Methods: 1.5 mm Pluronic F127 macrofibers were bridged by three-dimensional networks comprised of either 100-500 µm Pluronic F127 microfibers or 10-400 µm melt-spun Shellac microfibers. Networks were embedded in type I collagen, sacrificed and “intraluminally” seeded with 5x106 cells/mL human aortic smooth muscle cells (HASMC) followed by 5x106 cells/mL HUVEC. Constructs underwent 7 or 14 days of culture either static or dynamic. Both seeded and unseeded constructs were microsurgically anastomosed to rat femoral vessels and perfused in vivo. Architecture and cell viability were confirmed via hematoxylin and eosin (H&E) staining. Immunohistochemical (IHC) analysis was performed to determine the spatial relationship of seeded cells.
Results: Pluronic and Shellac/Pluronic three-dimensional constructs were successfully embedded and sacrificed in type I collagen, leaving patent microchannels ranging in size from 10 to 500 µm. The presence of a dense network of microchannels with adherent cells was confirmed via H &E after 7 and 14 days for both types of networks. Within microvessels 50 µm or less, CD31 expressing endothelial cells were identified, whereas larger microvessels demonstrated both CD31 and α-smooth muscle actin (SMA) expressing cells along luminal surfaces after 7 and 14 days. Additionally, following perfusion, CD31 expressing cells remained adherent along the walls of the micro- and macrochannels. Both types of constructs were successfully anastomosed to rat femoral vessels and perfused.
Conclusions: We have developed two techniques to create three-dimensional microvascular networks within tissue-engineered constructs using Pluronic F127 and Shellac sacrificial microfibers. Both techniques produce channels that support adhesion and growth of endothelial cells crucial to providing thrombosis-free flow. These results represent significant progress towards the fabrication of constructs with a hierarchical vascular network analogous to that seen in vivo, necessary for the production of human-scale engineered tissues.