## 4 Discussion
Although in a former study HA was used to promote hMSC chondrogenesis using a polylactide electrospun scaffolds [52], in our experiments the primary role of HA in the nanocomposite fiber scaffold was purely physico-mechanical. The biological results showed a good cell viability and high scaffold surface colonization also in HA/*PCL scaffolds, demonstrated by the intensity and localization of Neutral Red dye. No difference in metabolic activity of the cells and cellularity (i.e., cell number) was detected between hMSC/*PCL and hMSC/HA/*PCL samples after one week. In this latter case, the fibroblast surface antigen resulted well expressed. Differently, collagen type II was not as intensely expressed as in hMSC/*PCL constructs. An in silico tool was used to evaluate the von Mises stress occurring on the scaffold during its motion in the bioreactor, which is the stress experienced by the differentiating cells laying on the nonwoven surface. As a result of the different scaffold properties, in the plain *PCL scaffold, von Mises stress resulted about two-fold higher than that calculated in the HA/*PCL counterpart. We thus concluded that not only a dynamic culture designed for TM cell/scaffold culture is needed, but a sufficient mechanical stress is fundamental to induce collagen type II synthesis by fibroblast-differentiating hMSCs. Many authors have addressedthe influence of dynamic culture on stem cell fate, which has resulted to depend on several geometrical factors of the bioreactor/scaffold system, as a consequence of flow modification within the interspaces, and thus on the cell position and specific exposure to mechanical forces [53]. The key aspect of our device is its capacity of generating two stresses within the TM substitutes: pressure-induced (larger at the center) and hydrodynamic shear stresses (larger at the periphery). Such controlled mechanical stimuli, together with scaffold architectural cues and chemical stimuli experienced by the hMSCs during their maturation have been set-up to guide the TM tissue development from molecular scale (i.e., desired collagen type expression), up to possible microscale (i.e., appropriate spatial orientation of fibrils and fibers) [54]. For our specific system, we modeled a topographic map of the stress, which also varied in time according to the oscillatory motion of the bioreactor holder. This map highlighted areas of higher stress that may have produced circumferential-like aggregations of cells, visible by Neutral Red staining of hMSC/*PCL constructs (Figure 1c,d) and not found in hMSC/HA/*PCL counterpart (Figure 8a,b). This evidence deserves more investigation, as it could also underpin a zonal production of collagens, which may ultimately help in understanding how circular fibers have developed in the native TMs. We demonstrated that in our in vitro system, electrospun *PCL scaffolds provided suitable properties for TM regeneration, in line with other investigators who have highlighted interesting outcomes in vivo using PCL family members. In particular, electrospun PCL/silk fibroin/umbilical cord serum fiber meshes have been reported to enable an efficient in vivo regeneration of subacute TM perforations in guinea pig models [55]. Similarly to PCL, the long biodegradation time shown by *PCL in vivo is expected to allow a sufficient timeframe for TM regeneration [56]. Indeed, residual infections and inflammatory processes connected to eardrum perforations, which usually lead to an aggressive environment for biomaterials, could also be controlled by the application of nanoparticles incorporated into polymer fibers for drug release in the middle up to the inner ear compartments [57; 58]. Replicating a really biomimetic and autologous substitute for the eardrum via a tissue engineering approach will offer deaf people a better performing solution, thus contributing to improving their quality of life.