## 3 Results and Discussion
### Cell Viability and Proliferation Studies  
When developing materials for biomedical applications, a very important factor to consider is toxicity and this can be followed by using conventional MTS cytotoxicity tests [46]. Figure 7 shows the MTS cytotoxicity test performed on the chitosan films with 15% and 30% of sodium hyaluronate, crosslinked with glutaraldehyde and with different concentrations of PEGDE.  
From Figure 7 it can be clearly seen that the cell viability of the pristine chitosan scaffolds improved with 15% HNa but a slight decrease was observed when the concentration of HNa was increased up to 30%. At low HNa concentrations it is possible that more amino groups from CHT are available while at 30% HNa cell adhesion is reduced due to complex formation and reduction of free NH\({}_{2}\)[47]. This behavior was enhanced when low PEGDE concentrations were used (PEGDE1) suggesting that a more hydrophilic surface improved cell viability. However, the competitive effect between HNa and PEGDE is also clear, i.e., low PEGDE concentrations work better for low HNa concentrations [48].  
For GA crosslinked CHT/HNa films viability was lower than PEGDE crosslinked samples. However, when high HNa concentration were used osteoblast viability increased even when the surface roughness was similar to that of pristine CHT with more available free amino groups.  
[PERSON] et al. reported that the use of low concentrations of glutaraldehyde did not affect cell growth and proliferation. This is consistent with the findings in the present investigation. Despite the fact that osteoblast survival is observed in our samples with GA, they exhibited the lowest values of cell viability for both HNa concentrations (15% and 30%) [16].  
[PERSON] et al. [42] evaluated cell proliferation in chitosan scaffolds with hyaluronic acid. They reported a significant difference in cell behavior on scaffolds and indicated that  
Figure 7: Viability of osteoblast with Chitosan (CHT), Chitosan and HNa (CHT/HNa), glutaraldehyde crosslinked chitosan/HNa (CHT-HNa-GA), PEDGE crosslinked chitosan/HNa (CHT-HNa-PEDGE) at 15% and 30% of HNa.  
hyaluronic acid improves cell proliferation. Those results coincided with previous studies that suggest that chitosan-hyaluronic acid-based matrices (hydrogels, electrospun fibers) provide a favorable surface for cell adhesion and proliferation.  
Cellular Adhesion by Scanning Electron Microscopy (SEM)  
Cellular adhesion was observed by SEM by direct and indirect contact after 48 h (Figure 8). A better interaction and cellular morphology were observed in direct contact, since the cytoplasmic extensions and the organization of the osteoblasts in the form of clusters were observed.  
Chitosan films with 15% on HNa, showed a better adhesion when they were crosslinked with PEGDE; moreover, a better cell morphology was observed in scaffolds crosslinked with PEGDE 1 (low concentrations). The same effects were observed in scaffolds with HNa at 30% with a smaller number of well-shaped cells. Osteoblasts were scarce in CHT/HNa samples crosslinked with glutaraldehyde for both concentrations of HNa. [PERSON] et al. [19], reported that CHT/HA polyelectrolites exhibited a significant positive effect on cell adhesion in agreement with our results.  
According to [PERSON] et al., cells seem to prefer more homogeneous surfaces with smaller pores. We observe this effect in our samples, where all the films have a homogeneous surface [49].  
Our results not only showed that osteoblasts optimally adhered to our CHT/HNa samples exhibiting a good viability, but they also showed that osteoblast proliferated considerably in the PEGDE crosslinked scaffolds. Furthermore, osteoblasts aggregated into nodules consisted of large groups of spherical cells at the beginning and then their cytoskeleton is flattened, which would lead to the synthesis and secretion of components of the autologous matrix [28] and, eventually, to bone formation [31].  
Figure 8: SEM images of osteoblast seeded onto CHT scaffolds on direct and indirect contact.