Embedded cell-only bioprinting to engineer structurally aligned meniscal fibrocartilage

The engineering of functional meniscal grafts remains elusive, largely due to an inability to recapitulate the highly organized collagen architecture of the native tissue which is integral to its biomechanical function. In this study, we investigated whether external geometric confinement can direct collagen alignment in fibrocartilaginous tissues generated by mesenchymal stem/stromal cells (MSCs). First, MSCs were cast within non-adhesive agarose channels, which supported the development of fibrocartilaginous tissues with collagen fibres aligned parallel to the long axis of the confining agarose wells. To translate these findings into a scalable biofabrication platform, MSC-only bioinks were bioprinted into a methacrylated xanthan gum (XGMA) support bath to generate filaments of differing widths. It was found that reducing filament width enhanced collagen alignment and fibrocartilaginous matrix deposition. To elucidate the role of cellular mechanotransduction on the observed boundary induced collagen organization, YAP and ROCK pathways were inhibited during culture. While inhibition disrupted cytoskeletal and nuclear alignment, collagen organization remained highly aligned, with no observed differences in Brillouin frequency shift. Finally, this strategy was scaled to fabricate anisotropic fibrocartilage sheets and circumferentially organized meniscus-like constructs. Overall, the findings of this study establish external geometric confinement as a powerful and scalable strategy to engineer meniscal grafts with a more biomimetic collagen organization, which may pave the way for the future development of scaffold-free meniscal grafts.