Low-Dose Gamma Irradiation of Decellularized Heart Valves Results in Tissue Injury In Vitro and In Vivo Meghana R.K. Helder, MD, Ryan S. Hennessy, MD,

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Low-Dose Gamma Irradiation of Decellularized Heart Valves Results in Tissue Injury In Vitro and In Vivo Meghana R.K. Helder, MD, Ryan S. Hennessy, MD, Daniel B. Spoon, MD, Brandon J. Tefft, PhD, Tyra A. Witt, CVT, Ronald J. Marler, DVM, PhD, Sorin V. Pislaru, MD, PhD, Robert D. Simari, MD, John M. Stulak, MD, Amir Lerman, MD The Annals of Thoracic Surgery Volume 101, Issue 2, Pages 667-674 (February 2016) DOI: 10.1016/j.athoracsur.2015.07.080 Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Completed pulmonary valve replacement in a juvenile sheep model. The pericardium is shown reflected with silk sutures. Also visible are the venous and aortic cannulas for cardiopulmonary bypass. The porcine decellularized valve is shown after completion of both anastomoses. (PA = pulmonary artery; RVOT = right ventricular outflow tract.) The Annals of Thoracic Surgery 2016 101, 667-674DOI: (10.1016/j.athoracsur.2015.07.080) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Testing different doses of gamma irradiation. (A–D) The top panel shows culture results of cusp, sinus, and root of the valve construct. Nonsterilized valves and those exposed to gamma irradiation at 1,000 Gy show contamination in tissue culture (note cloudy culture media [B]) whereas those irradiated at 3,000 and 10,000 Gy remained sterile (note clear culture media [D]). The bottom panel shows dose-corresponding transmission electron microscopy images of cusps at an acceleration voltage of 80 kilovolts and at a magnification of ×4,000 for all images. The Annals of Thoracic Surgery 2016 101, 667-674DOI: (10.1016/j.athoracsur.2015.07.080) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Sterility testing of valves gamma irradiated with 3,000 Gy of gamma irradiation. (A) Culture of sinuses in tryptic soy broth, thioglycollate broth (duplicates), and Sabouraud dextrose broth showing no contamination. (B) Hematoxylin-eosin staining showing the lack of cells in the decellularized heart valves after gamma irradiation. (C) Modified gram staining (Brown and Brenn) of an irradiated sinus exposed to 3,000 Gy gamma irradiation showing no bacteria. (D) Fungal stain of an irradiated sinus exposed to 3,000 Gy gamma irradiation showing no evidence of fungal contamination. Original magnification 20x. The Annals of Thoracic Surgery 2016 101, 667-674DOI: (10.1016/j.athoracsur.2015.07.080) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Tensile strength testing of valve cusps exposed to neither low-dose nor high-dose gamma irradiation. Asterisk (*) indicates p values less than 0.5 when comparing the two groups (1,500 Gy and 3,000 Gy gamma irradiation). (A) The thickness of each group of cusps was comparable. (B) The stiffness of the cusps decreased with each dose of gamma irradiation compared with the control. (C) The ultimate strength of cusps with 3,000 Gy gamma irradiation was decreased significantly. (D) Tensile stress versus strain curves for decellularized porcine cusps sterilized with 0 Gy (n = 12 [red lines]), 1,500 Gy (n = 12 [green lines]), or 3,000 Gy (n = 12 [blue lines]) of gamma irradiation. Samples were tested in the circumferential direction. The Annals of Thoracic Surgery 2016 101, 667-674DOI: (10.1016/j.athoracsur.2015.07.080) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Images of echocardiography of the pulmonary valve in vivo: (A) immediately postoperatively; (B) at 1 week after implantation; (C) at 1 month after implantation; and (D) immediately before sacrifice. Note progressively worsening turbulent flow (arrows), starting in the right ventricular outflow tract (RVOT) just below the xenograft and extending into the main pulmonary artery (PA). Increasing gradients were noted by continuous wave Doppler consistent with progressive xenograft valve stenosis. The Annals of Thoracic Surgery 2016 101, 667-674DOI: (10.1016/j.athoracsur.2015.07.080) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 An explanted valve irradiated with 1,500 Gy gamma irradiation. (A) Gross image of the valve at explantation shows friable, thick cusps with vegetations (arrow). (B) Staining with hematoxylin-eosin shows abundant neutrophils (arrow). (C) Brown and Brenn stain shows bacteria characteristic of streptococcus. (D) Fungal stain shows no contamination with fungus. Original magnification 20x. The Annals of Thoracic Surgery 2016 101, 667-674DOI: (10.1016/j.athoracsur.2015.07.080) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 7 An explanted valve irradiated with 3,000 Gy gamma irradiation. (A) Gross image of the valve at explantation shows firm, rubbery cusps. (B) Staining with hematoxylin-eosin shows giant cell formation and abundant fibroblasts and macrophages (arrow) but few neutrophils. (C) Brown and Brenn stain shows no bacteria. (D) Fungal stain shows no contamination with fungus. Original magnification 20x. The Annals of Thoracic Surgery 2016 101, 667-674DOI: (10.1016/j.athoracsur.2015.07.080) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions