Seeding of Human Endothelial Cells on Valve Containing Aortic Mini-Roots: Development of a Seeding Device and Procedure Helmut Gulbins, MD, Anita Pritisanac,

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Presentation transcript:

Seeding of Human Endothelial Cells on Valve Containing Aortic Mini-Roots: Development of a Seeding Device and Procedure Helmut Gulbins, MD, Anita Pritisanac, MD, Antje Uhlig, Angelika Goldemund, Bruno M. Meiser, MD, Bruno Reichart, MD, Sabine Daebritz, MD The Annals of Thoracic Surgery Volume 79, Issue 6, Pages 2119-2126 (June 2005) DOI: 10.1016/j.athoracsur.2004.05.085 Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Homograft, fixed on the incubator tube and placed inside the incubator viewed through the graft in the direction of blood flow. With this tube, the cell suspension reached only the luminal surface of the graft, which was shorter than the incubator, thus allowing longitudinal movements of the graft inside during the rotation. This ensured an exchange of the cell suspension between both sides of the leaflets. The Annals of Thoracic Surgery 2005 79, 2119-2126DOI: (10.1016/j.athoracsur.2004.05.085) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 The incubator was fixed on the self-developed rotation device. Shown is the new tube placed inside and the closing mechanism. The incubator was filled with 120 mL cell suspension and the air was removed through the 3-way connector. The two controls at the base were used to choose the speed and time settings. The Annals of Thoracic Surgery 2005 79, 2119-2126DOI: (10.1016/j.athoracsur.2004.05.085) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Rotations and other movements of the tube inside the incubator during the rotation phase (the tube rotated axially and longitudinally, whereas the whole incubator was moved counterclockwise). These movements re-suspended those cells that had not attached to the graft during the resting phase. The Annals of Thoracic Surgery 2005 79, 2119-2126DOI: (10.1016/j.athoracsur.2004.05.085) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Homograft fixed to the new tube (groups 4 and 5). This new tube design allowed the grafts to be washed around by the cell suspension. This ensured that the outer surface was also cell coated, especially with fibroblasts. The Annals of Thoracic Surgery 2005 79, 2119-2126DOI: (10.1016/j.athoracsur.2004.05.085) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Scanning electron microscopy (magnification, ×200) of the luminal surface of the free wall of a homograft after cryopreservation before endothelial cell seeding. No endothelial cells were seen, but interstitial fibers were seen. The Annals of Thoracic Surgery 2005 79, 2119-2126DOI: (10.1016/j.athoracsur.2004.05.085) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Graphical demonstration of the seeding results in the different groups. The Annals of Thoracic Surgery 2005 79, 2119-2126DOI: (10.1016/j.athoracsur.2004.05.085) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 7 Scanning electron microscopic examination (magnification, ×500) of group 5 after fibroblast seeding. The fibroblasts formed a confluent cell layer. The cells could be identified by their typical long shape. The Annals of Thoracic Surgery 2005 79, 2119-2126DOI: (10.1016/j.athoracsur.2004.05.085) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 8 (A) Scanning electron microscopic examination (magnification, ×500) showing a leaflet of one of the grafts of group 5 after initial endothelial cell seeding. Also shows no defects within a homogenous cell layer. (B) Group 5, immunohistochemical staining for factor VIII (magnification, ×100, peroxidase reaction). Positive reaction (dark band) on the luminal surface indicating viable endothelial cells. The Annals of Thoracic Surgery 2005 79, 2119-2126DOI: (10.1016/j.athoracsur.2004.05.085) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 9 (A) Group 5, immunohistochemical staining for collagen IV (magnification, ×100, peroxidase reaction) revealed strong positive reaction (dark band) within the luminal surface, indicating synthesis of this component of the extracellular matrix. Pre-seeding with autologous fibroblasts allowed both cell types to synthesize their specific mixture of extracellular matrix proteins, which was therefore advantageous compared with pre-coating with singular components such as fibronectin or laminin. (B) Group 4, immunohistochemical staining for collagen IV (magnification, ×100, peroxidase reaction). The slight positive reaction on the luminal surface (dark band) was much weaker compared with group 5 (Fig 9A). The Annals of Thoracic Surgery 2005 79, 2119-2126DOI: (10.1016/j.athoracsur.2004.05.085) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions