Osteoarthritis (OA) is a multifactorial disorder affecting up to 15% of the adult population and involving the joint as a whole. The different therapeutic options proposed are predominately palliative and still far from being effective in reversing the pathology. We recently demonstrated that nasal chondrocytes (NC) can be used clinically for repair of traumatic articular cartilage defects in the knee. The possibility to extend indications of NC to stages of early osteoarthritis would represent a step forward in this area of research. The development of reliable OA in vitro models is thus of primary importance to advance the research into causes and to design potential therapeutics. Different innovative approaches have been proposed, relying on the generation of 3D cartilage/osteochondral constructs obtained through biomaterials and/or bioreactor. However, integrating all key components of OA environment in a single system still remains an open challenge. Working hypothesis. In the proposed research, we will use a microfluidic hydrogel platform to generate physiological and pathological models of osteochondral units. The model will be first exploited to elucidate mechanisms of cartilage/bone cross talk during the triggering of OA, and subsequently to screen novel OA disease-modifying compounds. Specifically, we hypothesize that factors released by nasal chondrocytes are potential novel candidates as OA disease-modifying compounds.Specific aims. We will first address the generation of a 3D microscale hydrogel model of a human osteochondral (OC) unit, consisting of a cartilaginous layer integrated with an endothelialized bone layer (Aim1). We will then switch the OC healthy model towards OA phenotype, by perturbing it through inflammatory treatments, pathological/physiological-like mechanical loadings and their combination (Aim2). The OA model will be then used to investigate the main molecular pathways involved in the cross talk between cartilage and bone cells during the triggering of the pathology (Aim3). Finally, we will exploit the generated model to test the anti-OA effect of factors released by NC, based on their capacity to reverse OA like traits (Aim4).Experimental design. A microscale platform will be developed, comprising a 3D spatially organized co-culture module, equipped with an integrated system for biochemical and mechanical stimulation. A healthy model of OC unit will be first established, consisting of a cartilaginous layer (generated by human articular chondrocytes, AC) integrated with a bone layer (generated by human mesenchymal stem/stromal cells - MSC - or osteoblasts). The feasibility of AC and MSC/osteoblasts to differentiate into phenotypically stable and integrated cartilage-bone composites and the ingrowth of a vascular-like structure within the bone layer will be evaluated. To switch from healthy to OA-like phenotype, the OC unit will be exposed to inflammatory factors (IL-1ß, TNF-a, IFN-?) and/or mechanical loadings. Responses of the system in terms of cartilage/bone matrix degradation, inflammatory and nociceptive markers will be assessed. Bone/cartilage cross talk will be assessed by exposing each layer to conditioned medium collected from the insulted complementary layer, and the role of key molecular pathways will be evaluated (i.e. TGFß/BMP and Wnt/ßCatenin). Finally, factors released by NC will be characterized and used to counteract inflammatory/pain/degrading responses previously induced, together with drugs demonstrated to have anti-inflammatory effects in animal studies. Expected value of the proposed project. Our study will lead to the establishment of the first on-chip in vitro OA model able to recapitulate the key-features of a human osteochondral joint, relevant for gaining insights into OA pathophysiology and anti-OA drug development. In details, thanks to our study we aim at elucidating the role of key molecular pathways in cartilage-bone cross talk during the triggering of OA. Moreover, new insights on the anti-inflammatory and OA-reversing capacity of nasal chondrocytes will be gained, leading to defining putative bioactive factors involved. In turns, this will give new insights for the definition of new therapeutic strategies.