Multicellular microtissues from fused ligament- and bone-cell spheroids relevant to enthesis repair

The enthesis, the point where a tendon or ligament attaches to bone, is a graded fibrocartilaginous interface that poorly regenerates after injury. Here, we present a modular and scaffold-free strategy for engineering microtissues relevant to enthesis repair by fusing anterior cruciate ligament-derived spheroids with spheroids from osteogenically differentiated human mesenchymal stromal cells. We show that the maturation state of the constituent spheroids governs fusion dynamics and spatial organization, enabling the controlled formation of ligament-, fibrocartilage-, and bone-like regions within a single, radially/concentrically organized construct. Within 10 days, the fused tissues display locally distributed lineage-specific markers and type X collagen localized at the interface between the osteogenically-derived core and ligamentous shell. The latter is indicative of the de novo formation of a fibrocartilage-like region between these regions. The system is scalable by simply adjusting the spheroid number. It supports external mechanical stimulation via ultrasound. The acoustic cues further promote extracellular matrix deposition and tissue growth while maintaining structural integrity. This readily implementable heterotypic spheroid platform offers an in vitro model for studying enthesis mechanobiology, screening therapeutic compounds, and evaluating microscale biomaterials, with translational potential as injectable or bioprintable building blocks for enthesis repair.