FIG. 3.

Dynamic revitalization of scaECM supports compartmentalized coculture of ECs and SMCs for at least 3 weeks. In vitro dynamic culture system was used to mimic the physiological setting and study the ability of the scaECM graft to support endothelial and SMCs. Primary psvECs and pcaSMCs (0.5–1 × 10⁵ cells/cm²) were seeded on decellularized scaECM luminal and external surfaces, respectively, and dynamically cultivated (50 mL/min and 80 mmHg) using our previously reported¹⁵ custom-designed bioreactor, whose perfusion chamber is shown (a). psvECs coated the luminal side, displayed cobblestone-like morphology as imaged with WETSEM (b), and remained on the surface, forming a pseudoendothelium as verified by CD31 immunohistochemical cross-sectional stain (c). In contrast, pcaSMCs penetrated deeper into the media layer, but did not penetrate the pseudoendothelium as visualized by immunofluorescent stain for αSMA (an SMC marker, Cy3, red) counterstained with DAPI [cell nuclei, blue (d) note the endothelium layer on the right-hand side is only stained with DAPI, but not with αSMA]. pcaSMC ability to remodel the dynamically cultured scaECM graft is demonstrated by qPCR quantification of the expression quantity of ECM remodeling-related genes (TIMP1, MMP2, and MMP14; normalized to GAPDH as housekeeping gene) 2 and 6 weeks postseeding, as indicated on graph (e). Chondroitin sulfate (a representative GAG) secretion by the SMC is demonstrated by immunohistochemical stain (f), and quantification of total GAG content through dynamic cultivation time (up to 6 weeks) is shown as percent of native porcine carotid artery tissue values (control) (g). Scale bars: (b) 50 μm; (c, d, f) 100 μm. EC, endothelial cell; pcaSMC, porcine carotid artery SMC; psvECs, porcine saphenous vein ECs; SMCs, smooth muscle cells. Color images available online at www.liebertpub.com/tea