Design and manufacturing of bioprinted gellan gum-based constructs representative of the articular cartilage

Articular cartilage (AC) is a highly specialized tissue which exhibit topographical heterogeneity in terms of matrix composition and mechanical properties. Due to its avascular nature AC shows limited regenerative ability, therefore representing an excellent subject for tissue engineering (TE). Particularly, bioprinting is an emerging additive manufacturing technology that has already demonstrated its potential use in regenerative medicine and cartilage TE. It allows to recapitulate the tissues microstructure by a controlled deposition of “bioinks”, suspensions of cells alone or encapsulated in biomaterials. As cells source, mesenchymal stem cells and chondrocytes, both naturally found in AC, are mainly selected. Hydrogels are largely used as biomaterials for their ability to resemble soft tissues extracellular matrix (ECM), providing an ideal micro-environment for the embedded cells survival, proliferation and differentiation. Hydrogels are produced from synthetic and natural polymers, including gellan gum (GG), a biocompatible polysaccharide that has gained interest in cartilage TE because of its structural similarity to cartilage glycosaminoglycans (GAGs) and chondrogenic potential. The aim of this work was the design and manufacturing of 3D constructs mimicking AC by extrusion bioprinting. Particularly, this thesis objectives (OBJ) were: the synthesis and characterization (physico-chemical, morphological, mechanical) of methacrylated GG-based hydrogels subjected to a dual physical and photo-chemical crosslinking (OBJ1); the subsequent biofabrication via Rokit INVIVO bioprinter of in vitro constructs (OBJ2) and biological characterization of cell-laden constructs in terms of cells viability and AC tissue formation (OBJ3). The final stage of this work dealt with the manufacturing of osteoarthritis (OA) in vitro models, via culturing healthy models in cytokine-enriched culture medium, for future analysis on novel OA therapeutic treatments. Firstly, the success of GG methacrylate (GGMA) synthesis was demonstrated through FTIR and XPS analysis. Then, 4 photo-curable hydrogels were prepared: pure GGMA 2% w/v (GG2) and 3% w/v (GG3), and GGMA (respectively 2% w/v and 0.75% w/v) combined with 5% w/v manuka honey (GG/MH) and 10% w/v gelatin (GG/GEL). Gelation analysis at room temperature showed that GG3 and GG/GEL underwent sol-gel transition in ~1 minute, while GG2 and GG/MH in ~3 minute. Water uptake (WU) analysis demonstrated the strong hydrophilic nature of these hydrogels, reaching WU values up to ~1700%. Morphological analysis evidenced that they had an interconnected porous morphology with a mean pore diameter in the range 100-200 μm, suitable for AC applications. Similarly, mechanical analysis showed that hydrogels had a compressive Young’s modulus between ~25 and ~16 kPa, comparable to other natural hydrogels found in literature. GG2 and GG/MH hydrogels were selected as bioinks encapsulating human TERT immortalised stem cells differentiated into chondrocytes (Y201-C; 7x106 cells/ml). The double-crosslinked bioinks were successfully printed into stable constructs. Live/Dead assay demonstrated high cell viability for both bioprinted constructs. The GAGs quantification assay showed that Y201-C GAGs production increased over time in both hydrogels. Finally, scanning electron microscopy analysis showed that cells exhibited a typical chondrocytes rounded-shaped morphology and tended to aggregate in both healthy and OA GG2 constructs .