# Mesenchymal Stem Cell Differentiation Driven by Osteoinductive Bioactive Nanoscale Topographies
## 3 Results  
### Preparation of Bioactive Nanostructured Samples  
Silicon nanopillars were fabricated on full wafers with high uniformity and reproducibility. Such characteristics were made possible due to the ability of PS-b-P2 VP to self-assemble into polymeric colloids able to further organize into hexagonally distributed templates, as reported by [PERSON] et al. (2018). Solutions of reverse micelles were spin-coated on the substrates at two distinct speeds to prepare polymeric templates of identical diameter, but different periodicity, as summarized in Table 2. The SiO\({}_{2}\) thin film was used as an intermediate mask to improve the geometric characteristics of the nanopillars, given its superior selectivity for Si etching than the initial block copolymer (BCP) film, while preserving the dimensions of the initial reverse micelles. SEM characterization (Figure 2) demonstrated that feature heights were close to the 80 nm targeted (Table 2). The success of the surface modification was confirmed by XPS after each step on flat silicon substrates (Figures 1 and 3) and extrapolated to the nanostructured samples, considering the chemical composition observed before and after peptide grafting. High resolution spectra for C1s and N1s at each step of surface functionalization are represented in Figure 3.  
Silicon substrates characterized before functionalization exhibited high silicon (56.4%) and oxygen (30.8%) percentages, characteristic of the native silicon oxide layer of the substrates. A slight carbon contamination (12.6%) is impossible to avoid, even if the samples were only exposed to air during mounting on XPS sample holder. Still, this value was within the same set of values reported in previous studies, therefore being in an acceptable range [2,27,35]. Nitrogen content was 0.2%, a value corresponding to measurements at the detection limits of the XPS system. After silanization, XPS surveys showed a decrease in silicon content (to 47.8%), a significant increase in carbon content (to 23.0%) and the appearance of nitrogen (1.7%) associated with the formation of an APTES layer on the surface. High resolution C1s spectrum indicated an increase in C-C bonds compared with the clean substrate. NH\({}_{2}\)-C bonds were observed in N1s high resolution spectrum, confirming the existence of the silane layer on the treated samples. The slight increase of oxygen (to 29.3%) after reaction of the amine-terminated surfaces with SMP  
Figure 3: Deconvolution of high resolution XPS spectra of C1s (**a**) and N1s (**b**) at each step of surface functionalization, namely, Si (control), APTES, SMP followed by grafting of RGD or BMP-2 on flat silicon with values as shown in Figure 1.  
indicates the presence of the crosslinker on the surfaces. Nitrogen remained constant after binding of SMP, which can be a consequence of the existence of only one nitrogen atom in a SMP molecule, which is not sufficient to contribute to a change in the overall percentages of elemental composition. A minor decrease in carbon content was verified, even if each crosslinker molecule has seven carbon atoms. This fact may be related with a reduced carbon contamination on the new surfaces than on silanized surfaces. Finally, peptides were bound to the maleimide group of SMP crosslinker via their cysteine. The significant decrease on silicon content observed after BMP-2 binding can be associated with the large dimensions of this molecule, preventing the possibility of interactions of the X-rays with the silicon substrate. An increase in carbon (to 27.4%) and nitrogen content (to 3.7%) are also linked to the immobilization of the mimetic peptide which is constituted by a large number of carbon and nitrogen atoms. Additionally, high resolution C1s spectra shows an increase in C-C bonds. The significant increase of N-C=O alongside with the appearance of N-C=Ox of higher energy in the high resolution N1s spectrum, further confirms the immobilization of the BMP-2 mimetic peptide on the surface. A similar tendency was detected after the grafting of RGD peptide.