PROJECT SUMMARY A wide range of skeletal conditions require assisted bone repair, including trauma, cancer resections, and bone augmentation for oral implant therapy. Current methods to treat these conditions rely on procedures to harvest and implant bone autografts, which are costly, invasive and difficult to scale up. The other alternatives are synthetic bone replacement materials, which show high failure rates and fail to mimic the native bone structure, composition and osteogenic properties. Stem cell-based tissue engineering has long been proposed as a promising alternative for the repair of bone defects. However, treating large bony structures remains problematic. It is generally believed that scaffold materials that closely approximate the characteristics of native bone represent improved materials for bone regeneration. However, the development of in-vitro scaffolds mimicking the highly vascularized, innervated, and mineralized cell-rich bone matrix down to the nanoscale has remained elusive to date. Here, we will develop a new bone scaffold biomanufacturing process where osteoprogenitor cells are three-dimensionally embedded in controlled nano-mineralized, pre-vascularized and innervated bone-like injectable microgels, thus mimicking the mineralized nanostructure, cellular and extracellular microenvironment of native bone. (aim 1) We will determine the mechanistic characteristics enabling the differentiation of hMSCs into osteogenic phenotypes as influenced by bone-like microenvironments, and engineer cell-laden mineralized injectable microgels that approximate the regenerative potential of autologous bone grafts. We will then adapt this strategy to engineer (aim 2) pericyte-supported vascular capillaries and (aim 3) neuronal networks, that are embedded in nanoscale mineralized hydrogels, to determine the mechanisms that enable vasculature and innervation enhancement of osteogenesis in-vitro and regeneration in-vivo. We argue that this multi-pronged strategy will enable the engineering of highly innovative bone scaffold materials and in-vitro bone model systems that will share great nanostructural and physical similarities to native bone. Ultimately, this will lead to biomaterials that closely approximate the regenerative potential of autologous bone in the clinic.