Dean et al.: Translation of vascular tissue engineering to pediatric CHD 233 in media are pipetted onto the outer lumen and into the inner lumen of the scaffold. The cells are allowed several hours to attach to the scaffold in vitro prior to implantation. The drawbacks of this process have been long recognized and include low seeding ef- ficiency and the potential for intra- and inter- operator variability. While many techniques have been proposed, including dynamic, magnetic, and electrostatic [37], none of these techniques have been evaluated in clinical trials or fulfill the characteristics of an ideal seeding system, which should be rapid, automated, and standardized, while maximizing cell viability and minimizing risk of contamination. In an effort to reach this ideal, our group utilizes a disposable vacuum-seeding device in which negative pressure is used to rapidly lodge cells in the pores of a scaffold fastened to a perforated tubular device connected to suction [38]. This vacuum-seeding device has been ap- proved for use in a pilot clinical study and achieves similar cell seeding viability and efficiency as that seen in static seeding, but with the advantage of automaticity and self- containment (Figure 1). BACk To BEn CH Large Animal Studies In an effort to better understand the mechanisms behind TEVG stenosis, further bench work studies are necessary in an effort to create improved second generation TEVGs. Using an ovine model, TEVGs have been implanted as interpositional IVC grafts in juvenile lambs and monitored with bi- monthly magnetic resonance angiography over a 6-month time course. At the end of the time course, all grafts were patent and demonstrated an increase in volume propor- tional to the native right pulmonary artery. Explanted grafts at 6 months resembled na- tive IVC in that they contained a similar wall thickness with an inner monolayer of en- dothelial cells surrounded by several layers of smooth muscle cells in concentric layers. These explanted grafts contained an extracel- lular layer with collagen, elastin, and gly- cosaminoglycan (GAG) contents comparable to native IVC and a similar biomechanical profile. Equally important, by 6 months, the TEVG expressed Eph-B4, a marker of ve- nous differentiation, suggesting that implan- tation of TEVG in venous circulation leads to formation of venous vascular neotissue [39]. Small Animal Studies While this ovine model marked the first time a TEVG demonstrated molecular evi- dence of normal vascular development, a mouse model was sought after to better char- acterize the process of neotissue formation. In this effort, sub-1mm tubular scaffolds composed of similar materials used in the pilot clinical trial were constructed. These TEVGs had the advantage of being im- plantable in immunodeficient SCID-beige mice, enabling the use of TEVGs seeded with human cells without the need for im- munosuppression [40]. TEVGs seeded with human BM-MNCs and implanted as IVC in- terpositional grafts remained fully patent and functional as venous conduits over a 6- month time course. By 6 months, TEVGs contained a mature vascular architecture consisting of a confluent endothelial cell lined intima and one to two layers of smooth muscle cell media, while the original scaf- fold material had degraded and was replaced by a supportive adventitial layer of collagen fibrils [41]. Extensive histological and immunohisto- chemical characterization was performed to in- vestigate the mechanism by which neotissue formation within the TEVGs occurred. Al- though it has been hypothesized that stem cells within the seeded BM-MNC population dif- ferentiate into the cells of the neotissue [42], by 1 week after implantation, human BM- MNCs were no longer detectable within the TEVGs. Rather, it is hypothesized that these seeded cells augment a host inflammatory process via a paracrine mechanism through the secretion of chemokines that recruit host cells to the scaffold, ultimately leading to neovas- cularization. When compared to unseeded grafts, seeded grafts demonstrated a higher concentration of macrophages during early graft development. Notably, interleukin 1-beta