## 4 Discussion
Therefore, such a unique tissue characteristic provided by nature to the ear could be entitled with a peculiar acousto-mechanical function and replicating such a structure could provide an optimally engineered replacement. Unfortunately, human TM fibroblasts cannot easily be used for self-transplant procedures due to low availability and resulting tissue morbidity; as such, current in vitro studies mostly rely on culturing animal explants [39]. In contrast, dermal fibroblasts can be easily collected and rapidly overgrow in vitro, giving consistent multilayer structures in post-confluent conditions, by abundantly producing ECM components like collagen types I and III [40]. However, no evidence of controlling other collagen types has been reported for such cultures. In this scenario, stem cells, such as hMSCs, can offer a brilliant alternative, even though a specific differentiation pathway has to be set-up for TM fibroblast differentiation. This study specifically aimed at identifying biochemical, physical, and mechanical signals to induce the differentiation of hMSCs into fibroblasts synthesizing collagen type II, in addition to other collagens found in the eardrum, such as types I, III, and IV, which can spontaneously be secreted by other fibroblasts. A TM-fibroblast differentiating CM was prepared with the purpose of promoting collagen type II expression, while maintaining the spindle-shaped morphotype proper of hMSCs, therefore their fibroblic character. Specifically, a hand-prepared serum-free CM previously used for chondrogenic differentiation of hMSCs [41], was modified by the addition of FBS. It is recognized that serum-free 3D cultures enhance chondrogenic differentiation of hMSCs, with the production of collagen type II [42]. On the other hand, the _lamina propria_ of the _pars tensa_ is made up by fibroblasts, not chondrocytes. FBS was thus used to initiate cellular attachment to the substrate, facilitate cell proliferation and avoid a classic chondrogenic differentiation route by sustaining the fibroblic morphotype [43]. In tissue culture plastics, hMSCs from the bone marrow express collagen type II gene, can synthetize the protein (mainly, tropocollagen II) in low amount, and do express the fibroblast surface antigen, as we also showed in this study (Figures 4d and 5d). To test the efficacy of the differentiating cocktail, the expression of genes associated to the collagens of interest was evaluated by RT-PCR. The results showed that the application of different static and dynamic culture conditions and the administration of the differentiating CM influenced the collagen gene expression. Collage type I gene was less expressed in the samples cultivated in a static way, while collagen type II gene showed high expression in presence of differentiating CM without being significantly affected by culture condition mode. The expression of collagen type IV-\(\alpha\)1 gene was higher in statically differentiated constructs, whereas type IV-\(\alpha\)2 gene showed no statistically significant differences in all evaluated conditions. The interaction with the scaffold is crucial for the cells to synthesize the ECM molecules, since it provides three-dimensional cues for their growth and differentiation. In this study, randomly oriented electrospun fibers based on *PCL were produced to obtain cell/scaffold constructs suitable for TM regeneration. In fact, the electrospinning process allows the fabrication of thin nonwovens made of ultrafine fibers that resemble the architecture of the fibrous ECM components of connective tissues, thus providing suitable physical stimuli for this specific application [27]. *PCL fiber meshes showed a Young's modulus of 3 MPa, whereas in normal TMs, it is found to be higher than 30 MPa, going up to 300 MPa, depending on the radial and circular direction and applied strain rates [44; 45]. It is important to point out that the fully developed TM replacement will be mechanically reinforced by the cell-produced collagens. Therefore, we considered this value an acceptable starting point.