Modular de- and re-construction of vascularized osteochondral tissues in an Organ-on-Chip dual-compartment platform.

BACKGROUND: Homeostasis at the cartilage-bone interface of articular joints depends on tightly orchestrated signalling among chondrocytes, osteogenic progenitors, and subchondral vasculature. Disruption of this crosstalk is considered one of the main drivers of osteoarthritis (OA), the most prevalent musculoskeletal disease worldwide. However, the timing, location, and mechanisms underlying the pathological onset of OA remain unclear, hindering the development of targeted regenerative strategies. This knowledge gap emphasises the need for in vitro models that replicate OA's multi-tissue crosstalk in a representative yet accessible format. METHODS AND RESULTS: Here, we present a modular, dual-compartment Organ-on-Chip (OoC) platform that enables the stepwise 'de- and re-construction' of the vascularized osteochondral unit, allowing systematic interrogation of cell-specific roles in homeostasis and inflammation. Through the side-by-side culture of human articular chondrocytes (hACs) and bone marrow-derived mesenchymal stromal cells (bmMSCs), we generated biphasic, compartmentalized constructs with a contiguous interface, in which bmMSCs exhibited osteogenic commitment without compromising the stable chondrogenic capacity of hACs. The addition of human umbilical vein endothelial cells (HUVECs) to the bmMSCs compartment at a finely tuned 3:2 ratio (bmMSCs:HUVECs) enabled the formation of lumenized vascular vessels surrounded by α-SMA-expressing cells and laminin sheaths, while preserving bmMSCs' osteogenic commitment. Under homeostatic conditions, the presence of a cartilage layer adjacent to such vascularized and mineralized tissue impeded vascular and stromal invasion, whereas exposure to IL-1β (1 ng/mL) allowed to override such chondrocyte "barrier," triggering endothelial and stromal penetration into the cartilage, thus mimicking inflammatory OA. CONCLUSION: The proposed platform combines ease of use, real-time imaging capabilities, and precise control over cellular modules, offering a versatile tool for future mechanistic studies in OA and related joint disorders. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This modular Organ-on-Chip platform offers a physiologically relevant and experimentally accessible model of the vascularized osteochondral interface, enabling systematic dissection of cell-specific roles in joint homeostasis and inflammation. By recapitulating key features of early osteoarthritic pathology-including barrier breakdown, stromal invasion, and endothelial remodeling- with a highly modular and technologically robust approach, this system holds translational promise for preclinical testing of disease-modifying OA therapies, biomarker discovery, and regenerative strategies targeting cartilage-bone crosstalk.