Tissue Engineering of Autologous Human Heart Valves Using Cryopreserved Vascular Umbilical Cord Cells Ralf Sodian, MD, Cora Lueders, PhD, Liv Kraemer,

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Tissue Engineering of Autologous Human Heart Valves Using Cryopreserved Vascular Umbilical Cord Cells Ralf Sodian, MD, Cora Lueders, PhD, Liv Kraemer, MD, Wolfgang Kuebler, MD, Mehdi Shakibaei, MD, Bruno Reichart, MD, Sabine Daebritz, MD, Roland Hetzer, MD The Annals of Thoracic Surgery Volume 81, Issue 6, Pages 2207-2216 (June 2006) DOI: 10.1016/j.athoracsur.2005.12.073 Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Tissue engineering technique for fabrication of autologous human heart valves. Fetal echocardiography will be performed in order to diagnose abnormal cardiac anatomy (eg, pulmonary valve malformation). Vascular-wall cells of the umbilical cord will be isolated, expanded in vitro, and cryopreserved. At the ideal time point of surgery the cryopreserved human umbilical cord cells will be recultivated and seeded on a biodegradable heart valve scaffold. The cell polymer construct will be transferred into a dynamic cell culture system (“bioreactor”) and grown in vitro. After maturation in the bioreactor system the tissue-engineered heart valve will be implanted as an autologous heart valve into the same patient. The Annals of Thoracic Surgery 2006 81, 2207-2216DOI: (10.1016/j.athoracsur.2005.12.073) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 (A) Three-dimensional reconstructed stereolithographic model from the inside of an aortic homograft. (B) Trileaflet heart valve scaffold from porous poly-4-hydroxybutyrate including sinus of Valsalva (seen from the aortic side) fabricated from the stereolithographic model. The Annals of Thoracic Surgery 2006 81, 2207-2216DOI: (10.1016/j.athoracsur.2005.12.073) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 The fixed cryopreserved human umbilical cord cells showed myofibroblast-like morphology. Immunofluorescence staining revealed intracellular expression of anti-ASO2 (A), fibronectin (B), anti-alpha actin (C), and collagen (D). The Annals of Thoracic Surgery 2006 81, 2207-2216DOI: (10.1016/j.athoracsur.2005.12.073) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Tissue-engineered heart valve after 7 days of static conditioning and an additional 7 days of dynamic conditioning in a bioreactor system. The Annals of Thoracic Surgery 2006 81, 2207-2216DOI: (10.1016/j.athoracsur.2005.12.073) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Hematoxylin and eosin staining of the tissue-engineered heart valves shows (A) layered tissue formation and cellular ingrowth in the dynamically conditioned constructs (black arrows). The white arrows show the degrading polymeric scaffold. (B) The controls showed hardly any cellular adherence and tissue formation with only a small number of cells attached to the scaffold and almost no cellular ingrowth (black arrows). The white arrow shows the degrading polymeric scaffold. The Annals of Thoracic Surgery 2006 81, 2207-2216DOI: (10.1016/j.athoracsur.2005.12.073) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Immunohistochemistry of the heart valves revealed positive expression for collagen (A, black arrows), fibronectin (B, black arrows), and actin (C, black arrows) in the dynamically conditioned heart valves. Static controls showed less organized extracellular matrix formation such as collagen (D, black arrows), fibronectin (E, black arrows), and actin (F, black arrows). The white arrows show the degrading polymeric scaffold. The Annals of Thoracic Surgery 2006 81, 2207-2216DOI: (10.1016/j.athoracsur.2005.12.073) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 7 Electron microscopy of the dynamically conditioned heart valves (A) showed layered tissue formation and a confluent surface. In contrast, static controls showed only few cells and no cell ingrowth (B). The Annals of Thoracic Surgery 2006 81, 2207-2216DOI: (10.1016/j.athoracsur.2005.12.073) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 8 Functional analysis showed an intact intracellular Ca2+ concentration after stimulation with histamine in the recultivated and cryopreserved cells (A and B) as well as in the dynamically conditioned constructs (C and D). The Annals of Thoracic Surgery 2006 81, 2207-2216DOI: (10.1016/j.athoracsur.2005.12.073) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 9 Mechanical properties of the conditioned constructs showed an increase of the tensile strength in longitudinal direction in dynamically conditioned heart valves compared with static controls. The Annals of Thoracic Surgery 2006 81, 2207-2216DOI: (10.1016/j.athoracsur.2005.12.073) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions