## 4 Discussion
### Chemico-Physical Characterization  
The hydration state of a hydrogel is crucial for its effectiveness in terms of its capability to stimulate cell proliferation, absorb fluids, and maintain moisture [34]. The capability of a hydrogel to swell starting from different dehydration percentages is a desirable property in order to potentially use scaffolds at different wound stages depending on the need to keep the underlying tissue more moisturized or, on the opposite hand, to absorb large amounts of exudate while maintaining ambient moisture [34]. After the production/ disinfection process, the scaffolds contained around 90% water, which is considered to be a high water content and is suitable for the biomimetic use of scaffolds directly on tissues. Interestingly, all of the scaffold types could be rehydrated by over 90% starting from hydration levels of19-26%. Furthermore, a three-dimensional architecture could be restored perfectly. High percentages of water (around 80%) could be re-taken up even from harder dehydration conditions (10-11% of hydration), but in this case, it was noticed that the 3D structure was damaged and did not return back to a fully turgid state. This can constitute a limitation in view of a potential scale up of the dressing, for which a fully dehydrated state could be preferrable for packaging.  
Materials that are clinically employed for skin regeneration purposes require a certain elasticity, both to favor their application and their adaptability in situ. For all of the conducted tests, the elastic modulus indicated that the material was significantly more elastic (about ten times higher) than human skin, that is, by about 1 MPa [35].  
Porosity is widely reported to be one out of the five most important requirements for a hydrogel scaffold for tissue regeneration [36]. An accurate analysis of SEM micrographs revealed that an interconnected porous network was present both on the surface and in the inner part of the scaffolds. Interconnected pores separated by thin membranes, as observable in Figure 3, can easily permit gas and small nutrient diffusion. Pores were shown to have dimension ranges compatible with human cell attachment and proliferation [37]. In particular, the cumulative pore size distribution reported in Figure 4 clearly shows that the VitE 1 pores were bigger on an average and presented wider size ranges compared to the VitE 0.1 scaffolds. This phenomenon could be explained by the higher concentration of the material inside the VitE 1 scaffolds. In fact, this means a lower thermic conductivity, and thus, a slower freezing rate. Since it is known in literature that freezing velocity has a direct impact on the formation of ice crystals and on their dimensions [37], provided that the pores are formed by ice formation, it is possible to conclude that an higher concentration of VitE may lead to the formation of slightly bigger pores.  
Three-dimensionally structured chitosan-based hydrogels, as reported in the previously work mentioned [27], might boost human fibroblast proliferation on/through the scaffold compared to bi-dimensional objects of the same material. As a consequence, the maintenance of the three-dimensional structure compared to the CAD model becomes of primary importance for the prediction of cell behavior. Data reported in Section 3.2.3 show evidence that the correspondence between the hydrogel and the model was quite well obtained and also depict the high reproducibility of the technique used as well as its fidelity in creating three-dimensionally structured chitosan-based hydrogels. Moreover, it is reported in the literature that three-dimensional patterned hydrogels with similar holes sizes stimulated angiogenesis and the formation of new blood vessels in vivo [37].