## 4 Discussion
HMSCs were cultured on *PCL fiber meshes in static and dynamic environments, including undifferentiated and differentiated modes. The cells were viable, colonized uniformly the scaffold surface in any conditions, as confirmed by the Neutral Red assay, and adhered to the scaffold surface showing a remarkable expression of \(f\)-actin by the constructs under differentiation. This finding supported the role of the selected CM as a whole, and not only of FBS, which is present in both growth and differentiating CM, towards the development of a mature fibroblast morphotype. The different samples showed a diverse morphology of _f_-actin filaments, appearing as parallel-stretched in statically differentiated, bundle-like in dynamically differentiated and disordered in the undifferentiated dynamic constructs. The difference in _f_-actin cytoskeleton between the statically and dynamically cultured samples was attributed to the dynamic forces present in the bioreactor. Regulation of cytoskeletal proteins, such as _f_-actin, enables physical cell processes, including cell shape and differentiation [46], which are greatly involved in the fibroblast-chondrocyte transformation [44]. The expression at a protein level of both differentiation markers, fibrobl surface antigen and collagen type II, evaluated via immunohistochemistry, was also affected by the different culture conditions. The intensity of the fibrobl surface antigen increased due to the differentiation cocktail and was further enhanced by the application of mechanical stimuli. Moreover, higher expression of collagen type II, with respect to other culture conditions, was only observed in the hMSC/*PCL constructs that underwent bioreactor culture, which ultimately resembled the expression found in human TM specimen. At a protein level, the expression of collagen types I and III was not modulated by the dynamic culture, resulting as intense as in static culture condition. Interestingly, in the differentiated constructs, collagen type IV localization resembled the one of the basal lamina, but it was more intensely expressed in statically cultured hMSC/*PCL constructs. This finding is in line with collagen type IV-\(\alpha\)1 expression detected at a gene level. Several mechanisms regulate the expression of collagen type VI in fibroblasts, with respect to collagen types I and III. Collagen type IV expression has been indeed correlated to cell shape changes involving collagen VI as a cell-binding protein [47]. Altogether, these outcomes highlighted that by the use of *PCL electrospun scaffold, a properly supplemented CM and a TM bioreactor, it is possible to modulate collagen type II expression by fibroblast-differentiated hMSCs, which still produced other collagen types, such as types I, III and IV. In order to better understand the cell response to the mechanical stimuli applied by the bioreactor, we produced a similar scaffold still based on *PCL, having slightly different physico-mechanical characteristics. This scaffold was obtained by adding HA nanoparticles to the polymer solution to be electrospun, which resulted in a lower density and lower elastic modulus (1.39 MPa), than those measured in the plain *PCL scaffold. Alteration of polymer solution parameters has a direct effect on fiber size, which has been found to result in a different porosity, thus affecting the nonwoven density [48; 49]. The decreased Young's modulus is suggestive that, in our experimental setting, due to insufficient interfacial properties, HA acted as a defect for the *PCL fibers, and not as a reinforcement, as reported in other nano-HA/PCL studies [22; 50], thus reducing the stiffness of the nanocomposite [51]. It has to be considered that, for the same molecular weight, a star-branched polymer, like *PCL, accounts for many shorter polymer chains than its linear counterpart [23; 24]. As a consequence, the polymer interaction with nanosized fillers could be reduced under mechanical loading. HA is naturally found in human body, so it is cytocompatible.