Longitudinal Use of In Vivo Imaging Techniques to Assess Vascularization, Hemodynamics, and Oxygenation within Implanted Engineered Tissues

Perhaps the largest obstacle to creating engineered tissues with significant clinical relevance is the difficulty associated with achieving adequate oxygen delivery following implantation into a host. One potential strategy to improve oxygen delivery following implantation is to prevascularize engineered tissues. We have previously demonstrated the feasibility of creating prevascularized tissues that anastomose with the host circulation following implantation. However, changes in vascular density, implant oxygenation, and intra-implant blood flow, in addition to the dynamics of the host-implant interactions, are not well understood. Such information is critical to ameliorating the design of engineered tissues to maximize their functionality and viability. To this end, we utilized a variety of imaging techniques to provide this information about implanted prevascularized tissues.;To quantify vascular density (VD) and functional vascular density (FVD) within implanted tissues, image acquisition and automated processing techniques were first developed. (F)VD quantifications could be performed with comparable accuracy to manual computation, but with a 12-fold increase in speed, thus making subsequent analysis of a large number of implants feasible.;Fibrin-based prevascularized tissues were then created in vitro and implanted into dorsal window chambers surgically prepared on severe combined immunodeficient mice. (F)VD, blood flow, and hemoglobin oxygen saturation were quantified within the implants using optical techniques. Although implanted vasculature anastomosed with the host circulation, thrombosis occurred within the implants in less than one hour. Thrombosis was not consistently identifiable using histology, highlighting the need for longitudinal optical analysis to fully understand the dynamic implant environment and the need to re-design comparable prevascularized tissues to maintain vascular functionality.;Cell-dense prevascularized spheroids composed of collagen and a collagen-fibrin composite were subsequently created to fill this need. These spheroids anastomosed with the host circulation and underwent thrombosis as observed previously, however, subsequent angiogenesis and vascular remodeling resulted in the establishment of a functional intra-implant vascular network. Phosphorescence lifetime measurements showed that the resulting oxygen tension within prevascularized spheroids was significantly greater than that in control spheroids. Finally, histological analysis demonstrated that the viable cell density within prevascularized collagen spheroids was comparable to that of metabolically active native tissue 14 days after implantation.