Differential Tensile Strength and Collagen Composition in Ascending Aortic Aneurysms by Aortic Valve Phenotype Joseph E. Pichamuthu, MS, Julie A. Phillippi,

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University of Pittsburgh Vascular Bioengineering Laboratory Departments of Surgery and Bioengineering and the McGowan Institute for Regenerative Medicine.

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Differential Tensile Strength and Collagen Composition in Ascending Aortic Aneurysms by Aortic Valve Phenotype Joseph E. Pichamuthu, MS, Julie A. Phillippi, PhD, Deborah A. Cleary, BS, Douglas W. Chew, BS, John Hempel, PhD, David A. Vorp, PhD, Thomas G. Gleason, MD The Annals of Thoracic Surgery Volume 96, Issue 6, Pages 2147-2154 (December 2013) DOI: 10.1016/j.athoracsur.2013.07.001 Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 (A) Representative image of tubular ascending thoracic aortic aneurysm tissue harvested. Suture indicates proximal end, and a tentative aorta axis is drawn in white dotted line. (B) Tubular tissue is cut open, and rectangular pieces of circumferentially (CIRC) and longitudinally (LONG) oriented specimens are harvested for tensile testing. The Annals of Thoracic Surgery 2013 96, 2147-2154DOI: (10.1016/j.athoracsur.2013.07.001) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 A representative fit of the model given by Equation 1 to the experimental stress-stretch data obtained from the tensile testing of an ascending thoracic aortic aneurysm tissue specimen. This particular data is from a longitudinally oriented specimen of an ascending thoracic aortic aneurysm with bicuspid aortic valve. The Annals of Thoracic Surgery 2013 96, 2147-2154DOI: (10.1016/j.athoracsur.2013.07.001) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 The average tensile strength of bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV) in circumferential (CIRC) and longitudinal (LONG) orientations. ¥The strength of circumferentially oriented specimen is significantly higher than longitudinally oriented specimen for both bicuspid aortic valve (p < 0.01) and tricuspid aortic valve (p < 0.01). **Bicuspid aortic valve is significantly stronger than tricuspid aortic valve in both orientations (p < 0.01). The Annals of Thoracic Surgery 2013 96, 2147-2154DOI: (10.1016/j.athoracsur.2013.07.001) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Three representative micrographs of Masson’s trichrome staining of bicuspid aortic valve (A, C, E) and tricuspid aortic valve (B, D, F) tissues. Note that bicuspid aortic valve shows more disrupted collagen (blue color, white arrows). Tricuspid aortic valve has less collagen (blue color, black arrows), but aligned in orientation. Scale bar = 50 μm. The Annals of Thoracic Surgery 2013 96, 2147-2154DOI: (10.1016/j.athoracsur.2013.07.001) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Collagen content, as estimated by hydroxyproline (HYP), as a function of tensile strength for each tricuspid aortic valve (TAV; A) and bicuspid aortic valve (BAV; B) specimen tested. Note a moderate correlation (r = 0.359) between the collagen content and strength for tricuspid aortic valve, but not for bicuspid aortic valve (r = 0.014). The Annals of Thoracic Surgery 2013 96, 2147-2154DOI: (10.1016/j.athoracsur.2013.07.001) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 The extrapolated bicuspid aortic valve (BAV) tensile strength for the various tricuspid aortic valve (TAV) patient ages. *The extrapolated tensile strength of bicuspid aortic valve is significantly higher than tricuspid aortic valve in both orientations (circumferential [CIRC], p < 0.01; longitudinal [LONG], p < 0.01). The Annals of Thoracic Surgery 2013 96, 2147-2154DOI: (10.1016/j.athoracsur.2013.07.001) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions