Biological Characterization of Human Autologous Pericardium Treated with the Ozaki Procedure for Aortic Valve Reconstruction

Figures

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  Figure 1. Histological characterization of pre-Ozaki and post-Ozaki samples. (A) Representative H/E image of the pre-Ozaki pericardium showing an intact layer of serosa (s) made up of mesothelial cells and a thicker fibrosa (f) layer consisting of collagen bundles and elastic fibers produced by its constituent fibroblasts. (B) H/E staining of the post-Ozaki pericardium shows preservation of the native ECM; nevertheless, the serous layer appears interrupted, whereas the fibrous layer is denser and compact. (C) Representative image of native pericardium after Weigert van Gieson staining shows variously oriented and wave-like collagen fibers (in red) with interspersed small elastic fibers (in black). (D) Weigert van Gieson staining of GA-treated pericardium shows changes in the architectures of collagen and elastin, which appear more stretched.

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  Figure 2. SEM morphological analysis of pre-Ozaki and post-Ozaki samples. (A,C) Representative images of the serosa layer in the pre-Ozaki pericardium show a continuous layer of mesothelial cells, whose surface is covered by short and abundant microvilli. (B,D) This layer appears interrupted in some areas (red box) in the post-Ozaki pericardium. (E,G) Representative images of the surface of the fibrosa layer in the pre-Ozaki pericardium display overlapping wavy collagen and elastic fibers. (F,H) These fibers assume a more parallel and compact arrangement in the post-Ozaki pericardium. Representative images at 1500× (A,B,E,F) and 7000× (C,D,G,H) magnifications.

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  Figure 3. Evaluation of cell viability in peri cardial samples after treatment with GA (post-Ozaki) in comparison to native pericardium (pre-Ozaki). (A) MTT assay shows that residual cell viability in post-Ozaki samples is 33.32 ± 1.29% when compared to that of pre-Ozaki samples. **** p < 0.0001; n = 15. (B,C) Representative images of pre-Ozaki and post-Ozaki samples, respectively, showing live (green) and dead (red) cells, and their quantification (graphs in panel (B) and panel (C), respectively). **** p < 0.0001; n = 15. (D) Representative images of nuclear morphology following Hoechst staining (blue) and evaluation of nuclei dimension (left graph), circularity factor (center graph), and Hoechst intensity (right graph). * p < 0.05, ** p < 0.01; n = 15.

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  Figure 4. Citotoxicity assay, endothelialization, and expression of pro-inflammatory mediators in the post-Ozaki pericardium and BP control membranes. Morphology of L929 mouse fibroblasts cultured (A) without tested materials (blank), (B) in the presence of 0.6% GA (positive control), or grown in contact with (C) the commercial BP membrane (negative control) and (D) post-Ozaki samples. (E) Quantification of cytotoxicity by the MTT assay. Data are expressed as a percentage of viability referred to control cells growing without tested materials. *** p < 0.001, **** p < 0.0001; n = 15. (F) Quantification of endothelial cell number on the fibrous side of pericardium 4 days after seeding. **** p < 0.0001; n = 15. Representative images of HUVEC, labelled with the vital dye calcein-AM (in green) and the nuclear dye Hoechst (in blue), adhering on the BP membrane (left) and on post-Ozaki pericardium (right). Quantification of (G) HMGB1 and (H) IL-6 from cell culture supernatant after 4 days from seeding. * p < 0.05, **** p < 0.0001; n = 15.