## 3 Results and Discussion
### Surface Properties of Crosslinked CHT
#### 3.2.2 Surface Topography by AFM  
Atomic force microscopy was used to assess the surface topography of sodium hyalurone chitosan films crosslinked with glutaraldehyde and PEGDE at different concentrations. Figure 6 shows the 3D topography while Table 5 presents the corresponding roughness. The addition of 15% HNa to uncrosslinked CHT increased surface roughness (64.7 nm) but reduce it (31.7 nm) when 30% HNa was used. Crosslinking with PEGDE or GA reduced surface roughness for CHT/HNa 15% but tend to increase it for CHT/HNa 30%.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & **HNa** & **\%C** & **\%O** & **\%N** & **\%Na** & **\%Si** & **\%Ca** \\ \hline CHT & \multicolumn{3}{c}{70.13 \(\pm\) 1} & 22.23 \(\pm\) 2 & 4.64 \(\pm\) 0.6 & - & 1.27 \(\pm\) 1 & 1.74 \(\pm\) 0.7 \\ CHT/HNa & 15\% & 69.14 \(\pm\) 3 & 24.67 \(\pm\) 2 & 4.79 \(\pm\) 2 & 0.27 \(\pm\) 0.2 & 0.88 \(\pm\) 0.8 & 0.25 \(\pm\) 0.1 \\  & 30\% & 76.6 \(\pm\) 5 & 18.4 \(\pm\) 1 & 3 \(\pm\) 0.2 & 1.26 \(\pm\) 0.5 & 0.74 \(\pm\) 0.3 & - \\ CHT-PEGDE1 & 15\% & 76.2 \(\pm\) 2 & 20.56 \(\pm\) 1 & 2.72 \(\pm\) 0.9 & 0.52 \(\pm\) 0.4 & - & - \\  & 30\% & 74.93 \(\pm\) 4 & 20.11 \(\pm\) 2 & 3.21 \(\pm\) 0.8 & 0.73 \(\pm\) 0.1 & 1.02 \(\pm\) 0.9 & - \\ CHT-PEGDE2 & 15\% & 69.3 \(\pm\) 1 & 25.67 \(\pm\) 3 & 3.7 \(\pm\) 0.9 & 0.8 \(\pm\) 0.2 & - & 0.55 \(\pm\) 0.2 \\  & 30\% & 75.22 \(\pm\) 2 & 22 \(\pm\) 1 & 1.85 \(\pm\) 1 & 0.93 \(\pm\) 0.3 & - & - \\ CHT-PEG23 & 15\% & 66.6 \(\pm\) 5 & 27.8 \(\pm\) 3 & 4.10 \(\pm\) 1 & 0.83 \(\pm\) 0.2 & 1.27 \(\pm\) 1 & - \\  & 30\% & 74 \(\pm\) 3 & 20 \(\pm\) 2 & 3.9 \(\pm\) 1 & 0.96 \(\pm\) 0.1 & 0.79 \(\pm\) 0.3 & 0.35 \(\pm\) 0.5 \\ CHT-GA & 15\% & 75.5 \(\pm\) 1 & 21.2 \(\pm\) 2 & 2.62 \(\pm\) 0.8 & 0.68 \(\pm\) 0.2 & - & - \\  & 30\% & 75 \(\pm\) 2 & 22 \(\pm\) 3 & 3 \(\pm\) 1 & - & - & - \\ \hline \hline \end{tabular}
\end{table}
Table 4: XPS elemental composition of chitosan/HNa films crosslinked with GA and PEGDE.  
Figure 6: 3D topography of GA crosslinked and PEGDE crosslinked chitosan/HNa films. HNa 15% (Top, **a**-**e**) and HNa 30% (bottom, **f**-**j**); (**a**) CHT/HNa15, (**b**) CHT/HNa15%/PEDGE1, (**c**) CHT/HNa15%/PEDGE2, (**d**) CHT/HNa15%/PEDGE3, (**e**) CHT/HNa15%/GA, (**f**) CHT/HNa30%, (**g**) CHT/HNa30%/PEDGE1, (**h**) CHT/HNa30%/PEDGE2, (**i**) CHT/HNa30%/GA.  
Assessment of topography by AFM is an important parameter, since it is known that cells tend to adhere to rougher surfaces features at the micro and nanoscale [19]. [PERSON] pointed out that the repulsion forces and/or the electrostatic interactions between the components of this type of mixtures can lead to an increase in the size of the microdomain. These interactions play an important role in the structure and properties of binary and ternary polymer blends. The biopolymers used, that is, hyaluronic acid and chitosan are polyelectrolytes, in which the properties are strongly associated with the electrostatic interactions that determine the shape of the macromolecule. In the aqueous solution of a polyelectrolyte, the macromolecules are stretched due to electrostatic repulsive forces between charges on the functional groups. Therefore, in these mixtures, interactions by hydrogen bonds and repulsive forces mainly predominate. These factors can lead to decrease surface homogeneity and increased roughness as in these compositions [43]. Furthermore, it is possible that phase separation take place due to the slow acetic acid evaporation. This was only perceived by AFM in CHT-HNa films crosslinked as some HNa islands can be observed in CHT/HNa 15% PEGDE1 films.