## 3 Fn Application and Involvement in Biomaterials and Medical Device Engineering
### Fn in Complex Coted Interfaces  
Surface biofunctionalization can occur via Fn 2D simple coatings, but increasingly, studies have focused on complex interfaces in order to form bioengineered interfaces. In the context of biomedical applications, coatings formed by layer-by-layer (LbL) assemblies are of growing interest due to their high versatility [79]. This technique is based on the alternate deposition of oppositely charged polyelectrolytes [80], which results in the formation of thin coatings, ranging from a few nanometers to a few microns, which are easy to fabricate and can be deposited on any type of surface irrespective of their geometry and chemistry, making them attractive for implant functionalization. Thin films' properties can be easily modulated in terms of thickness, rigidity, and roughness and can be functionalized with bioactive molecules or serve as reservoirs for the controlled delivery of biomolecules [81, 82, 83, 84, 85, 86]. Fn can be considered a polyampholyte with negatively and positively charged patches and an acidic isoelectric point (5.5-6) (Figure 2). It is thus negatively charged at physiological pH and can be used as a polyanion to functionalize the surfaces of polyelectrolyte multilayer films or directly incorporated during film assembly. Polyelectrolyte multilayer films often present low mechanical rigidity, which is not favorable for cell adhesion and spreading. To overcome this drawback, several authors decided to functionalize an LbL film with Fn adsorbed on top of the film. [PERSON] et al. showed that it is possible to adsorb Fn as a top layer on a poly-L-lysine (PLL) (+)/dextran sulfate (\(-\)) nanofilm and that it positively affects cell spreading [87]. They noted that the charge of the terminated layer could influence Fn adsorption, with a higher amount of protein adsorbed on films exhibiting a positively charged terminated layer. Surprisingly, it is also possible to adsorb Fn on poly(sodium 4-styrenesulfonate) (PSS) in a higher amount than on non-functionalized surfaces. The nature of the terminated layer, thus, has an impact on Fn adsorption and could also impact the protein conformation. [PERSON] et al. studied the adsorption of Fn on PSS (\(-\)) and poly(allylamine hydrochloride) (PAH, (+)) thin films. They found that, on PAH-terminated films, the Fn adsorption, density, and thickness are higher than those on a PS-terminated layer. However, integrin-binding sites are more accessible on PSS-terminated thin films, suggesting a potential higher bioactivity [88]. It has been clearly shown that the adsorption of an Fn monolayer on the top of thin films positively influences cell adhesion and spreading [87, 89, 90]; the properties of the underlying thin films (i.e., thickness and roughness) are also of great importance [90]. Recently, hybrid cell adhesive materials (hCAMs) have been developed where a bilayer of Fn and PAH is deposited on top of polyelectrolyte multilayer films. hCAMs allow the long-term anchorage of P19 cells, and pluripotent cells are able to differentiate into neuronal skeletal muscle and cardiac muscle. Their proliferation and differentiation into neuron-like cells have been studied under dielectrophoretic conditions and continuous fluid flow [91]. hCAM constructs have also been used to develop an in vitro model to study tumor-endothelial cell interactions. The co-culture of hepatocarcinoma cells (HepG2) and endothelial cells (HUVECs) on hCAMs shows that HepG2 tends to form 3D structures while endothelial cells form cords [92]. When hCAMs are treated with transglutaminase, known to play a role in the cross-linking of ECM proteins, HepG2G cells change from 3D to 2D morphology, suggesting that Fn remodeling plays a key role in 3D structure morphology formation. Fn has also been directly integrated in polyelectrolyte multilayer films during film assembly in place of an anionic polyelectrolyte in order to generate interfaces biomimetic of the ECM.