This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Tissue engineering has the potential to revolutionize regenerative medicine. It is well known that nutrient availability is a primary factor in engineered tissue viability. Without an internal vascular network, only thin avascular tissues can remain viable following implantation. With controlled release of proangiogenic factors within the engineered tissue, a robust microvascular network can develop, albeit in a relatively slow fashion. Recent experiments by our research team have demonstrated that prevascularization of engineered tissue prior to implantation, accelerates the formation of functional anastomoses with host vasculature. What is not known are 1) the precise time and preceding events required to achieve perfusion of the implanted tissue, and 2) the host tissue characteristics that promote perfusion. Lack of such knowledge represents an important problem because, until the knowledge becomes available, timely development of optimized strategies to maximize viability of implanted engineered tissues, is unlikely. Our long-term goal is to develop a functional, thick engineered tissue which can be adapted to specific regenerative medicine applications. The overall objective of this application, which is the next step toward attainment of our long-term goal, is to develop an intravital imaging approach to determine the time course of perfusion of the implanted tissue. Our central hypothesis is that, with our current protocols to develop fibrin gel tissue implants, we expect perfusion of the engineered tissue to develop within 48 hours after implantation in our animal model. This hypothesis was formulated on the basis of histological findings previously published by our group. The major testable hypotheses are: 1. Focal regions of implant perfusion occur within 24 h after implantation. 2. Perfusion of the entire implant occurs within 48 hours after implantation, depending on the endothelial cell type. 3. To achieve perfusion of the entire implant, the number of perfused vessels will increase in an abrupt manner, due to a minimal number of anastomic sites between host and implant vasculature. We plan to test our hypotheses and accomplish the overall objective of this application by pursuing the following Specific Aims: 1. Develop a robust animal model that enables real-time visualization of host and implant microvasculature 2. Establish the time course of perfusion dynamics within the host and implant microvasculature