Project Summary Traditional treatments for bone injuries have significant limitations. While over one million allogenic and autologous bone grafting procedures are performed each year, significant incidences of medical complications - often involving modest viability, poor integration, or an immune response - still occur. Therefore, the flexibility provided by an in vitro cultured, engineered tissue provides an excellent avenue to repair and replace damaged bone tissue. This approach involves seeding and growing a cell source on a scaffold and implanting the cell-laden construct into the injury site. However, the culture of large volume engineered tissues - and particularly cell viability, expansion, proliferation, and differentiation in these large tissues - is limited by current culture techniques. To address this concern, TR&D1 aims to develop a 3D printed (3DP) bioreactor as a dynamic culture system to control cellular microenvironment and therefore promote cell viability, expansion, proliferation, and differentiation within large engineered constructs. To this end, we have recently developed a tubular perfusion system (TPS) bioreactor that enables the expansion of human mesenchymal stem cells, the differentiation of these cells into osteoblasts, and the subsequent formation of boney tissue. Based on our earlier TPS bioreactor, we will use 3D printing to fabricate specialized bioreactor chambers with variable architecture, controlled flow environments, and spatially located cell populations; thus, we can ensure adequate availability of nutrients and oxygen for the expansion of stem cells within these large constructs. Furthermore, 3D printing control of the spatial location of cell populations will allow us to determine interactions between multiple cell populations, such as mesenchymal stem cells and endothelial cells. Finally, we will utilize the strategies developed in the in vitro 3D bioreactor chambers to fabricate removable, biodegradable scaffolds of engineered bone tissues that are suitable for in vivo application. The results of these studies will deliver a 3DP bioreactor system that can support the growth of large engineered tissues, while also providing a set of tools to develop other, similarly designed, tissue specific bioreactor systems.