Temporal enzymatic treatment to enhance the remodelling of multiple cartilage microtissues into a structurally organised tissue

AbstractScaffold-free tissue engineering strategies aim to recapitulate key aspects of normal developmental processes as a means of generating highly biomimetic grafts. Cartilage and fibrocartilaginous tissues have successfully been engineered by bringing together large numbers of cells, cellular aggregates or microtissues and allowing them to self-assemble or self-organize into a functional graft. Despite the promise of such approaches, considerable challenges still remain, such as engineering scaled-up tissues with predefined geometries, ensuring robust fusion between adjacent cellular aggregates or microtissues, and directing the (re)modelling of such biological building blocks into a unified scaled-up graft with hierarchical matrix organisation mimetic of the native tissue. In this study, we first demonstrate the benefits of engineering cartilage via the fusion of multiple cartilage microtissues compared to conventional scaffold-free approaches where (millions of) individual cells are allowed to aggregate and generate a cartilaginous graft. Key advantages include the engineering of a tissue with a richer extracellular matrix, a more hyaline-like cartilage phenotype and a final graft which better matched the intended geometry. A major drawback associated with this approach is that individual microtissues did not completely (re)model and remnants of their initial architectures where still evident throughout the macrotissue. In an attempt to address this limitation, the enzyme chondroitinase ABC (cABC) was employed to accelerate structural (re)modelling of the engineered tissue. Temporal enzymatic treatment supported robust fusion between adjacent microtissues, enhanced microtissue (re)modelling and supported the development of a more biomimetic tissue with a zonally organised collagen architecture. Additionally, we observed that cABC treatment modulated matrix composition (rebalancing the collagen:glycosaminoglycans ratio), tissue phenotype, and to a lesser extent, tissue mechanics. Ultimately, this work demonstrates that microtissue self-organisation is an effective method for engineering scaled-up cartilage grafts with a pre-defined geometry and near-native levels of ECM accumulation. Importantly we have demonstrated that key limitations associated with tissue engineering using multiple cellular aggregates, microtissues or organoids can be alleviated by temporal enzymatic treatment during graft development.