At the beginning of the project a bibliographic research was performed on the state-of-the-art about the use of benign solvents for the fabrication of scaffolds for tissue engineering and in particular for the combined use with other scaffolds fabrication techniques for the development of multilayered graded scaffolds. After the characterization of the single layer scaffolds obtained from the electrospinning with acetic acid and formic acid, composites fibers were obtained after the addition of bioactive glass (BG) particles in the solution before the electrospinning. BG particles were selected because of their well-known effects on osteogenesis, angiogenesis and their antibacterial activity. For the selection of the polymers, a synthetic polymer, poly(epsilon-caprolactone) (PCL), and a natural polymer, chitosan, and their blends were chosen as biomaterials for the scaffolds fabrication, because of their biocompatibility and documented use for scaffold fabrication. The focus for the replication of subchondral bone was oriented on the development of composites fibers, having a polymer matrix (constituted by PCL and PCL/chitosan blends) with the addition of BG particles having different glass compositions and particles size. Positive results were obtained, confirming the incorporation of the BG particles inside the polymeric matrix and the preservation of the BG bioactivity, confirmed after the immersion of the samples in simulated body fluid (SBF) solution. The mineralization observed on the composite fibers confirmed their suitability in mimicking the subchondral bone side in multilayered scaffolds. For mimicking the cartilage side a polymeric electrospun matrix was selected. Positive results were obtained by using neat PCL microfibers and nanofibers and PCL/chitosan blended nanofibers with acetic acid and formic acid as solvents for this layer. Electrospun fibrous mats could present the disadvantage of lack or reduced cell infiltration inside the scaffolds because of the high density of the fiber nets with scare porosity and pore size not compatible with complete scaffold colonization. For this reason, electrospun PCL fibers with a regular macropattern, improving cell infiltration in the scaffold, were fabricated. This layer was used for the fabrication of stratified multilayered scaffolds. Another typology of multilayered scaffold was fabricated by integrating the electrospinning process with foam replica method and dip-coating. In fact, BG porous scaffolds, used as substrate, resembling the bone side, were fabricated by sponge replica method and reinforced by dip-coating in a solution of PCL in acetic acid. These BG-based scaffolds were used as target for fiber collection during the electrospinning process. PCL monolayer or bilayer of composite and polymers electrospun fibers were deposited on the BG-based scaffolds. Positive results were obtained from the characterization of these samples. In particular, a selective bioactivity was reported on the multilayered electrospun scaffolds, demonstrating high potential for the osteochondral segment regeneration. On the contrary, all the multilayered scaffolds based on BG porous scaffolds showed mineralization even in the neat polymeric layers. Cell biology tests were relevant because they were the focus of the researcher´s training during “BIOeSPUN scaffolds” project, completing the interdisciplinarity of her profile. Positive and promising results were obtained in terms of cell viability and morphology on the seeded multilayered scaffolds.
