234 Dean et al.: Translation of vascular tissue engineering to pediatric CHD on the proposed role for mono- cytes/macrophages in the process of neovessel formation and the develop- ment of graft stenosis. These studies were performed by isolating mono- cytes from the bone marrow of syn- geneic C57BL/6 mice followed by seeding onto the synthetic conduits and implantation [43]. Histological and immunohistochemical character- ization revealed that this model suc- cessfully recapitulated the results of the human trial, with stenosis remain- ing the primary graft-related compli- cation. Early stenosis was observed in 80 percent of unseeded TEVGs, but only 20 percent of seeded TEVGs, with all stenoses occurring within 2 weeks of implantation. Additionally, luminal diameter was significantly higher in the seeded grafts. Quantifi- cation of F4/80-positive macrophages revealed significantly higher levels of macrophage infiltration in the un- seeded grafts compared to seeded grafts. To determine whether these re- lationships were causative or correla- tive, additional TEVGs were implanted into CD11b-DTR trans- genic mice treated with diphtheria toxin to inhibit macrophage infiltra- tion [44-46]. This conditional knock- out decreased the total number of macrophages in the graft, increased rates of patency, and increased lumi- nal diameters. Macrophage inhibition resulted in a decrease in neovessel formation, with histological analysis revealing the absence of both the endothelial and smooth muscle layers, as well as reduced collagen dep- osition. These experiments illustrate a direct correlation between the degree of macrophage infiltration and the formation of stenosis. The data also highlight the well-recognized role of macrophages in the processes of vascular re- pair and remodeling. This model of seeded cells augmenting the host inflammatory response rather than playing a direct role in the formation of neo- tissue contrasts with the classic tissue engi- neering theory of seeded cells “as building f igure 2. TEVg development in mouse model. Early pulse of cytokine (MCP-1) release from seeded BM-MNC enhances monocyte recruitment to the scaffold. Monocytes infiltrate the scaffold and di- rect neotissue formation, leading to the recruitment of smooth muscle cells from neighboring native ves- sel wall. This process results in concentric layers of smooth muscle cells embedded in an extracellular matric with a monolayer of endothelial cells lining the luminal surface. (Adapted with permission from Roh (2010) [41]). (IL-1B) and monocyte chemoattractant pro- tein-1 (MCP-1) were both found in abundant quantity. Later experiments utilized alginate microspheres containing MCP-1. Implanted TEVGs with alginate microspheres incorpo- rated into their walls developed and functioned similar to human BM-MNC-seeded scaffolds with an internal lumen containing organized vascular neotissue [41] (Figure 2). Regenerative Model of Neotissue Formation The early abundance of host-derived in- flammatory cells led to further investigations