Matrices and strategies for complex tissue regeneration

In the field of Tissue Engineering, a new concept has been developed in the last few years. The formation of new tissue induced by a tissue engineered system needs to be accompanied by the achievement of a complete tissue functionality and scaffold properties have to be designed following the principles of biomimetics, i.e. the complexity of the physiological environment has to be translated and reproduced in the cell-scaffold construct. This approach is especially challenging when the interface between two tissues has to be restored. In this case, the scaffold has not only to sustain the regeneration of two different tissues, but also to ensure the regeneration of a functional interfacial zone between them. Therefore, scaffold properties must reflect the complexity of tissue boundary structures, in terms of controlled gradients in morphological, chemical and mechanical properties. The aim of this research work was the application of these advanced principles to the regeneration of the osteochondral defect, which is a degenerative pathology involving both cartilage and bone tissue, whose current treatments are uneffective in the long term. In this work, a multiphasic scaffold for osteochondral Tissue Engineering was produced and characterized. Silk fibroin-based 3D sponges were employed for the chondral and subchondral components for cartilage and bone regeneration, respectively, to exploit the biocompatibility and versatility of silk fibroin in Tissue Engineering applications. For the restoration of a functional interface, a nanometric net was used to separate the two components, in order to allow a dialogue among cells between the two phases thanks to a physiological solute flow, while preventing cell migration towards the chondral site, especially of blood cells which may cause mineralization of the non-calcified cartilage. For the chondral component, two different strategies were explored. First, pure silk fibroin sponges produced by salt leaching were combined to static or dynamic culture conditions to evaluate the chondrogenic potential of adipose- derived stem cells (ASCs). These cells have indeed many advantages for cartilage Tissue Engineering applications, such as abundance, easy accessibility, ability of self-renewal and stability during in vitro culture. The best differentiation of ASCs towards chondrocytes was achieved after 28 days of culture in a static environment and chondrogenic media, in terms of higher chondrogenic gene expression, new cartilage extracellular matrix deposition and increase of compressive mechanical properties. ASC/scaffold constructs were then implanted in vivo in a rat xiphoid critical size defect for 8 weeks and also in this case, the best outcomes in terms of new tissue volume and quality were obtained when static conditions and chondrogenic medium were employed during pre-culture. The aim of the second strategy presented in this work was to modify silk fibroin (SF) sponges with the addition of hyaluronic acid (HA). Besides hyaluronic acid is a natural component of cartilage and contributes to its biomechanics thanks to its ability to retain a remarkable amount of water, it has been shown to modulate chondrocyte phenotype when employed in scaf- folds for cartilage regeneration. Therefore, we exploited its properties producing silk fibroin/hyaluronic acid scaffolds by salt leaching at different HA concentrations, eventually cross-linked by genipin to improve HA retention. SF/HA sponges were completely characterized in terms of physical, chemical and mechanical properties and then used to culture primary chondrocytes in vitro. Results demonstrated that the scaffolds with the highest amount of hyaluronic acid both with and without cross-linking elicited better responses in cartilage cells with respect to pure silk fibroin sponges, in terms of chondrogenic phenotype enhancement and new cartilage extracellular matrix deposition. The nanometric net of the multicomponent scaffold for osteochondral regeneration was produced by electrospinning of poly-d,l-lactid acid/polyethylene glycol (PdlLA/PEG) blends. PdlLA was employed since it is a well-known biocompatible polymer and it is easy to process with this technique, while PEG was added to avoid fiber shrinkage in an aqueous environment. Nets were characterized in terms of morphology and thermal properties, then assembled to a silk fibroin sponge without any modification to their geometry. To preliminarily evaluate the biological properties of PdlLA/PEG electrospun nets, a system to co-culture chondrocytes on scaffolds with net and osteoblasts was designed and validated, so that the biochemical communications between cells could take place through the net fibers. In the future, this system will be employed to evaluate how osteoblasts can improve chondrocyte response in terms of phenotype maintenance and new cartilage tissue deposition. The results reported in this research work will be the basis for the final design of a multicomponent scaffold which comprises the best outcomes obtained. Hence, SF/HA scaffolds which elicited the best responses on chondrocytes will be used in combination with ASCs, in order to verify their potential to sus- tain chondrogenesis in vitro. Then, they will be assembled to the nanometric net and, before moving to an appropriate in vivo study, the co-culture system will be employed to assess how the cellular dialogue with osteoblasts can have beneficial effects on the chondrogenic differentiation of adipose-derived stem cells.