Nerve regeneration and elastin formation within poly(glycerol sebacate)-based synthetic arterial grafts one-year post-implantation in a rat model

Abstract

The objective of this study was to evaluate the long-term performance of cell-free vascular grafts made from a fast-degrading elastic polymer. We fabricated small arterial grafts from microporous tubes of poly(glycerol sebacate) (PGS) reinforced with polycaprolactone (PCL) nanofibers on the outer surface. Grafts were interpositioned in rat abdominal aortas and characterized at 1 year post-implant. Grafts remodeled into "neoarteries" (regenerated arteries) with similar gross appearance to native rat aortas. Neoarteries mimic arterial tissue architecture with a confluent endothelium and media and adventita-like layers. Patent vessels (80%) showed no significant stenosis, dilation, or calcification. Neoarteries contain nerves and have the same amount of mature elastin as native arteries. Despite some differences in matrix organization, regenerated arteries had similar dynamic mechanical compliance to native arteries in vivo. Neoarteries responded to vasomotor agents, albeit with different magnitude than native aortas. These data suggest that an elastic vascular graft that resorbs quickly has potential to improve the performance of vascular grafts used in small arteries. This design may also promote constructive remodeling in other soft tissues.

Keywords: Degradation; Elastin; Elastomer; Nerve; Vascular grafts.

Figure 1. Gross morphology and tissue architecture of neoarteries resemble native arteries

A. Gross morphology of neoarteries. Top left: transformation of graft into neoartery in situ over the course of 1 year. Nondegradable sutures (black) mark the graft location. Top Right: Transverse view of explanted neoarteries resembles that of native aortas. Bottom: Longitudinal view of explanted neoarteries resembles the adjacent native aorta. All ruler ticks are 1 mm. B. H&E stained transverse sections of the middle of neoarteries show similar tissue architecture with native aortas, with no visible graft material residues. Scale bar 100 μm. C. Neoartery sections immunostained for von Willebrand factor (vwf, red) and α-smooth muscle actin (α-SMA, green). The luminal surface of neoarteries is completely covered by vWF positive cells (red), suggesting a confluent endothelium. Neoarteries contain a media-like middle layer of the vascular wall rich in α-SMA positive cells with circumferentially elongated nuclei, similar to vascular smooth muscle found in native aortas. The outermost layer of neoarteries lacks α-SMA, resembling native adventitia. Some cells in the media-like layer are negative for α-SMA, and some cells adjacent to the endothelium are α-SMA positive but not circumferentially elongated. Scale bar 100 μm. L indicates vessel lumen. Nuclei stained with DAPI (blue). D. En face view of the luminal surface of neoarteries shows complete coverage by CD31 positive cells with cobblestone-like morphology and alignment parallel to the direction of blood flow, an arrangement similar to that found in native aortas. Neoarteries were cut open longitudinally and imaged as whole mounts using confocal microscopy and z-stack flattening. Arrow indicates the direction of blood flow. Scale bar 100 μm. E. Immunostaining for smooth muscle myosin heavy chain 11 (MHC-11, red). Many elongated cells in the media-like layer are positive for MHC-11, suggesting a mature, contractile smooth muscle phenotype. The distribution of MHC-11 appears similar to that of α-SMA. MHC-11 positive cells in neoarteries stain brighter than those in native aortas at the same exposure. Scale bar 100 μm. L indicates vessel lumen. Nuclei stained with DAPI (blue).

Figure 2. Neoarteries resist common modes of late-term graft failure

A. Neoartery inner diameter is not significantly different from that of age-matched native aortas, suggesting resistance to dilation and narrowing caused by neointimal hyperplasia. B. Neoarteries are negative for von Kossa staining (dark brown indicates a positive stain), suggesting resistance to calcification. Bone (rabbit ulna) was stained with von Kossa as a positive control for calcified tissue. Scale bar 500 μm. C. Neoarteries contain cells positive for macrophage marker CD68, concentrated mostly in the outermost layer of the neoartery wall. Scale bar 100 μm. Nuclei stained with DAPI (blue).

Figure 3. Regeneration of nerves in neoarteries

A. Schematic of en face imaging of the adventitial surface of neoarteries. Neoarteries were cut open, immunostained for protein gene product 9.5 (PGP 9.5) and β-Tubulin, and imaged as whole mounts using confocal microscopy with z-stack flattening. B. Nerves cover the adventitial surface of neoarteries with similar morphology to those in native aortas. Nerves appear yellow from the overlap of nerve-specific marker PGP 9.5 (red) and nerve non-specific marker β-Tubulin (green), which stains nerves brightly but is also expressed ubiquitously. The co-staining technique was used to reduce effects of nonspecific staining which are amplified in whole mount imaging. Scale bar 100 μm.

