The successful translation of tissue engineering therapies into clinical application that will benefit veterans requires overcoming a number of technical, biological and surgical challenges. The volume of tissue that can be engineered is limited by the extent to which stable blood vessels can be stimulated to form. An extensive, stable blood supply is required to meet mass transport demands in the new tissues and most methods are optimized for engineering tissues in small volume pre-clinical models. We have shown that implantation of a chamber containing model tissue engineering therapies against the periosteum can lead to the generation of three-dimensional vascularized bone of clinically appropriate shape and volume.4 This technique has been translated into clinical application but required an autologous source of bone for the chamber components. Broad application of this approach requires the identification of alternative, non- autologous tissue sources. Tissue engineering has the potential to provide alternative sources for chamber components. In our previous MERIT grant we investigated and optimized the design of porous hydrogel scaffolds for vascularized tissue formation. In the previous cycle we developed techniques for polymer synthesis and design, evaluated vascularization and cellular response to these biomaterial scaffolds in vitro, in vivo, and in silico, and investigated new imaging techniques for the evaluation of tissue engineering strategies. These studies illustrate our ability to promote and influence vascular ingrowth into engineered tissues. Challenges remain in regards to achieving vascular ingrowth sufficient for engineering large volumes of bone, coordinating vascularization and bone formation and engineering complex structures suitable for clinical application. The broad goals of this proposal are to 1) investigate and optimize the design of biosignal-embedded poly(ethylene glycol)-based hydrogels for engineering vascularized bone and 2) apply these materials for engineering vascularized bone for reconstruction of large, complex craniofacial defects. In order to achieve our goals we will complete the following specific aims: Objective 1: Investigate and optimize the generation of gradients scaffolds for stimulating vascularized tissue invasion into porous hydrogels. Objective 2: Investigate porous hydrogel systems for coordination of vascularization and bone formation in porous hydrogels in vitro and in vivo. Objective 3: Develop topological optimization methods for applying the clinically-translatable large animal model to engineer vascularized bone of appropriate volume and structure for clinical application. This is an ambitious proposal focused on the optimization of techniques that will bring new reconstructive techniques closer to the clinic.