## 3 Results and Discussion
### Surface Properties of Crosslinked CHT
#### 3.2.3 Measurement of Contact Angle  
The contact angle (\(\theta\)c) formed by a drop of water on a flat solid surface is a macroscopic manifestation of surface-water interactions at the molecular level and is used to characterize surface wetting phenomena [44]. The wettability of a surface is determined by the chemistry of the surface and its topography. For an ideal film surface that is flat, smooth, and chemically homogeneous, the contact angle will decrease as the surface polarity increases.  
The contact angle of the surfaces of the crosslinked CHT films with the different content of HNa are shown in Table 5. CHT films showed, in general, water contact angles lower than 72\({}^{\circ}\), which were classified as weakly hydrophilic [37]. The incorporation of HNa and PEGDE reduced the contact angles in water and culture media in agreement with current literature [30]. This may be due to a greater amount of oxygen, given by PEGDE, on the surface of the films. In addition, this effect can also be explained by the fact that chitosan films with hyaluronate possess a complex chemical structure with amide, amine, and carboxyl groups, so the contact angle on a CHT-HNa surface must reflect the presence of these groups. Perhaps, this integration results in increased polarity at the CHT-AH film surface, reflecting as a lower contact angle. When DMEM was used as the sessile drop the contact angle was, in general, lower suggesting also possible interaction of proteins with this surface.  
For GA crosslinked CHT/HNa films there was also a reduction in water contact angle which was more marked when HNa was added at 30%. This implies that GA is a more effective crosslinker for CHT rendering a surface richer in HNa. Improved hydrophilicity when hyaluronate is incorporated into chitosan can have positive effects on cytocompatibility [45].  
[PERSON] et al. [19], measured contact angle with HEPES buffer and acetate buffer, in chitosan/hyaluronic acid polyelectrolyte multilayers, found that the value of contact angle  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & **Ra (nm)** & \multicolumn{5}{c}{**Contact Angle (\({}^{\circ}\))**} \\ \hline
**Films** & & & **H\({}_{2}\)O** & **DMEM** & **H\({}_{2}\)O** & **DMEM** \\ \hline CHT & \multicolumn{2}{c}{\(45.8\pm 5\)} & \multicolumn{2}{c}{\(62.9\pm 3\)} & \multicolumn{2}{c}{\(72\pm 4\)} & \\ \hline  & 15\% & 30\% & 15\% & & 30\% \\ \hline CHT-HNa & 64.7 + 7.3 & \(31.7\pm 2.6\) & \(73^{\circ}\) & \(74^{\circ}\) & \(74^{\circ}\) & \(66^{\circ}\) \\ \hline CHT-HNa-PEGDE1 & 85.3 \(\pm\) 9.6 & 106.2 \(\pm\) 8.9 & \(66^{\circ}\) & \(52^{\circ}\) & \(52^{\circ}\) & \(55^{\circ}\) \\ \hline CHT-HNa-PEGDE2 & 56.8 \(\pm\) 5.8 & 87.8 \(\pm\) 6.9 & \(60^{\circ}\) & \(56^{\circ}\) & \(56^{\circ}\) & \(47^{\circ}\) \\ \hline CHT-HNa-PEGDE3 & 61.2 \(\pm\) 5.2 & 134.3 \(\pm\) 14.2 & \(51^{\circ}\) & \(54^{\circ}\) & \(54^{\circ}\) & \(44^{\circ}\) \\ \hline CHT-HNa-GA & 44.6 + 5.9 & \(52.6\pm 7.2\) & \(68^{\circ}\) & \(42^{\circ}\) & \(42^{\circ}\) & \(18^{\circ}\) \\ \hline \hline \end{tabular}
\end{table}
Table 5: Roughness and contact angle of GA crosslinked and PEGDE crosslinked chitosan/HNa films.  
decreases from 30.2\({}^{\circ}\) to 20.6\({}^{\circ}\) after annealing. So, they conclude that PEMs are hydrophilic despite thermal annealing. [PERSON] et al. [44] reported that surface functionalization with PEG inhibits the adsorption of biomolecules, a property that is due in part to the ability of PEG to participate in interactions of hydrogen bonds with surrounding water molecules, i.e., hydrophilicity of the surface. PEGDE films have a more hydrophilic active layer reflected by a lower contact angle. Hydrogen bond accepting groups (i.e., O atoms) in ethylene glycol monomers enhanced the affinity of water for amino-PEGE surfaces.