Figure 1.
Graphic representation of the two types of experiments.
A) A pig autologous end-to-end peritoneal implant was used to study patency, eventual thrombosis, endothelization and arterializations of the graft, 2 weeks after surgery. B) A human-to-rat peritoneal graft approach was set up to study remodeling of the peritoneum and to determine the host vs donor origin of cells that contributed to the process of endothelization and eventual arterialization.
Figure 2.
Macroscopic representation of graft preparation, implantation and removal in pigs.
A–B) Tailoring of the peritoneal graft on a tutor. C) Coverage of the peritoneal graft with the metal mesh. D) Proximal anastomosis showing the adhesion of the peritoneum to the metal mesh when pulsatile flow is established into the graft. E) Graft harvested (A, native aorta; P, peritoenal graft) with no signs of overt dilatation. F) Fresh transversal equatorial cut of the peritoneal graft showing patency and (G) thickening of the peritoneal graft at 15 days from the implant. H) Intraluminal appearance at the proximal anastomosis (A, native aorta; P, peritoenum graft). Scale bars 1 cm.
Figure 3.
Histopathological analysis of the peritoneal patch explanted from pigs.
Peritoneal graft pre-innestum (A), native aorta (B, C) and graft after two weeks (D–J). A) normal peritoneum, double staining with alfa-SMA (blue) and elastic von Gieson (black-brown) magnification 80×, scale bar 40 µm. B) native aorta, double staining showing the media with elastic fibres in black and smooth muscle cells in blue, magnification 50×, scale bar 50 µm. C) Native aorta, von Willebrand factor staining of the endothelium, magnification 160×, scale bar 20 µm. D) Graft and metallic scaffold (arrows indicate some of the holes around the graft), magnification 12,5×, scale bar 250 µm. E) graft remodeling, magnification 31× scale bar 100 µm. F) Arterialization of graft, magnification 62,5×, scale bar 50 µm. G) Intimal hyperplasia, magnification 125×, scale bar 25 µm. H, I) Endothelization of the graft, von Willebrand factor staining magnification 160× (scale bar 20 µm) and 320× (scale bar 10 µm), respectively.
Figure 4.
Histopathological analysis of the aortic peritoneal patch explanted from rats.
Sections of the native aorta (top panels) and the patched aorta (middle panels and details in the bottom panels), were stained with hematoxylin and eosin, Masson's trichrome and using immunofluorescence for endothelium (vWf) and smooth muscle cells (alpha-SMA). The peritoneal patch (at the top) was covered by an endothelial layer, but there was no tunica media (red staining). Compared with the native aorta, the transversal sections containing the peritoneal patch showed extensive thickening, formed by a densely cellular reactive tissue. Detail panels in the bottom of the figure show that such tissue contained elongated cells, collagen, as well as capillaries and arterioles.
Figure 5.
Origin of peritoneal patch remodeling cells.
The remodeled patch developed as a new artery wall, with an endothelial layer. We asked whether cells that contributed to patch remodeling were of host (rat) or peritoneal (human) origin, by double staining with vWf (green in B–F) or alpha-SMA (green in H–M)) and human mitochondri (red). A–C) Within the densely cellulated reactive tissue that thickened the peritoneal patch, a few capillary-lining endothelial cells (green in B) were of human origin (red, higher magnification in panel C). D–F) Within the neo-formed endothelial layer (green in E), some cells (red in D) were of human origin (higher magnification in panel F). G–J) In the remodeled patch tissue, rare alpha-SMA staining cells (green in H and J) co-stained with human mitochondri (red). K–M) The same was in the close vicinity of the native aorta-peritoneal patch junction, where a medial smooth muscle layer (green in K, L) is still present.
Figure 6.
Progenitor cell markers in normal and patched aortic sections.
A–C) Aortic sections including the peritoneal patch (C, scale bar 500 µm) were co-stained for c-kit and CD140b, also known as PDGFRB (A, scale bar 100 µm), or c-kit and NG2 (B, scale bar 100 µm). Panel D (scale bar 100 µm) shows c-kit staining of an endothelial area overlying the peritoneal patch (scale bar 100 µm). Panel E shows c-kit staining in a section of a normal aorta (scale bar 100 µm). In panel D and E, there is no c-kit signal from nucleated cells above the green background attributable to elastic fibers autofluorescence.