Natural-based hydrogel as a 3D platform for cell co-culture towards cartilage regeneration.

Articular cartilage (AC) is an avascularised and highly specialized tissue, populated by only one cell type, the chondrocytes. Behind its simple and homogenous appearance, this tissue hides a heterogeneous composition with a high level of organisation and biomechanical properties that makes it difficult to be repaired when there are failures. Hydrogels, 3D polymeric networks, are identified as ideal structures for their ability to recapitulate the cartilage solid/liquid ratio and to provide an appropriate niche, scaffolding and environmental bioactive signals for cells. Several natural polymers have been analysed for hydrogel manufacturing. Among them, chitosan, a biocompatible and biodegradable polysaccharide, has shown promising features for reproducing cartilage Extracellular Matrix (ECM) for its structural similarity to cartilage glycosaminoglycans (GAGs) and its mechanical properties, inducing mesenchymal stem cell (MSCs) chondrogenic differentiation. Another regenerative approach consists into autologous MSCs harvesting, expansion and injection, as a strategy to regenerate cartilage, due to their reliable potential to differentiate into soft tissue when subjected to specific environmental stimuli. The aim of this work was the combination of tissue engineering and stem-cell based therapy to obtain an innovative 3D platform for cell co-culture towards cartilage. Particularly this thesis investigated the manufacturing and characterisation (physico-chemical, mechanical and biological) of a thermosensitive chitosan (CH)-based hydrogel, ionically crosslinked with the addition of β–glycerophosphate (BGP), a chemical compound used in the body as a minerals transporter, able to cause the CH sol/gel transition at physiological conditions. Rheological and gelification analysis showed that the sol/gel transition of the CH/BGP hydrogel occurred in the range of 31-33°C within 5±1 minutes. Mechanical tests evidenced a compressive and equilibrium Young’s modulus of respectively 37±4kPa and 17.0±0.8kPa. Furthermore, the Scanning Electron Microscopy analysis demonstrated that the gel possess an excellent interconnected porosity with pores diameter of 0-30µm, suitable for cells attachment, intracellular signalling and nutrients diffusion. In fact, nutrients diffusion and release test was successful throughout the structure. Then, the Live/Dead and Prestoblue assays confirmed that the hydrogel exhibited an ideal environment for MSCs viability and metabolic activity. Finally, it was studied the influence of a close-contact co-culture of chondrocytes (hAChs) spheroid on MSCs-laden gel on the formation of new cartilage. Immunofluorescence and histological analysis assessed up to 28 days of co-culture, revealed the production of type II collagen and GAGs, compared with controls (hAChs spheroid seeded on the gel and MSCs spheroid seeded on a MSCs-laded gel in chondrocytes proliferation medium or chondrogenic medium). Also, CD44 expression, chondrogenic marker, showed higher ability of MSCs to differentiate when co-cultured with hAChs. Further investigation on CH/BGP system will be done for osteoarthritis (OA) disease treatment, the most common form of joint disease nowadays, applied as injectable thermo-sensitive gel for minimal invasive articular joints surgery as well as for the development of 3D bioprinted in vitro models simulating OA or healthy cartilage tissue for new therapeutic drugs screening.