**3. GO effects on cell adhesion**

In general, it has been shown that the addition of GO favors the interaction between a cellular component and a material substrate, thus ensuring a positive effect on cell adhesion. Several studies [32, 33, 1] have demonstrated that bone marrow mesenchymal stem cells (BM-MSCs) developed a fusiform phenotype with multiple elongations and focal adhesion points in contact with graphene derivatives. These observations support the idea that GO favors cytoskeleton development and enhances cell adhesion to the material that contains GO. Experimental conditions used for 3D scaffolds based on chitosan ± GO or nylon ± GO [34, 2, 6] also concluded that osteoblasts or preosteoblasts adhered better in the presence of GO to the substrate materials. The mechanism underlying GO enhancement of cell adhesion has not been elucidated yet, but Kim et al. [35] suggested that the initiation of focal adhesions is in direct correlation with the nanotopography conditioned by GO.

From our experience, GO also induced a positive effect on murine preosteoblasts adhesion to polysulfone/GO biofilms [25]. A more developed F-actin cytoskeleton has been identified in the presence of 3 wt% GO by confocal microscopy, as compared to the cell cytoskeleton observed for pure polysulfone or plysulfone with 0.5–1 wt% GO addition.

To support this hypothesis, a substrate based on collagen and GO was developed and tested together with rat BM-MSCs for bioactivity in terms of cell viability, cell adhesion, and cell differentiation to bone cells [36]. An obvious dependency of F-actin fiber distribution with the GO content in the biomaterial was reported in this case, confirming our observations.

Other studies [37] described an increased cell adhesion when using GO in conjunction with fibronectin and titanium substrates. In this case, adhesion was evaluated by looking at focal adhesion molecules expression and localization. Vinculin was found to be highly active in the central and peripheral contact area of the cells cultivated in contact with fibronectin and GO.

Good adhesion of cells to their substrate is crucial for cellular processes such as survival, growth, and activation of molecular pathways involved in proliferation. In particular, it has been shown several times that adhesion to the material is essential to induce the molecular program underlying osteogenic differentiation and maturation to functional osteoblasts and osteocytes capable to produce bone-specific extracellular matrix.
