In situ formation of osteochondral interfaces through "bone-ink" printing in tailored microgel suspensions

Osteochondral tissue has a complex hierarchical structure spanning subchondral bone to articular cartilage. Biomaterials approaches to mimic and repair these interfaces have had limited success, largely due to challenges in fabricating composite hard-soft interfaces with living cells. Biofabrication approaches have emerged as attractive methods to form osteochondral analogues through additive assembly of hard and soft components. We have developed a unique printing platform that is able to integrate soft and hard materials concurrently through freeform printing of mineralized constructs within tunable microgel suspensions containing living cells. A library of microgels based on gelatin were prepared, where the stiffness of the microgels and a liquid "filler" phase can be tuned for bioprinting while simultaneously directing differentiation. Tuning microgel stiffness and filler content differentially directs chondrogenesis and osteogenesis within the same construct, demonstrating how this technique can be used to fabricate osteochondral interfaces in a single step. Printing of a rapidly setting calcium phosphate cement, so called "bone-ink" within a cell laden suspension bath further guides differentiation, where the cells adjacent to the nucleated hydroxyapatite phase undergo osteogenesis with cells in the surrounding medium undergoing chondrogenesis. In this way, bone analogues with hierarchical structure can be formed within cell-laden gradient soft matrices to yield multiphasic osteochondral constructs. This technique provides a versatile one-pot biofabrication approach without harsh post-processing which will aid efforts in bone disease modelling and tissue engineering. STATEMENT OF SIGNIFICANCE: This paper demonstrates the first example of a biofabrication approach to rapidly form osteochondral constructs in a single step under physiological conditions. Key to this advance is a tunable suspension of extracellular matrix microgels that are packed together with stem cells, providing a unique and modular scaffolding for guiding the simultaneous formation of bone and cartilage tissue. The physical properties of the suspension allow direct writing of a ceramic "bone-ink", resulting in an ordered structure of microscale hydrogels, living cells, and bone mimics in a single step. This platform reveals a simple approach to making complex skeletal tissue for disease modelling, with the possibility of repairing and replacing bone-cartilage interfaces in the clinic using a patient's own cells.