## 4 Discussion  
### Stability Evaluation of VitE through Scaffold Production and Disinfection  
This work aimed to set up a scaffold formulation procedure through 3D printing in order to obtain three-dimensionally structured chitosan hydrogels loaded with the antioxidant VitE at defined dosages. Liquid formulations based on chitosan were already successfully employed for the production of 3D hydrogels that resulted in hydrogels that are potentially employable for skin tissue regeneration purposes from in vitro and in vivo tests [27]. The modification of the material used as ink for 3D printing by its enrichment with biologically active compounds, such as supplementation with VitE, might ameliorate the general effectiveness of the scaffold by acting on multiple fronts. Moreover, the intrinsic antimicrobial activity of chitosan-based systems represents a desirable feature for prototypes of devices that can be easily contaminated in clinical settings by multi-drug resistant bacteria.  
The preparation of dispersions intended for pharmaceutical purposes generally implies the conduction of investigations on their physical and chemical stability over time. In particular, the dispersions developed as inks in this work were conceived to be prepared by avoiding the use of surfactants as stabilizing agents since the majority of them often show toxic effects when in contact with human cells [33]. Gel viscosity was the only element counteracting the coalescence of the droplets: for this reason, the developed materials were only intended for use as pre-formulations for scaffold production through 3D printing to be used within 24 h of their preparation.  
The scaffold production process implies the exposition of VitE to strong acidic and alkaline environments: these potentially critical chemical conditions needed to be investigated before its inclusion in the chitosan inks. In particular, acid degradation of VitE was assayed in order to evaluate its compatibility with the acidic chitosan solution. The obtained results indicated that VitE was chemically stable over 24 h even at a pH lower (pH 2.4) than the pH of the chitosan solution (pH 5). Afterwards, alkaline degradation tests showed that VitE was stable for up to one hour after exposure to ammonia vapors followed by a very fast degradation: for this reason, 60 min was selected as the maximum gelation time. The chosen time had already been demonstrated to be sufficient in order to obtain reproducible chitosan hydrogels with the right properties to be employed as scaffolds for soft tissue regeneration (i.e., biocompatibility, water content, elastic modulus) [32].  
The overall stability and total drug content in the ink formulations of VitE 1 and VitE 0.1 were evaluated over a period of 48 h to ensure the physical stability and the homogeneity of the dispersion for a sufficient time for usage. VitE was homogeneously dispersed in the ink for 48 h in both formulations, and the VitE content was homogeneous in the scaffolds after the disinfection process.  
Disinfection was also taken into consideration as a possible passage affecting the VitE content of the scaffolds. Due to the sensitivity of VitE to light, common sterilization under UV light could not be exploited, so disinfection using ethanol aqueous solutions was explored. VitE, as is the case with most oils, is soluble in alcohols, and given this fact, VitE content could be significantly lost. Stability tests demonstrated that after one hour, drug loss was not statistically significant compared to the drug content at time 0.