Composite Spheroid-Laden Bilayer Hydrogel for Engineering Three-Dimensional Osteochondral Tissue

A combination of hydrogels and stem cell spheroids has been used to engineer three-dimensional (3D) osteochondral tissue, but precise zonal control directing cell fate within the hydrogel remains a challenge. In this study, we developed a composite spheroid-laden bilayer hydrogel to imitate osteochondral tissue by spatially controlled differentiation of human adipose-derived stem cells. Meticulous optimization of the spheroid-size and mechanical strength of gelatin methacryloyl (GelMA) hydrogel enables the cells to homogeneously sprout within the hydrogel. Moreover, fibers immobilizing transforming growth factor beta-1 (TGF-beta 1) or bone morphogenetic protein-2 (BMP-2) were incorporated within the spheroids, which induced chondrogenic or osteogenic differentiation of cells in general media, respectively. The spheroids-filled GelMA solution was crosslinked to create the bilayer hydrogel, which demonstrated a strong interfacial adhesion between the two layers. The cell sprouting enhanced the adhesion of each hydrogel, demonstrated by increase in tensile strength from 4.8 +/- 0.4 to 6.9 +/- 1.2 MPa after 14 days of culture. Importantly, the spatially confined delivery of BMP-2 within the spheroids increased mineral deposition and more than threefold enhanced osteogenic genes of cells in the bone layer while the cells induced by TGF-beta 1 signals were apparently differentiated into chondrocytes within the cartilage layer. The results suggest that our composite spheroid-laden hydrogel could be used for the biofabrication of osteochondral tissue, which can be applied to engineer other complex tissues by delivery of appropriate biomolecules. Impact statement This research developed a bilayer hydrogel encapsulating two types of composite spheroids, with each layer engineered to induce osteogenic or chondrogenic differentiation. The hydrogel was prepared by adjusting the size of spheroids and mechanical strength of hydrogel to provide a desired microenvironment of encapsulated cells, leading to homogeneous sprouting. Spatially confined delivery of osteogenic or chondrogenic signals in each layer enabled the specific differentiation to each layer. Thus, our spheroid-laden bilayer hydrogel could be a potential model or therapeutic platform as three-dimensional (3D) osteochondral tissue.