Scaffolded spheroids as building blocks for bottom-up cartilage tissue engineering show enhanced bioassembly dynamics

Due to the capability of cell spheroids (SPH) to assemble into large high cell density constructs, their use as building blocks attracted a lot of attention in the field of biofabrication. Nevertheless, upon maturation, the composition along with the size of such building blocks change, affecting their fusiogenic ability to form a cohesive tissue construct of controllable size. This natural phenomenon remains a limitation for the standardization of spheroid-based therapies in the clinical setting. We recently showed that scaffolded spheroids (S-SPH) can be produced by forming spheroids directly within porous PCL-based microscaffolds fabricated using multiphoton lithography (MPL). In this new study, we compare the bioassembly potential of conventional SPHs versus S-SPHs depending on their degree of maturation. Doublets of both types of building blocks were cultured and their fusiogenicity was compared by measuring the intersphere angle, the length of the fusing spheroid pairs (referred to as doublet length) as well as their spreading behaviour. Finally, the possibility to fabricate macro-sized tissue constructs (i.e. cartilage-like) from both chondrogenic S-SPHs and SPHs was analyzed. This study revealed that, in contrast to conventional SPHs, S-SPHs exhibit robust and stable fusiogenicity, independently from their degree of maturation. In order to understand this behavior, we further analyze the intersection area of doublets, looking at the kinetic of cell migration and at the mechanical stability of the formed tissue using dissection measurements. Our findings indicate that the presence of microscaffolds enhances the ability of spheroids to be used as building blocks for bottom-up tissue engineering, which is an important advantage compared to conventional spheroid-based therapy approaches. STATEMENT OF SIGNIFICANCE: The approach of using SPHs as building blocks for bottom-up tissue engineering offers a variety of advantages. At the same time the self-assembly of large tissues remains challenging due to several intrinsic properties of SPHs, such as for instance the shrinkage of tissues assembled from SPHs, or the reduced fusiogenicity commonly observed with mature SPHs. In this work, we demonstrate the capability of scaffolded spheroids (S-SPH) to fuse and recreate cartilage-like tissue constructs despite their advanced maturation stage. In this regard, the presence of microscaffolds compensates for some of the intrinsic limitations of SPHs and can help to overcome current limitations of spheroid-based tissue engineering.