Project Summary Advances in tissue engineering have been limited, in part, due to challenges in controlling the biomolecule signals important for regeneration. In particular, tissue engineering approaches for bone repair have been inhibited by the supraphysiological doses of the growth factor bone morphogenetic protein-2 (BMP-2) required for adequate healing due to suboptimal delivery. In the native extracellular matrix (ECM), BMP-2 is tightly bound by proteins and glycosaminoglycans with its presentation regulated in space and time to enhance bioactivity. Furthermore, researchers have shown significant synergies with other signaling molecules, such as cell-matrix or cell-cell adhesion ligands. To mimic these interactions, a number of elegant approaches based on photopatterning and orthogonal chemistries have been developed for presenting biomolecules. However, these approaches rely on highly customized chemical reactions (which may be difficult to implement with multiple classes of biomolecules) and are generally restricted to 1-2 signals. They are also often based on photocleavage reactions for temporal control, which precludes their reversibility over multiple cycles. Given the complexity of the extracellular environment (e.g. the stem cell “niche”) in controlling the fate of cells, a general in vitro platform that can control three or more signals, as well as multiple types of biomolecules, in both space and time would be invaluable for teasing apart the factors that control behavior like cell proliferation, migration, differentiation, and new tissue formation. We propose to develop a biomaterial-based in vitro platform capable of independently, and reversibly, controlling the spatiotemporal presentation of the growth factor bone morphogenetic protein-2 (BMP-2) in combination with the RGDS peptide to mimic cell-matrix adhesion and the HAVDI peptide to mimic cell-cell adhesion. Thus, the specific aims of the work are: (1) Investigate the temporal effect of immobilized BMP-2 on osteogenesis and (2) Determine the spatiotemporal role of cell-cell and cell- matrix interactions during BMP-2-induced osteogenesis.