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. 2012 Jul;18(7):1148-53.
doi: 10.1038/nm.2821.

Fast-degrading elastomer enables rapid remodeling of a cell-free synthetic graft into a neoartery

Wei Wu  1 , Robert A Allen, Yadong Wang
Affiliations

Fast-degrading elastomer enables rapid remodeling of a cell-free synthetic graft into a neoartery

Wei Wu et al. Nat Med. 2012 Jul.

Abstract

Host remodeling is important for the success of medical implants, including vascular substitutes. Synthetic and tissue-engineered grafts have yet to show clinical effectiveness in arteries smaller than 5 mm in diameter. We designed cell-free biodegradable elastomeric grafts that degrade rapidly to yield neoarteries nearly free of foreign materials 3 months after interposition grafting in rat abdominal aorta. This design focuses on enabling rapid host remodeling. Three months after implantation, the neoarteries resembled native arteries in the following aspects: regular, strong and synchronous pulsation; a confluent endothelium and contractile smooth muscle layers; expression of elastin, collagen and glycosaminoglycan; and tough and compliant mechanical properties. Therefore, future studies employing large animal models more representative of human vascular regeneration are warranted before clinical translation. This cell-free approach represents a philosophical shift from the prevailing focus on cells in vascular tissue engineering and may have an impact on regenerative medicine in general.

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Figures

Figure 1
Figure 1
Characterization of the composite graft and host remodeling of the graft in vivo. (a) Schematic representation of direct implantation of the cell-free graft and the proposed remodeling process of the graft into a biological neo-artery. (b) SEM images of composite grafts to show surface topology, scale bar 100 μm. Inset: top view of the graft, scale bar 500 μm. (c) Lumen of the PGS tube, scale bar 100 μm. (d) The PCL fibrous sheath, scale bar 20 μm. (e) SEM of heparin soaked PGS after incubation in platelet rich plasma to show platelet adhesion and fibrin deposition. Scale bar 10μm. (f) SEM of unheparinized PGS after incubation in platelet rich plasma, scale bar 10 μm. An adherent platelet is circled in e and f to highlight difference in platelet morphology. Arrow indicates fibrin in (f). (g) Platelet adhesion on PGS presoaked in varying heparin concentrations, quantified by lactate dehydrogenase assay (n = 5). P < 0.0001 between all groups. (h) Suture retention and elastic moduli of grafts. Break force of 9–0 suture indicated by &. P < 0.05: * composite graft vs. 9–0 suture,+ composite graft vs. rat aorta, # composite graft modulus (n = 8) vs. that of PGS (grafts without the PCL sheath, n = 3). n = 5 for other groups. (i) Gross appearance of the graft during host remodeling to assess integration with host tissue. From left to right: day 0, day 14, day 90, and day 90 (top and bottom). Scale bars 2 mm, all ruler ticks 1 mm. Non-degradable sutures (black) mark the graft location. (j) Mechanical properties of neo-arteries. Left: The burst pressure of the neo-artery (n = 3) is statistically the same as native aorta (n = 4). Middle: Stress-strain curves of arteries and new grafts. “New grafts” represents unimplanted composite grafts. “PGS core” represents new grafts without the PCL sheath. Right: Compliance of arteries and new grafts. Standard errors for new grafts and the PGS core are very small and barely visible at the plotted scale. For j, n = 3 for neo-arteries and n = 4 for native aortas, new grafts, and PGS cores. Data represent mean ± standard deviation for e–g and mean ± standard error for j.
Figure 2
Figure 2
Smooth muscle cell infiltration and organization at 14 days. (a) Smooth muscle cell distribution (α-SMA, green) within the remodeled graft wall. The tissue was split longitudinally, half of which is shown. Native aorta is on the right, its border with the graft is indicated by the dashed line, scale bar 500 μm. L = lumen. (b) Magnified view of the mid-graft shows distribution of both α-SMA positive (green) and α-SMA negative cells. Nuclei counterstained by DAPI, scale bar 250 μm. (c) Further magnification of the mid-graft to view the complicated smooth muscle cell distribution (α-SMA, green), scale bar 50 μm. (d) Distribution of endothelial cells (vWF, red) and smooth muscle cells (α-SMA, green) in the graft wall. Immunofluorescent images merged with the brightfield image (darkened so as not to overwhelm the fluorescent images). Dark spots (*) in the brightfield image might be residual graft material. Scale bar 100 μm. brightfield images of original brightness are in Supplementary Fig. 2.
Figure 3
Figure 3
Remodeling of grafts. (a) H&E staining of the grafts during the transition into a neo-artery. An area of the neo-artery wall containing inflammatory cells is marked by (*). The top of the 14-day sample was trimmed to remove the adjoining vein. Image merged from a panel of 100 μ micrographs, scale bar 250 μm. (b) Magnified view of the vessel wall shows ECM content and alignment, scale bar 50 μm. (c) Luminal area of remodeling grafts to assess stenosis and aneurysm formation. There is no statistical difference between the two groups. (d) Changes in number and organization of macrophages (CD68, red). (e) Distribution of M2 macrophages (CD163, red). (f) Number of CD68 and CD163 positive cells quantifies total macrophage number and the proportion of M2 macrophages. (g) Changes of smooth muscle cell (α-SMA, green) organization over time. (h) Distribution of contractile smooth muscle cells (myosin heavy chain, red). All the immuofluorescent micrographs were counterstained for nuclei by DAPI (blue), scale bar 50 μm. Data represent mean ± standard deviation for c and f.
Figure 4
Figure 4
ECM organization and quantification at 90 days. (a) Verhoeff's, Masson's trichrome, and safranin O staining show elastin (black), collagen (blue), and glycosaminoglycansin (red). Immunofluorescent staining shows distribution of elastin (red), collagen I (green), and collagen III (red). Top: day 90 explant, bottom: native aorta. (b) Quantification of elastin (n = 4) and total collagen (n = 4). Data represent mean ± standard deviation.
Figure 5
Figure 5
Endothelialization of the neo-artery and vascular patency at day 90. (a) Laser Doppler ultrasound imaging assessment of graft patency and synchronization of pulsation with adjacent host aorta. Synchronization of pulsation also shown in Supplementary Video 1. (b) Angiography assessment of graft patency. Arrows indicate the graft location. (c) SEM of day 90 explant. Vessel split longitudinally, scale bar, 1 mm. Higher magnification micrographs (right) show lumenal surface at the anastomosis (suture indicated by the arrowhead, scale bar, 50 μm) and mid-graft (scale bar 5 μm). (d) Coverage of lumen by endothelial cells (vWF, red). Nuclei counterstained by DAPI (blue), scale bar, 500 μm. (e) Distribution of endothelial cells (vWF, red) and smooth muscle cells (α-SMA, green), scale bar 100 μm. (f) Transmission electron microscopy of interface between endothelial cells (EC) and smooth muscle cells SMC. Arrowheads denote a basement membrane. Scale bar 2 μm.
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