Figure 4. Extracellular matrix and mechanical properties of neoarteries resemble native arteries

A. Elastin quantification using a Fastin Elastin assay showed that neoarteries contain the same amount of cross-linked elastin per tissue wet weight as native aortas. B. Transverse cross-sections of neoarteries immunostained for vascular extracellular matrix proteins. Neoarteries contain elastin, fibrillin-1, collagen III, and collagen I. Elastin and fibrillin-1 are less uniformly distributed in the media-like layer of neoarteries than in native aortas. Collagen III appears ubiquitously distributed in neoarteries, whereas in native arteries it localizes to vascular media. Collagen I is less bright in the neoartery adventitia compared with adventitia in native aortas. L indicates vessel lumen. Scale bar 100 μm. Nuclei stained with DAPI (blue). C. In vivo dynamic mechanical compliance of neoarteries shows no statistically significant difference from native aortas. Compliance was measured in vivo using ultrasound to record neoartery inner diameter and a manometer implanted in the aorta to record pressure. Compliance was taken as % change in diameter normalized to the difference between systolic and diastolic pressure.

Figure 5. Elastin and collagen architecture in neoarteries and native aortas

All images are en face views of matrix autofluorescence imaged using multiphoton microscopy. Detailed depiction of en face view shown in Supplementary Fig. 1. Scale bars 25 μm. A. Elastin architecture within the neoartery wall, imaged > 10 μm from the luminal surface (left), or within 10 μm of the lumen (right). Elastin is arranged as a fibrous network in neoarteries, in contrast to the fenestrated lamellae seen in native aortas. “Fe” indicates fenestrations in elastic lamellae in native aortas. “Fold” notes where the lamellae drops below the viewing plane (schematic at bottom of A). B. Collagen architecture within the neoartery wall, viewed from the luminal side. Collagen is less aligned and less crimped than collagen seen in native aortas. Two regions from the same vessel segment (Region 1 and Region 2) are shown to demonstrate variation in collagen architecture within the same neoartery. Regions 1 and 2 represent different circumferential locations at the same longitudinal position near the middle of the regenerated vessel. In both regions, collagen at > 10 μm from the luminal surface is oriented in the circumferential direction, as seen in the vascular media of native aortas. However, collagen at < 10 μm from the lumen can either retain its orientation (Region 1) or substantially shift its orientation (Region 2), the latter contrasting with collagen near the intima of healthy native aortas. Arrows indicate the circumferential direction (white) and direction of orientation (blue). C. Collagen and elastin architecture near the abluminal surface of neoarteries. Left: Neoarteries contain little elastin near the abluminal surface, similar to the adventitia of native aortas. Right: Neoartery collagen near the abluminal surface is undulated resembling native adventitial collagen, but has higher undulation frequency than native arteries. Neoartery collagen alignment is stronger than that in native adventitia.

Figure 6. Neoarteries display sensitivity to vasodilators and vasoconstrictors

A. Response to physiologic vasoconstrictors. Neoarteries were freshly harvested, mounted on a standard dual pin myograph, resting tension established, then assessed in their response to vasoconstrictors. Neoarteries demonstrated constriction in response to both the selective α1-adrenergic receptor agonist phenylephrine (PE) and the 5-hydroxytryptamine receptor activator serotonin (5-HT). However, the magnitude of constriction was less than native arteries (32.5 +/− 2.78 vs. 1616 +/− 836.6 mg and 42.2 +/− 25.8 vs. 3485 +/− 1161 mg for PE and 5-HT, respectively). Results are the mean ± SEM of n=12 rings from 3 animals per treatment group. * P > 0.05. B–D. Response to physiologic vasodilators. Neoarteries were mounted as described in A, preconstricted with serotonin, then assessed in their response to vasodilators. Results are the mean ± SEM of n=12 rings from 3 animals per treatment group. * P < 0.05. B. Neoarteries relax in response to the endothelial specific activator acetylcholine (Ach, 10 μM), suggesting signaling capability between endothelial cells and contractile smooth muscle. C. Neoarteries relax in response to the cAMP activator (NO-independent) forskolin (10 μM). D. Neoarteries respond to the vascular smooth muscle cell specific activator sodium nitroprusside (SNP) in a dose-dependent manner.