## 4 Discussion  
TM is the anatomic component of the ear, responsible for collecting the sound waves and transmitting them to the auditory bones. The repair and regeneration of the TM, as a consequence of traumas or infective pathologies, is still a subject of investigation. Until now, only a very limited number of studies have developed a straightforward tissue engineering approach for TM regeneration, but rather patches or stem cell injections have widely been promoted for in vivo applications, due to the fact that the TM is easily accessible and the damaged areas may be relatively small [17; 18]. However, if the structural (i.e., collagenous) portion of the TM, i.e., the _lamina propria_ of the _pars tensa_, has not properly healed, as occurs in large perforations, suboptimal hearing is observed [34]. At least two key factors have been identified to tightly link anatomy to physiology in the eardrum [1], and should be considered to design an effective tissue engineering strategy for TM regeneration: (1) the peculiar spatial arrangement of collagen fibers--radial and circular; and (2) the specific collagen types present in the native TM. Mastering both factors would allow a fully biomimetic and functional TM substitute to be generated in vitro, as the final outcome would be consistent with the natural tissue, from supramolecular down to molecular level. In this respect, the specific acousto-mechanical properties of collagen molecules could contribute to an optimal hearing process [35]. The first key factor has been investigated by recent studies, which demonstrated the feasibility of fabricating 3D printed eardrums with specific macroscale anatomic features, replicating some traits of radial and circular collagen pathways [19; 20]. However, a route toward obtaining TM histological similarity in terms of collagen type expression by a cell source has not been reported so far. Our study aimed at modulating collagen type expression using the tissue engineering paradigm to address TM regeneration, thus facing the second key factor. Immunohistochemical studies of the TM showed that, in the _lamina propria_ of the _pars tensa_, the outer radial and inner circular fiber layers are formed of collagen types II and III to a larger extent with respect to the most common collagen type I [1]. The specific role of collagen type II in the eardrum is still unknown. Indeed, in other body domains collagen type II is not abundantly secreted by fibroblasts, but chondrocytes. For example, in tendons, collagen type II is found at the intersectoral zones with cartilage, whereas collagen type I accounts for 60-80% of collagens produced by specialized fibroblasts, called tenocytes [36]. In the cartilaginous tissue, the biomechanical role of collagen type II has been better described and is associated with the high resistance to deformation and fracture \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Collagens** & ***PCL, Static** & ***PCL, Dynamic** & **HA/*PCL, Static** & **HA/*PCL, Dynamic** \\ \hline
**Type I** & +++ & +++ & - & - \\
**Type II** & + & ++ & + & + \\
**Type III** & + & + & - & - \\
**Type IV** & ++/+++ & ++ & - & - \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Semi-quantitative evaluation of the immunopositivity for TM collagens as expressed by different hMSC/scaffold (*PCL or HA/*PCL) constructs after differentiation for seven days in different culture conditions (static or dynamic). by virtue of collagen type II fiber interaction with proteoglycans [37]. Collagen type II fibers in the cartilaginous tissue have lower organization degrees than the ones usually found in type I fibers present in tendons and bone. Conversely, well organized collagen type II fibrils and fibers, as a result of fibroblast synthesis, have solely been found in the TM and in some other tissue structures of the inner ear, such as the vectorial membrane, the basilar membrane and other accessory structures [12; 38].