Intrathoracic esophageal replacement by in situ tissue-engineered esophagus Yuen Nakase, MD, PhD, Tatsuo Nakamura, MD, PhD, Shuichi Kin, MD, PhD, Susumu.

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Intrathoracic esophageal replacement by in situ tissue-engineered esophagus Yuen Nakase, MD, PhD, Tatsuo Nakamura, MD, PhD, Shuichi Kin, MD, PhD, Susumu Nakashima, MD, PhD, Tetsuji Yoshikawa, MD, PhD, Yoshiaki Kuriu, MD, PhD, Chohei Sakakura, MD, PhD, Hisakazu Yamagishi, MD, PhD, Junji Hamuro, PhD, Yoshito Ikada, PhD, Eigo Otsuji, MD, PhD, Akeo Hagiwara, MD, PhD The Journal of Thoracic and Cardiovascular Surgery Volume 136, Issue 4, Pages 850-859 (October 2008) DOI: 10.1016/j.jtcvs.2008.05.027 Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Surgical technique of esophageal replacement. A, The graft did not show any infection or adhesion to the abdominal cavity. B, The tube stent could be easily removed, and the scaffold developed into soft tubular tissue. C, The thoracic esophagus was mobilized, and a 3-cm length was resected and replaced by the graft. The anastomosis was completed with 3-0 Vicryl suture (Ethicon). D, Metal clips (arrows) were placed at the cut ends of the native esophagus as markers. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 850-859DOI: (10.1016/j.jtcvs.2008.05.027) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Histologic and immunohistochemical features of the oral epithelial cell sheets. A, Hematoxylin and eosin staining. B, Immunostaining for cytokeratin MNF116. C, Immunostaining for α-smooth muscle actin (α-SMA) of autologous oral mucosal epithelial sheets. (Original magnification: ×200.) Fibroblasts penetrated the amniotic membrane, and the keratinocytes were well differentiated and stratified into 4 to 5 layers. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 850-859DOI: (10.1016/j.jtcvs.2008.05.027) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Histologic features of the grafts of the KF(+) group (dog B-3), in situ tissue-engineered esophagus (ITEE). Hematoxylin and eosin staining is shown in A, B, and D. C, Immunostaining for cytokeratin MNF116. E, Immunostaining for α-SMA. B to D, High-power views of the boxed regions in A. (Original magnifications: A, ×40; B and C, ×400; D and E, ×200.) The surface of ITEE was covered by 4–5 layers of stratified, well-differentiated cells. The connective tissue of ITEE was similar to the smooth muscle tissue. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 850-859DOI: (10.1016/j.jtcvs.2008.05.027) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Postoperative contrast study: macroscopic findings and histologic features of the KF(–) group (dog A-2). A and B, This esophagogram is the side view of the intrathoracic esophagus. The right upper corner indicates the oral side and the left lower corner indicates the anal side. The arrows indicate metal clips at the cut ends of the native esophagus. A, At 1 week after esophageal replacement, the graft showed a stricture. B, At 22 days after esophageal replacement, the graft showed almost complete obstruction. C, No infection or adhesion of the thoracic cavity noted. D, The surface of the ITEE was rough and showed a cicatricial stricture. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 850-859DOI: (10.1016/j.jtcvs.2008.05.027) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Postoperative contrast study: macroscopic findings of the KF(+) group (dog B-5) at 350 days after esophageal replacement. A, Metal clips (arrows) at the cut ends of the native esophagus were detected, and placement of the ITEE was confirmed by radiography. B to D, Sequential photographs of the contrast study. ITEE showed good distensibility, and esophageal peristalsis smoothly transferred food into the stomach. E, X-ray image of F. Metal clips at the cut ends of the native esophagus were detected by radiography. F, The ITEE shows a smooth surface similar to the native esophagus. The surface showed no signs of stricture or esophagitis. Histologic and immunohistochemical features of the ITEE at the broken line in F are shown in Figure 6. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 850-859DOI: (10.1016/j.jtcvs.2008.05.027) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 Histologic and immunohistochemical features of the KF(+) group (dog B-5). A, Hematoxylin and eosin staining at the broken line in Figure 5, F. ∗ITEE; original magnification, ×4. B to D, High-power views of the boxed region in A; original magnification, ×100. E, High-power view of the boxed region in C; original magnification, ×400. Hematoxylin and eosin staining is shown in A, B, C, and E. D, Immunostaining for α-SMA (B) showed the histologic features of the native esophagus. The arrow indicates the glandular structure. The ITEE surface was covered by well-differentiated, stratified (12–15 layers) keratinocytes and showed thick smooth muscle–like tissue that was similar to that of the native muscle layer, but no glandular structures. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 850-859DOI: (10.1016/j.jtcvs.2008.05.027) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions