Cell characterization of porcine aortic valve and decellularized leaflets repopulated with aortic valve interstitial cells: the VESALIO project (vitalitate.

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Cell characterization of porcine aortic valve and decellularized leaflets repopulated with aortic valve interstitial cells: the VESALIO project (vitalitate exornatum succedaneum aorticum labore ingenioso obtenibitur) Barbara Bertipaglia, PhD, Fulvia Ortolani, PhD, Lucia Petrelli, ScD, Gino Gerosa, MD, Michele Spina, MD, Paolo Pauletto, MD, Dino Casarotto, MD, Maurizio Marchini, PhD, Saverio Sartore, PhD The Annals of Thoracic Surgery Volume 75, Issue 4, Pages 1274-1282 (April 2003) DOI: 10.1016/S0003-4975(02)04706-9

Fig 1 Electrophoresis and Western blotting (a, b, and c) of aortic valve leaflet (AVL) and aortic smooth muscle (SM) tissue extracts examined with a panel of antibodies. (A) 12.5% sodium dodecyl sulfate (SDS) gel. (B) 5% SDS gel. Lane 1, aortic extract; lane 2, AVL extract. Note in AVL extracts the actin, vimentin, and SM22 band (A), the absence of SM myosin band and the presence of platelet myosin isoforms with different intensity (B). (MyHC-Apla1 = type-1 platelet myosin heavy chains; MyHC-Apla2 = type-2 platelet myosin heavy chains.) The Annals of Thoracic Surgery 2003 75, 1274-1282DOI: (10.1016/S0003-4975(02)04706-9)

Fig 2 Immunocytochemical staining of aortic valve leaflet from the left coronary cusp (middle part, ventricularis and spongiosa) reacted with a panel of antibodies (A–I). Valve interstitial cells are all positive for vimentin, MyHC-Apla1, and -Apla2. Differences in the distribution of labeled cells among the different cusps can be seen for smooth muscle (SM) α−actin and SM22. Arrows in F indicate SM myosin+ cells. Note that myofibroblasts (*) and SM cells are localized to the outermost layer of the cusp. Bar = 150 μm. (e = endothelium; MyHC-Apla1 = type-1 platelet myosin heavy chains; MyHC-Apla2 = type-2 platelet myosin heavy chains; niIgG = non-immune IgG; TM = thrombomodulin; vWf = von Willebrand factor.) The Annals of Thoracic Surgery 2003 75, 1274-1282DOI: (10.1016/S0003-4975(02)04706-9)

Fig 3 Immunofluorescence staining (A–F, H) of valve interstitial cell cultures from aortic valve leaflet explants obtained at the third passage. Note that although most cells are vimentin, MyHC-Apla1, and SM22 positive, none express smooth muscle (SM) myosin or von Willebrand factor (vWf). A proportion of cells are stained for actin and only very weakly for MyHC-Apla2. (G) Phase contrast of panel H. The red line encircles a typical cluster of epithelioid cells. Bars in A–F = 50 μm; bars in G–H = 30 μm. (MyHC-Apla1 = type-1 platelet myosin heavy chains; MyHC-Apla2 = type-2 platelet myosin heavy chains.) The Annals of Thoracic Surgery 2003 75, 1274-1282DOI: (10.1016/S0003-4975(02)04706-9)

Fig 4 Hematoxylin-eosin (H&E) staining (A, C, E) and Movat staining (B, D, F) of intact (A, B), decellularized (C, D), and decellularized+repopulated (E, F) aortic valve leaflets (AVL). Decellularization procedure was carried out in the presence of benzonase. Note that Movat stains the elastic fibers purple, the collagen fibers pink, and the proteoglycans blue. Bar = 100 μm. Box a in panel (E) indicates the valve region used for ultrastructural studies shown in Fig 7. The Annals of Thoracic Surgery 2003 75, 1274-1282DOI: (10.1016/S0003-4975(02)04706-9)

Fig 5 Electron micrograph illustrating a decellularized aortic valve leaflet treated with benzonase revealing normal appearing collagen fibrils (c), elastic fibers (e), and fibrillin microfibrils (f). Bar = 0.5 μm. The Annals of Thoracic Surgery 2003 75, 1274-1282DOI: (10.1016/S0003-4975(02)04706-9)

Fig 6 (A–I) Immunocytochemical identification of cells repopulating aortic valve leaflet bioscaffolds from left coronary cusp (middle part, ventricularis and spongiosa; images were taken from a region similar to that illustrated in Fig 4E, square “a”). Note that the majority of cells are stained for vimentin whereas fewer cells are actin+, rare are smooth muscle (SM) myosin+ (arrow) and none are stained for the two anti-MyHC-Apla isoforms. Anti-SM22, anti-von Willebrand factor (anti-vWf) and anti-TM antibody labeled a thin rim of transplanted cells. The latter also stains inner cells (asterisk). Bar = 110 μm. (MyHC-Apla1 = type-1 platelet myosin heavy chains; MyHC-Apla2 = type-2 platelet myosin heavy chains; niIgG = non-immune IgG; TM = thrombomodulin.) The Annals of Thoracic Surgery 2003 75, 1274-1282DOI: (10.1016/S0003-4975(02)04706-9)

Fig 7 Ultrathin sections of decellularized/repopulated aortic valve leaflet. (A) Two endothelial-like cells interacting with the bioscaffold join together (arrow) overpassing a furrow lined by basal lamina (arrowheads). (B) Endothelial-like cells closely adhering to basal lamina-like layer (double arrows). (C) A cell junction (arrow) is developing between two endothelial-like cells (double arrows). (D) Cell junctions between two myofibroblasts, or smooth muscle-like cells (arrowheads). (E) Oblong myofibroblast. (F) Myofibroblast demonstrating abundant microfilaments. (G –I) Fibroblast-like cells; note fibrillin microfibrils surrounding developing elastin fibers (double arrowheads) and collagen-fibril-forming channels (arrowheads). Bars: A, B, D, H, and I = 1 μm; C and F = 0.5 μm; E and G = 3 μm. The Annals of Thoracic Surgery 2003 75, 1274-1282DOI: (10.1016/S0003-4975(02)04706-9)