOR & 0.0 & 3.7 & 0.0 & 3.2 & 0.0 & 3.6 \\  & COORH & 0.6 & 0.0 & 0.7 & 0.0 & 0.5 & 0.0 \\ \hline \multirow{3}{*}{N} & NH\({}_{2}\)C, N-C=O & 0.0 & 3.2 & 0.0 & 3.0 & 0.0 & 2.9 \\  & N-C-O\({}_{x}\) & 0.3 & 0.2 & 0.2 & 0.4 & 0.3 & 0.5 \\ \hline \multirow{3}{*}{O} & O-C & 0.4 & 6.5 & 0.3 & 4.7 & 0.0 & 6.0 \\  & O-C & 27.3 & 17.7 & 28.4 & 21.7 & 28.5 & 19.9 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Atomic percentages obtained from deconvolution of high resolution XPS spectra after sample cleaning (clean) and after BMP-2 grafting (BMP-2) for the flat control (F) and the nanostructured Samples A and B. (Atomic % represented as % of total peak area). flat samples was approximately half the signal observed on nanoarrays of Type A. Runx2 expression on bare B nanodopographies was similar to the level observed on plain flat surfaces. The same was noticeable for Surface B grafted with RGD peptide and flat modified with this peptide. Yet, grafting of BMP-2 mimetic peptide or the combination of the two peptides lead to a significant increase in expression of Runx2 on Nanostructures B (2-fold). Runx2 with Silicon with Nanopillars A (diameter 105 \(\pm\) 14 nm, periodicity 141 \(\pm\) 12 nm, height 75 \(\pm\) 6 nm) with or without peptide functionalization is clearly overexpressed in comparison with flat silicon and silicon with Nanopillars B.  
Lastly, N-C=O components could be observed on all samples in C1s and in N1s spectra, further indicating that BMP-2 mimetic peptide was immobilized on the surfaces. Contrarily, OPN expression appeared to be dependent not only on the topography but also strongly on  
Figure 4: hMSCs cultured for 2 weeks on flat silicon (F) and silicon with Nanopillars A and B (Table 2) before and after functionalization with RGD and/or BMP-2 peptides (RGD alone, BMP-2 alone or combined RGD/BMP-2 peptides). Cells were stained for the early osteogenic (Runx2) marker in green, the late osteogenic (OPN) marker in red and cell nucleus in blue. An example is given here with RGD + BMP-2 peptides immobilization (**above**) (scale bar: 40 um). Quantitative analyses of the nuclei immunofluorescence intensity of Runx2 and OPN in hMSCs (**left**). Tables (**right**) summarize significant differences between conditions (grey: non-significant, green: significant differences. \({}^{*}\) represents \(p<0.05\), \({}^{**}\)\(p<0.01\), \({}^{***}\)\(p<0.001\)). surface chemistry, with higher fluorescence signal being detected on non-modified flat and on nanopillars with reduced pitch (Condition A). The change of surface chemistry of these two topographies (flat and Nanoarray A), achieved via peptide grafting induced a decrease in OPN expression to roughly two thirds of the signal on plain surfaces.