In Vitro Fabrication of a Tissue Engineered Human Cardiovascular Patch for Future Use in Cardiovascular Surgery Chao Yang, MD, Ralf Sodian, MD, Ping.

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In Vitro Fabrication of a Tissue Engineered Human Cardiovascular Patch for Future Use in Cardiovascular Surgery Chao Yang, MD, Ralf Sodian, MD, Ping Fu, MD, Cora Lüders, PhD, Thees Lemke, MD, Jing Du, MD, Michael Hübler, MD, Yuguo Weng, MD, Rudolf Meyer, MD, PhD, Roland Hetzer, MD, PhD The Annals of Thoracic Surgery Volume 81, Issue 1, Pages 57-63 (January 2006) DOI: 10.1016/j.athoracsur.2005.07.013 Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Schema illustrating the principle of metal rings used in cell seeding. The scaffold was positioned between two metal rings to concentrate the cell suspension on the seeded area of patch. The lower metal ring had a gap, which was used for deairing of the space under the patch and allowed the cell medium subsequently to enter through it because two sides of the patch were seeded. The upper metal ring with no gap fixed the polymer and circumscribed the cell suspension within the seeding area. The Annals of Thoracic Surgery 2006 81, 57-63DOI: (10.1016/j.athoracsur.2005.07.013) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 (A) Elongated cells with fibroblast-like morphology reached confluence after 10 to 14 days. The cultivated cells are bipolar in shape and look like a fish shoal. Fluorescence immunohistochemistry of the cultivated cells showed positive staining for (B) alpha smooth muscle actin and (C) myosin heavy chain. (D) Cultivated cell cultures showed negative immunofluorescent staining of CD31; cell nuclei are stained with 6-diamidino-2-phenylindole. (A: magnification 100×; B, C, D: magnification 400×.) The Annals of Thoracic Surgery 2006 81, 57-63DOI: (10.1016/j.athoracsur.2005.07.013) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Hematoxylin and eosin staining of bioreactor conditioned tissue-engineered patch. (Magnification 200×.) The Annals of Thoracic Surgery 2006 81, 57-63DOI: (10.1016/j.athoracsur.2005.07.013) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Hematoxylin and eosin staining of static control. (Magnification 200×.) The Annals of Thoracic Surgery 2006 81, 57-63DOI: (10.1016/j.athoracsur.2005.07.013) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 (A) Positive immunohistochemical staining for collagen I of a tissue-engineered (TE) patch seeded with human vascular cells. Open arrow shows stained collagen I. Polymeric scaffold (solid arrow) was partially absorbed. (B) Immunohistochemical staining for alpha smooth muscle actin (open arrow) of TE patch was positive. (C) Staining for fibronectin (open arrow) of TE patch was positive. (Magnification 200×.) The Annals of Thoracic Surgery 2006 81, 57-63DOI: (10.1016/j.athoracsur.2005.07.013) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 (A) Scanning electron microscopic examination of the conditioned tissue-engineered patch demonstrated homogenous tissue and confluent smooth surfaces with cell orientation in the direction of flow exposition. (B) The static controls showed a less confluent, inhomogenous surface without cellular orientation. (Acc.V = accelerating voltage; WD = working distance.) (Magnification 500×.) The Annals of Thoracic Surgery 2006 81, 57-63DOI: (10.1016/j.athoracsur.2005.07.013) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 7 The DNA content of the conditioned patches versus the static controls. The Annals of Thoracic Surgery 2006 81, 57-63DOI: (10.1016/j.athoracsur.2005.07.013) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 8 (A) Collagen concentration of the dynamic versus the static tissue-engineered patch. (B) Quantitative biochemical assays of elastin of the tissue-engineered patch. The Annals of Thoracic Surgery 2006 81, 57-63DOI: (10.1016/j.athoracsur.2005.07.013) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions