(a–c) Pre-implantation histology of TEBV (1.5 mm ID). (a) H&E staining reveals a decellularized internal membrane (IM), the living layer (LL) and the lumen (L) of TEBV. Lumens of vessel were seeded with syngeneic rat endothelial cells. Note that the wall is scalloped because this vessel was an unused surgical sample that contracted prior to fixation. (b) Movat staining reveals the large proteoglycan (aqua green) content of the TEBV at the time of implantation. (c) Verhoff-Masson staining indicates the high collagen (blue) content of the TEBV. (d) 90 days after implantation, H&E staining of the perfusion-fixed graft shows complete tissue integration with minimal inflammatory/immune response and a modest neomedia formation. VC: vena cava. (e) Verhoff-Masson staining clearly indicates that the IM was largely 16 intact and still acellular. The neomedia was rich in collagen and cells and appeared to have elastic fibers (black). (f) At higher magnification, a Movat staining reveals the forming elastic fibers and a developing internal elastic lamella-like structure forming under a confluent endothelium. Note that proteoglycans were present in the neomedia and subendothelial space but not no longer in the IM (yellowish staining indicates collagen). (g–h) Immunohistochemical staining for von Willebrand factor and smooth muscle α-actin reveals the confluent endothelium and the SMC layers (respectively) of the TEBV and adjacent vena cava. Note the presence of SMC-specific α-actin positive cells at the IM/adventitia interface.

(a) Computed tomography angiogram after 8 weeks of implantation showing a patent TEBV (red) and the adjacent vena cava (blue). Note that the initial diameter mismatch is still visible in the middle of the graft and that the curved shape is typical of an interpositional graft. (b) Perfusion-fixed TEBV at 8 weeks shows complete tissue integration and a smooth lumen with no signs of thrombosis, stenosis or mechanical failure. Arrow indicates suture line. VC: vena cava. (c) Histology (H&E) of the graft at 8 weeks shows multiple leukocyte infiltrations (arrows) and rare sites of limited luminal growth (arrow heads). (d–e) At higher magnification, Verhoff-Masson and Movat stainings show an intact and still acellular IM. Note that proteoglycans are no longer present in the TEBV. Vasa vasorum formation was noted (arrows in D). The significant immune response observed (arrows in e) may be responsible for 18 incomplete tissue integration. (f) Immunolabeling for von Willebrand factor reveals the confluent endothelium and subendothelial matrix (nuclei counterstained in blue). (g) Immunolabeling for SMC-specific α-actin stains a cell population at the IM/adventitia interface (blue counterstain). Arrow indicates small blood vessel. (h) TEBV internal diameter was measured at the center of the graft by weekly ultrasound-Doppler examination of all animals. After an increase of about 5% associated with initial pressurization, the diameter remained constant for the duration of the study.

(a) Age- and risk-matched TEBV before implantation (4.2 mm ID) being removed from its temporary tubular support. (b) TEBV, anastomosed as end-to-end interpositional femoral graft, immediately after removal of the cross-clamps. (c) Doppler-ultrasound imaging at 2 weeks shows a patent vessel with uniform lumen and normal flow (arrows indicate anastomoses). TEBVs were endothelialized with autologous canine endothelial cells.

(a–b) After 180 days, Verhoff-Masson and Movat stainings of the perfusion-fixed TEBV wall demonstrate a largely intact and acellular IM as well as abundant elastic fiber formation in the neomedia. Vasa vasorum formation was common (arrow in a). (c) Movat staining of native rat aorta. Note the resemblance with the remodeled wall of the TEBV shown in (b). (d) At the latest time point (225 days), grafts appear fully integrated in the surrounding tissue and show no signs of thrombosis, stenosis or mechanical failure. (e–g) Transmission electron micrographs (N=nucleus). (e) Flat endothelial cells with accompanying basement membrane (arrow) line the lumen of the TEBV 17 (arrow head: cell-cell junction). Underlying SMCs are rich in microfilaments and dense bodies suggesting a contractile phenotype. The dark staining material along the surface of the SMCs, as revealed by ruthenium red, resembles forming elastic fibers. (f) Higher magnification of the endothelium reveals tight endothelial cell-cell junctions (arrow) and close apposition to a continuous basement membrane. (g) Multi-directional bundles of collagen fibers in the IM. Note the periodic banding pattern typical of collagen fibers.