Introduction: Vascular interactions are essential for developing tissues during embryogenesis, particularly in skeletal development through endochondral ossification. During this process, mesenchymal cells undergo condensation to give rise to skeletal elements, while simultaneously interacting with newly formed vascular networks. These skeletal elements are later invaded by the vasculature, initiating bone marrow formation. While we and other groups established an in vivo model for endochondral ossification using human embryonic stem cell (hESC)-derived sclerotome (osteochondral progenitors) implanted in immunodeficient mice have successfully recapitulated key aspects of it (Cell Rep. 2023). However, given the physiological differences between mouse and human systems, there remains a need for in vitro platforms that can more precisely model early-stage, human-specific vascular-mesenchymal interactions during endochondral ossification.

Methods: To bridge this gap, we utilized a commercial organ-on-a-chip platform based on a 64-chip microfluidic device (OrganoPlate® Graft) to replicate vascular-mesenchymal interactions relevant to early endochondral ossification (Fig.1). Each chip consists of 1 in-gel culture channel, 2 perfusion channels, and an open graft chamber. These perfusion channels mimic physiological flow conditions critical for vascular sprout formation. Out of the tested extracellular matrices, fibrin gel supported significantly more robust vascular-like network formation by mCherry-labeled human umbilical vein endothelial cells (HUVECs) compared to conventional collagen-I gel. The perfusability of these networks was demonstrated using fluorescein isothiocyanate (FITC)-dextran and fluorescent microbeads, indicating the establishment of functional vascular-like networks.

Results: To model mesenchymal condensation during the initial stage of endochondral ossification and its interaction with vascular networks, we mixed hESC-derived sclerotome and enhanced green fluorescent protein (EGFP)-labeled HUVECs and introduced this mixture into the chip, which we termed SH organoid. These SH organoids sustained vascular-like networks formed by mCherry-HUVECs with greater longevity than sclerotome spheroids alone. Notably, EGFP-HUVECs migrated outward from the organoids and contributed to the formation of secondary vascular-like structures, which partially connected with the pre-existing mCherry-labeled vascular-like networks (Fig.2). After 7 days in culture, SH organoids exhibited SOX9 expression in the condensed mesenchymal core and type I collagen in a perichondrial-like outer layer, recapitulating early events of physiological endochondral ossification.

Conclusion: This SH organoid-on-a-chip model represents an in vitro system that mimics the early vascular microenvironment of human skeletal development. Beyond developmental studies, this platform holds potential for investigating the impact of vascular dynamics on early ossification and for modeling genetic skeletal disorders such as campomelic dysplasia (SOX9 mutations) and thus may contribute to the discovery of targeted therapeutic strategies.