Vascularization remains the principle obstacle that impedes the translation of most bone tissue engineered constructs to clinical practice. Pre-vascularization of large constructs has great potential to improve functional vasculature throughout the scaffold to rapidly facilitate integration with the surrounding tissue and circumvent necrosis in the core of the scaffold. In our previous studies, we have developed microengineered gels to co- culture human endothelial progenitor cells (hEPCs) and human mesenchymal stem cells (hMSCs) to engineer microvasculature, but observed limited anastomotic potential. We have also developed a number of approaches in which engineered hydrogels can be assembled into graded tissue scaffolds to generate vascularized constructs, and determined the need to protect structural integrity of microvasculature using mechanically-robust scaffolds. A major remaining challenge in our work is that despite the initial remodeling of the cells into functioning microvessels, efficient integration to major vessels has not been observed. The main premise of this renewal proposal is that by incorporating a perfusable vessel graft that connects the microvasculature of the engineered construct to large host vessels, we will be able to address this challenging issue. In this renewal project, we propose to engineer a bone scaffold that integrates microvasculature with a surgically-anastomizable large vessel graft to heal critical size segmental bone defects. This construct will be developed in vitr such that the microvascular capillaries in gels connect with the large vessel graft and subsequently to the host vessel in vivo. Our hypothesis is this construct can substantially improve on the integration to the host tissue by restoring blood supply immediately after transplantation. To test our hypothesis, our team comprised of experts in tissue engineering, biomaterials, microengineering, and orthopedics proposes the following aims: Aim 1. Develop components for fabrication of surgically-anastomizable pre- vascularized bone construct. Aim 2. Engineer and characterize surgically-anastomizable pre-vascularized bone construct in vitro. Aim 3. Evaluate the efficiency of surgically-anastomizable pre-vascularized bone construct in repair of critical size segmental bone defects. The completion of this work will be a paradigm shift and a landmark achievement in efforts to clinical treatments for bone critical size defects.