Towards the joint on a chip: double layered directly interfaced tissues to mimic the Osteoarthritic cartilage-subchondral interface

Osteoarthritis (OA) is the most diffused musculoskeletal disease and a worldwide cause of pain and disability. Although OA’s most evidently affected tissue is cartilage (AC), pathological changes regard the whole joint. An altered biomechanic at the interface between AC and bone, namely widening of a layer of mineralized hypertrophic cartilage (HC), has been suggested among pathological causes. Most OA in vitro models, however, do not account for joints’ biomechanics nor for the multiplicity of tissues affected by OA. In this work we present an OoC model of OA joint enabling to culture two directly interfaced 3D-tissues differentiated in AC and HC and coupled with a mechanism to provide both vertically aligned 3D constructs with mechanical compression. Theory and Experimental procedure Starting from a recently developed OA cartilage on chip model, we first investigated culture conditions to properly differentiate 3D micro-constructs into AC and HC within the miniaturized model. Articular chondrocytes (CH) were used as cell source for AC, mesenchymal stromal cells (MSCSs) for HC. Tissues were cultured either singularly or in coculture where a direct interface between 3D micro-construct was achieved through removable PDMS molds. A new device was then designed to provide vertically interfaced micro-constructs, with mechanical stimulation. ECM deposition and gene expression were analyzed both in single and coculture. Results A tailored culture medium allowed to reach differentiation of both AC and HC. Aggrecan and Collagen type-II(Col2a1) characterized AC matrix, while HC matrix, still Col2a1 positive, was also rich in collagen type-X and calcium deposits, hallmarks of the hypertrophic tissue. MSCSs and ACs maintained different expression levels of distinctive markers (e.g. ACAN, COL10A1 and OPN) in both cultures. Moreover, a proof-of-concept of the possibility to stack the two tissues in a knee joint-like vertical configuration was achieved by means of a new device. Notably it was possible to couple it with an actuation layer for OA induction through mechanical overload. Conclusions and discussion Successful differentiation of both tissues and achievement of a direct interface between AC and HC opens the path for a more representative Joint-like model. Mechanical stimulation, furthermore, allows for the study of biomechanics role in OA onset. Indeed, these results could allow an in vitro representation of the tidemark and for investigation on the causes (e.g. mechanical injuries) of the transition of AC to HC which distinguish OA’s onset. Acknowledgments Fondazione Cariplo (#2018-0551) MSCA IF (#841975). SNF (310030_175660). PoliFab CleanRoom