# Mesenchymal Stem Cell Differentiation Driven by Osteoinductive Bioactive Nanoscale Topographies
## 3 Results
### RT-qPCR Assays  
After two weeks of cell culture, total RNA was extracted, and RT-qPCR was performed to investigate the expression of the selected markers. Considering the large number of conditions to be compared, for each gene, results were normalized to a flat, bare silicon control. Hence, differences between nanotopographies and/or surface modifications can be easily distinguished. These results are summarized in Figure 5. It is interesting to note that similar levels of gene expression could be obtained on all topographies when RGD peptide and BMP-2 mimetic peptide were co-immobilized on the surfaces, and that such level of expression was the maximum observed. For the case of non-functionalized surfaces, Nanostructures A appeared to be the most relevant for promotion of osteogenic differentiation of hMSCs, as the highest expression levels for the genes studied was observed for this pattern. In particular, the expression of the three markers on nanopillars of Type A was significantly higher than their expression on flat substrates (as high as four-fold in the case of Runx2). Functionalization with RGD peptide caused an increase in expression, especially of the early differentiation marker Runx2. COL1A1 and OCN levels suffered a significant increase on flat surfaces after functionalization. Yet, for the other topographies, OCN expression did not change significantly.  
BMP-2 mimetic peptide grafting had distinct effects on gene expression on each topology. Regarding Runx2 expression, it caused a significant increase in expression for cells cultured on flat samples (comparing with no modification or grafting of RGD peptide). However, Runx2 expression on Nanostructures A was similar to the one observed after functionalization with RGD peptide. Oppositely, BMP-2 functionalization on B samples lead to a significant decrease of Runx2 expression compared with RGD functionalization. The impact of BMP-2 on the remaining markers was rather different. Expression of COL1A1 and OCN was not as increased on all topographies as observed for Runx2 after surface functionalization with BMP-2 mimetic peptide. COL1A1 expression on cells cultured on A structures with BMP-2 peptide was even significantly lower than Bare A, or A grafted with RGD peptide. Oppositely, a significant increase in expression of COL1A1 was observed on B topographies.  
Finally, co-immobilization of the two molecules had a positive impact on all topographies. Concerning flat surfaces, a significant increase on expression of all markers was noticeable when comparing with bare surfaces (14-fold for Runx2, 1.5-fold for COL1A1, 2-fold for OCN). However, gene expression of flat surfaces grafted with RGD and/or BMP-2 mimetic peptide did not show significant differences between themselves. The same tendency was observed for Nanotopography B with the diameter 104 \(\pm\) 13 nm, the periodicity 201 \(\pm\) 23 nm and height 82 \(\pm\) 6 nm. Contrarily, gene expression of cells cultured on Topography A (diameter 105 \(\pm\) 14 nm, periodicity 141 \(\pm\) 12 nm, height 75 \(\pm\) 6 nm) benefited from the co-immobilization of peptides for the enhancement of osteogenic differentiation. Runx2 level for this last functionalization was 3.5 times higher than for non-modified surface, and 1.5 times higher than for the nanostructures functionalized with only one peptide (RGD or BMP-2). COL1A1 expression was similar to the levels observed on plain and RGD-grafted Nanostructures A. Conversely, OCN expression was significantly higher than non-modified nanoarrays for all topographies.