# Poly(vinyl alcohol)/Gelatin Scaffolds Allow Regeneration of Nasal Tissues
## 4 Discussion
In addition to providing a scaffold suitable for diverse nasal tissues, the second key point of this study was to suggest a unique cell source for regenerating those tissues, and in particular, cartilage, pre-vascularized bone and fibrous tissue. We thus proposed the use of MPCs isolated from the human bone marrow, as they are able to give rise to pre-endothelial cells and to MSCs, the latter capable of differentiating into several mesodermal lineages [37; 38; 41]. In a previous work, we tested PVA/G scaffolds for the regeneration auric cartilage, finding that it was promising for elastic cartilage engineering [34]. In fact, we obtained elastic fibers by applying mechanical forces during the chondrogenic differentiation of MSCs. In this study, rMSCs seeded on the PVA/G sponges were differentiated towards the chondrogenic phenotype aimed to obtain a hyaline cartilage model. MSCs proliferated and chondro-differentiated efficiently in PVA/G scaffolds. The obtained results showed that the cells colonized the scaffold also in depth. Moreover, differently from other cell sources, like dental pulp [43], the bone marrow MSCs in this scaffold massively assumed a rounded morphology and acquired the ability to aggregate into clusters within its pores already after 10 days, but mainly after 20 days of chondrogenic differentiation. Differentiated MSCs also gave rise to the production of molecules typical of hyaline cartilage, such as sulphated acidic GAGs and collagen type II. As aggregation into isogenic groups is a hallmark of hyaline cartilage, the positivity to these specific chondrogenic markers indicates that this scaffold is advantageous to support chondrogenic differentiation of bone marrow MSCs [46]. We thereafter used hMSCs to generate a panel of 3D nasal tissue models. Unlike cartilage, bone is vascularized tissue. The key steps towards optimized clinical applications of tissue-engineered bone substitutes rely on neovascularization of the engineered construct after implantation [47]. For this reason, strategies enabling subsequent in vivo vasculogenesis, such as the addition of vascular endothelial cells are being investigated [35]. In the pre-vascular osteogenic model, we aimed to exploit the angiogenic and osteogenic potential of a specific subpopulation of bone marrow cells, recently known as MPCs. In parallel cultures, MPCs were differentiated either into pre-endothelial cells or into MSCs, the latter further seeded on the scaffolds upon osteogenic differentiation. The pre-endothelial cells differentiated in 2D culture were then seeded on the 3D bone model generated in the meantime for a final coculture step, as similarly reported in a previous study [35]. In the obtained construct, the production of bone ECM and the expression of osteogenic markers, such as ALP and OCN, along with the strong immunopositivity for the VEGF receptor Fik-1 highlighted in some cells, suggested that the PVA/G scaffold allowed both osteogenic and endothelial differentiation of MPCs. This evidence is confirmed by the comparison with MPCs differentiated in 2D culture, in which this protein was poorly expressed. On the other hand, PECAM and the phosphorylated form of the Fik-1 receptor, weakly expressed in the 2D culture, were absent in our 3D model, indicating the need to further investigate the role of the PVA/G sponge in the endothelial differentiation of MPCs, due to their early angiogenic potential [38]. This pre-vascularized bone model retraced the findings previously obtained using hUVECs and hMSCs by instead using a single cell source, patient-derived, which better opens up translational perspectives [35]. PVA/G sponges are mechanically soft materials, although the addition of G is able to increase the sponge elastic modulus by virtue of the chemical crosslinking [34,48]. In general, for PVA scaffolds, a low mechanical strength has been reported, which may not be suitable for skeletal bone engineering [49].