Rapid remodeling observed at mid-term in-vivo study of a smart reinforced acellular vascular graft implanted on a rat model

Abstract

Background: The poor performance of conventional techniques used in cardiovascular disease patients requiring hemodialysis or arterial bypass grafting has prompted tissue engineers to search for clinically appropriate off-the-shelf vascular grafts. Most patients with cardiovascular disease lack suitable autologous tissue because of age or previous surgery. Commercially available vascular grafts with diameters of < 5 mm often fail because of thrombosis and intimal hyperplasia.

Result: Here, we tested tubular biodegradable poly-e-caprolactone/polydioxanone (PCL/PDO) electrospun vascular grafts in a rat model of aortic interposition for up to 12 weeks. The grafts demonstrated excellent patency (100%) confirmed by Doppler Ultrasound, resisted aneurysmal dilation and intimal hyperplasia, and yielded neoarteries largely free of foreign materials. At 12 weeks, the grafts resembled native arteries with confluent endothelium, synchronous pulsation, a contractile smooth muscle layer, and co-expression of various extracellular matrix components (elastin, collagen, and glycosaminoglycan).

Conclusions: The structural and functional properties comparable to native vessels observed in the neoartery indicate their potential application as an alternative for the replacement of damaged small-diameter grafts. This synthetic off-the-shelf device may be suitable for patients without autologous vessels. However, for clinical application of these grafts, long-term studies (> 1.5 years) in large animals with a vasculature similar to humans are needed.

Keywords: 3D printing; Electrospinning; Medium-term performance; Nanofibers; Rat abdominal aorta replacement model; Tissue regeneration; Vascular graft.

Fig. 1

Structural characterization of small-diameter grafts, surgical procedure overview, and remodeling at 3 months post-implantation. A As-fabricated PCL/PDO grafts have an internal diameter of ~ 1.7 mm and length of 15 mm. B Cross-sectional scanning electron microscope images shows wall thickness of ~ 100 μm (magnified in the inset). C Scanning electron microscope image shows a side view of graft with 3D reinforcement and a microfibrous outer layer. D Gross image of isolated infrarenal abdominal aorta and corresponding PCL/PDO graft prior to implantation. E Gross surgical view of PCL/PDO graft anastomosis. The rat’s head is situated beyond the upper left quadrant of the image. F Graft remodeling at 3 months post-implantation. White arrows indicate the formation of tiny visible blood vessels on the engineered graft. Scale bar, 5 mm. G A 1.7-mm graft explanted from a rat at 3 months post-implantation demonstrates the formation of endothelium (black arrow, a) and an adventitial layer (black arrow, b). H A 1.7-mm PCL/PDO graft was explanted from the rat aorta at 3 months post-implantation (arrow indicates anastomotic suture line)

Fig. 2

Monoplanar (anteroposterior) projection images of digital subtraction angiography at 12 weeks after implantation (n = 3). A Normal rat abdominal aorta (control). B PCL/PDO graft indicated by red lines. C B-mode ultrasonography with a patent PCL/PDO graft indicated by the blue dotted line. D Color Doppler showing blood flow through the graft. E Pulse Doppler

Fig. 3

Remodeling of the PCL/PDO graft at 12 weeks after implantation compared with native aorta. Top and bottom rows show the native vessel and PCL/PDO graft, respectively. A and F H&E staining. B and G Masson’s trichrome staining reveals collagen in the explanted graft and native aorta. C and H Safranin O staining. D and I Verhoeff-Van Gieson staining. E and J Orcein staining for Elastic fibers. Scale bars: top row, 200 μm (inset, 20 μm); bottom row, 500 μm (inset, 20 μm). L = lumen

Fig. 4

Tissue remodeling and extracellular matrix deposition in explanted grafts at 3 months compared with the native aorta. A Cross-sectional images of native vessels and regenerated grafts (top and bottom rows, respectively) were immunostained to examine endothelial cells (green), smooth muscle cells (red), and the distribution of contractile smooth muscles (myosin heavy chain [red]). Endothelial cells were stained with anti-vWF antibody (first column; green, DAPI is stained blue; P = 0.007). Smooth muscle cells were stained with anti-α-SMA antibody. B L = Lumen area (± standard deviation) of PCL/PDO grafts measured from histological images in millimeters (P = 0.01). C Mean fluorescence intensity of vWF-positive cells measured from immunofluorescence images. Mean fluorescence intensity of myosin heavy chain (MHC)-positive cells measured from immunofluorescence images (P = 0.0026). Data are mean ± standard deviation. *P < 0.05; n = 5

Fig. 5

A Representative fluorescence micrographs of immunostaining for CD68 (pan-macrophage marker) at 3 months, indicated by the yellow arrows (left column). CD68 was stained red, and nuclei were counterstained with DAPI (blue). Immunostaining of collagen I/III (right column). Collagen type I was stained green, collagen type III was stained red, and cell nuclei were counterstained with DAPI (blue). Images are representative of at least three independent samples. L = lumen. B Wall thickness differed between native vessels and PCL/PDO grafts at 3 months

Fig. 6

Scanning electron micrographs of the luminal surface of explanted PCL/PDO grafts at 12 weeks compared with native vessels. A Representative image of a neovessel (PCL/PDO graft). B Anastomotic site; the green dashed line represents the site of anastomosis. C Native vessel. D High magnification of the area within the brown dashed-line box in A. E Magnified portion of the anastomotic site, with suture holes indicated by yellow arrows; green line indicates anastomotic site and the transition from native vessel to neovessel). F Magnified image of autogenous rat aortic arch wall (green dashed-line box in C)