Cell-only bioprinting of articular cartilage progenitor cells within a physically constraining support bath to engineer structurally organized grafts.

Engineering functional articular cartilage (AC) grafts is one of the greatest challenges in tissue engineering. Recapitulating the arcade-like collagen organisation of AC, which is integral to the tissues' strength and stiffness, is necessary to engineer truly functional grafts. This motivates the need for innovative strategies to control collagen alignment in engineered tissues in a programmable manner. Emerging 3D bioprinting strategies can provide spatially defined cues to guide tissue growth. Therefore, the goal of this study was to use embedded bioprinting to provide spatially defined boundary conditions to AC progenitor cells (ACP) to direct collagen organization and support the development of biomimetic cartilage tissues. ACPs were isolated through differential adhesion to fibronectin and demonstrated superior chondrogenesis to donor matched chondrocytes. Two different approaches (casting and 3D bioprinting) were used to physically constrain ACPs with external boundaries of differing widths (250, 500, or 750 μm). For both approaches, thinner boundaries promoted greater collagen alignment along the long axis of the developing tissue. Building on this, ACPs were bioprinted into a sheet, with collagen fibers aligning parallel to the print direction. Finally, a multi-layered graft was bioprinted with horizontal filaments (XY plane) overlaying vertical filaments (Z-axis). The bioprinted tissue had an arcade-like collagen organization with horizontal collagen fibres overlaying vertical collagen fibres. These findings demonstrate how support baths can be used to provide spatially defined physical boundary conditions to bioprinted cells to guide matrix organization, enabling the engineering of anisotropic AC grafts.