A self-renewing, tissue-engineered vascular graft for arterial reconstruction Kei Torikai, MD, Hajime Ichikawa, MD, PhD, Koichiro Hirakawa, MS, Goro Matsumiya,

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A self-renewing, tissue-engineered vascular graft for arterial reconstruction Kei Torikai, MD, Hajime Ichikawa, MD, PhD, Koichiro Hirakawa, MS, Goro Matsumiya, MD, PhD, Toru Kuratani, MD, PhD, Shigemitsu Iwai, MD, PhD, Atsuhiro Saito, PhD, Naomasa Kawaguchi, PhD, Nariaki Matsuura, MD, PhD, Yoshiki Sawa, MD, PhD The Journal of Thoracic and Cardiovascular Surgery Volume 136, Issue 1, Pages 37-45.e1 (July 2008) DOI: 10.1016/j.jtcvs.2007.06.039 Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 A, Scaffold design of polyglycolic acid/poly-l-lactic acid graft. Graft wall had three-layered structure: luminal side, knitted polyglycolic acid compounded with collagen microsponge; middle layer, polycaprolactone; outside, woven poly-l-lactic acid. B, Implantation of graft into porcine aorta. Scale bar represents 10 mm. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 37-45.e1DOI: (10.1016/j.jtcvs.2007.06.039) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Upper row, Macroscopic findings of polyglycolic acid/poly-l-lactic acid grafts at 1 (A), 2 (B), 4 (C), 6 (D), and 12 (E) months after implantation. All explants were patent, with no sign of thrombus or aneurysm formation, and all surfaces were completely endothelialized. Lower row: macroscopic findings of expanded polytetrafluoroethylene grafts at 2 (F), 4 (G), and 6 (H) postoperative months. Endothelial defects were always detected. Scale bar represents 5 mm. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 37-45.e1DOI: (10.1016/j.jtcvs.2007.06.039) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Scanning electron micrographs of luminal surfaces of polyglycolic acid/poly-l-lactic acid grafts 2 (A), 6 (B), and 12 (C) months after grafting. Surfaces were covered with confluent endothelial cell lining parallel to direction of blood flow. D, Scanning electron micrograph of endothelial defect in expanded polytetrafluoroethylene graft. Direct exposure of its fibrils to graft lumen was observed. Original magnification ×800; scale bar, 20 μm. Arrows indicate direction of blood flow. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 37-45.e1DOI: (10.1016/j.jtcvs.2007.06.039) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 A, Histologic findings in each model. Left: immunohistochemical double staining for von Willebrand factor (vWF) and α–smooth muscle actin (alpha-SMA). Right, Double staining with hematoxylin and eosin (H&E) and Victoria blue stain. Immunohistochemical staining showed endothelial monolayers and parallel alignment of smooth muscle cells beneath endothelium in all polyglycolic acid/poly-l-lactic acid graft models. Victoria blue–positive fiberlike structures consistent with elastic fibers were detected in long-term models. With expanded polytetrafluoroethylene (ePTFE) grafts, good tissue regeneration was not observed. Original magnification ×100; scale bar, 100 μm. B (2-month model) and C (12-month model) show all layers of implanted polyglycolic acid/poly-l-lactic acid grafts stained immunohistochemically. Asterisks represent space occupied by remaining poly-l-lactic acid layers. Original magnification ×40; scale bar, 250 μm. D, Immunohistochemical findings in 2-month model. Arrowheads indicate endothelial monolayer. Original magnification ×200; scale bar, 100 μm. E, Hematoxylin and eosin stain of polyglycolic acid/poly-l-lactic acid graft 1 month after implantation. Mild infiltration of inflammatory cells and phagocytic giant cell were observed around scaffold remnants in subendothelial layer. Original magnification ×200; scale bar, 100 μm. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 37-45.e1DOI: (10.1016/j.jtcvs.2007.06.039) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 A, Qualitative study of vasomotor responsiveness with neotissue obtained from 2-month model. Suspended arch-shaped sample exhibited series of formation changes suggestive of relaxation and contraction on administration of sodium nitroprusside and norepinephrine, respectively. B, Contractile activities of 2-month (triangles) and 12-month (squares) models in isometric studies. Both models exhibited dose-response curves to norepinephrine. Asterisk indicates P < .05 versus 2-month model. C, Relaxation responses of 2-month (triangles) and 12-month (squares) models and native aortas (circles) in isometric studies. Data represent percentage reduction in precontraction with 10−5-mol/L norepinephrine. In each model, dose-dependent relaxation to sodium nitroprusside was demonstrated. Asterisk indicates P < .05 versus 2- or 12-month model. Double asterisk indicates P < .05 versus 12-month model. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 37-45.e1DOI: (10.1016/j.jtcvs.2007.06.039) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Wall thickness of neotissue formed in implanted PGA/PLLA grafts Wall thickness of neotissue formed in implanted PGA/PLLA grafts. Thickness was measured at three points: 5 mm from proximal and distal anastomoses, respectively, and at midpoint of graft. In all groups, mean value at each measured point was approximately 1.5 mm. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 37-45.e1DOI: (10.1016/j.jtcvs.2007.06.039) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Immunohistochemical examination for collagen type IV 1 (A), 2 (B), 4 (C), and 6 (D) months after implantation. Positively stained thin layers just below endothelium and scattered positive staining in subendothelial layers were seen. Original magnification ×100; scale bar, 100 μm. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 37-45.e1DOI: (10.1016/j.jtcvs.2007.06.039) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions