Impact of Cryopreservation on Extracellular Matrix Structures of Heart Valve Leaflets Katja Schenke-Layland, PhD, Navid Madershahian, MD, Iris Riemann,

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Impact of Cryopreservation on Extracellular Matrix Structures of Heart Valve Leaflets Katja Schenke-Layland, PhD, Navid Madershahian, MD, Iris Riemann, PhD, Barry Starcher, PhD, Karl-Jürgen Halbhuber, MD, Karsten König, PhD, Ulrich A. Stock, MD The Annals of Thoracic Surgery Volume 81, Issue 3, Pages 918-926 (March 2006) DOI: 10.1016/j.athoracsur.2005.09.016 Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Macroscopical appearance of representative specimens of (A) fresh, (B) with antibiotics treated and (C) thawed cryopreserved heart valves. No macroscopical cracks or other apparent damages were noticed after thawing. The Annals of Thoracic Surgery 2006 81, 918-926DOI: (10.1016/j.athoracsur.2005.09.016) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Images of representative parts of fresh (A) and cryopreserved (B) leaflets (longitudinal sections). (A, B) Both specimens show a normal interstitial cellularity (cell nuclei: dark magenta) and the expected layered structure of the leaflet with the ventricularis (v) as inflow side, spongiosa (s) as inner layer, followed by fibrosa (f) and arterialis (a) as the outflow side. (B) Only a few collagen-containing structures (yellow) are detectable within the ventricularis layer of the thawed cryopreserved leaflets. Furthermore, a more loose structure with a slight degree of partial detachment of the fibrosa from the spongiosa layer is detectable within these specimens. (Movat-pentachrome stain for visualization of the major matrix proteins: collagen, yellow; glycosaminoglycans, green-blue; elastin, black.) Magnification is ×10. Scale bar equals 100 μm. The Annals of Thoracic Surgery 2006 81, 918-926DOI: (10.1016/j.athoracsur.2005.09.016) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Immunohistochemical staining of representative cross-sections (ventricularis; z = 25 μm to 30 μm) of fresh (A, B) and cryopreserved specimens (C, D) with an antibody against elastin at two different magnifications. Magnification of A, C is ×40; magnification of B, D is ×63. Scale bars equal 100 μm. The Annals of Thoracic Surgery 2006 81, 918-926DOI: (10.1016/j.athoracsur.2005.09.016) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Transmission confocal laser scanning microscopy images, converted by gray value inversion, show cross-sections of the fibrosa of fresh leaflets (A, B, C), and the fibrosa (D, E, F) and ventricularis (G, H, I) of cryopreserved leaflets. Extracellular matrix structures stained positive for collagen type I (A, D, G), type III (B, E, H), and type IV (C, F, I) appear yellow (pseudofluorescence). The images are representative for all fresh and cryopreserved specimens. Magnification is ×63. Scale bar equals 100 μm. The Annals of Thoracic Surgery 2006 81, 918-926DOI: (10.1016/j.athoracsur.2005.09.016) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Autofluorescence images of the elastin-rich ventricular side of fresh and cryopreserved heart valve leaflets induced with 760 nm laser pulses. Single cells with fluorescent mitochondria based on two-photon excited reduced coenzyme NAD(P)H, and nonfluorescent nuclei are visible especially at the surface of fresh tissues (z = 0 μm). Leaflet cells of the cryopreserved specimens appear to be damaged or non-viable, indicated by the total strong-fluorescent cellular structures, including cell nuclei. Arrows indicate cell debris within the cryopreserved tissue. Compared with the distinct elastin network of the fresh tissue, just a few elastic fibers are detectable at depths of 10 to 15 μm of the cryopreserved leaflets. The images shown in this figure are representative for all monitored leaflet tissues. Scale bar equals 30 μm. The Annals of Thoracic Surgery 2006 81, 918-926DOI: (10.1016/j.athoracsur.2005.09.016) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Multiphoton imaging of the outflow side of representative fresh and cryopreserved leaflets induced with 840 nm near-infrared femtosecond laser pulses show substantial ultrastructural deterioration and disintegration of most collagenous structures in cryopreserved tissues. Second harmonic generation was only inducible in fresh tissue structures. Scale bar equals 30 μm. The Annals of Thoracic Surgery 2006 81, 918-926DOI: (10.1016/j.athoracsur.2005.09.016) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions