Development and validation of human in vitro articular cartilage models

Abstract

Osteoarthritis (OA) is a multifactorial disease characterised by the whole joint degeneration. The low availability, reproducibility, and reliability of early-stage in vitro disease models are currently limiting research into novel treatments. The aim of this PhD project was to develop new in vitro models and evaluate their effectiveness. Firstly, a scaffold-free spheroids-based model of Articular Cartilage (AC) was developed, taking inspiration from the clinically approved Chondrosphere® technique. This approach utilised a bankable cell type (immortalised mesenchymal stem cells differentiated in chondrocytes (Y201-C)) and produced a standardisable and reproducible AC-like construct, showing high expression of chondrogenic markers and AC matrix production. By optimising a cocktail of proinflammatory mediators (Interleukin-1β, 6 and TNF-α) the main features of OA pathogenesis at early-medium disease stage could be introduced. The obtained in vitro OA model was used as platform for evaluating a novel microRNA-based polyplex treatment, showing potential for further therapeutics testing. The main limitation of scaffold-free models is to adequately recapitulate the heterogeneous composition and high level of AC zonal organisation, and the influence of subchondral bone (SB) in OA pathogenesis. Therefore, the second part of this PhD was focused on the design, manufacturing, and optimisation of a four-zones osteochondral model by combining multiple manufacturing techniques (electrospinning, bioprinting, soft lithography and fusion deposition modelling); different materials (natural-based hydrogels for the AC side and a thermo-plastic polymer for the SB side) and two cells type (Y201 for SB and Y201-C for AC). The physico-chemical properties and biological performances of each individual layer were studied in healthy and OA conditions, before their assembly as whole zonal model. Each layer reproduced features of the corresponding native zone in terms of matrix and cells organisation, gene expression and matrix production. Also, the main OA features (e.g. degradation of collagen from the surface, chondrocytes hypertrophy within the deep layer) were observed in OA condition. In conclusion this project has successfully developed and validated human in vitro models closely mimicking AC with characteristic pathological features of early-stage OA. These models provide novel insights to inform future production of reliable and scalable human in vitro models for testing novel OA treatments.