Rapid Endothelialization of Off-the-Shelf Small Diameter Silk Vascular Grafts - PubMed (original) (raw)

Rapid Endothelialization of Off-the-Shelf Small Diameter Silk Vascular Grafts

Elysse C Filipe et al. JACC Basic Transl Sci. 2018.

Abstract

Synthetic vascular grafts for small diameter revascularization are lacking. Clinically available conduits expanded polytetrafluorethylene and Dacron fail acutely due to thrombosis and in the longer term from neointimal hyperplasia. We report the bioengineering of a cell-free, silk-based vascular graft. In vitro we demonstrate strong, elastic silk conduits that support rapid endothelial cell attachment and spreading while simultaneously resisting blood clot and fibrin network formation. In vivo rat studies show complete graft patency at all time points, rapid endothelialization, and stabilization and contraction of neointimal hyperplasia. These studies show the potential of silk as an off-the-shelf small diameter vascular graft.

Keywords: ECM, extracellular matrix; PCL, polycaprolactone; PCNA, proliferating cell nuclear antigen; SMC-α, smooth muscle cell actin; ePTFE, expanded polytetrafluoroethylene; rat aortic interposition; silk; small diameter; vWF, von Willebrand factor; vascular graft.

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Graphical abstract

Figure 1

Figure 1

Mechanical Properties of Electrospun Silk Scaffolds for Vascular Grafting (A) Scanning electron micrograph of electrospun silk. Scale bar = 10 μm. (B) Cross section of electrospun silk (white), demonstrating scaffold porosity (black). (C) Representative amine I Fourier transform infrared spectroscopy spectra of silk prior to electrospinning (red), post-electrospinning (blue), and after cross-linking (black). (D) Proportion of β-sheet crystallinity in electrospun silk prior to electrospinning, post-electrospinning, and after cross-linking. Mechanical properties of silk, compared with expanded polytetrafluoroethylene (ePTFE) and fresh rat aorta as control scaffolds. (E) Youngs moduli, (F) ultimate tensile strength (UTS), and (G) percentage of elongation. Data are mean ± SEM; n = 9 to 12 (n = 2 for rat aorta). (H) Macroscopic images of electrospun silk graft. Scale bar = 1 mm. (I) Quantification of the maximum pressure before failure. Data are mean ± SEM; n = 6 (n = 3 for rat aorta). (J) Suture pull-out data of silk graft, with rat aorta as a control. Data are mean ± SEM; n = 7 (n = 3 for rat aorta).

Figure 2

Figure 2

Endothelial Cell and Blood Compatibility With Electrospun Silk Scaffolds for Vascular Grafting Representative scanning electron micrograph images of endothelial cell attachment on electrospun silk scaffolds (A) and expanded polytetrafluoroethylene (ePTFE) control scaffolds (B). Scale bar = 20 μm. Representative scanning electron micrograph images of endothelial cells on electrospun silk (C) and ePTFE scaffolds (D) at 1, 3, and 6 days post-seeding. Scale bar = 20 μm. (A to D) White arrows demonstrate cells and black arrows show scaffold surface. (E) Representative cross-sectional image of an endothelial cell monolayer on the surface of electrospun silk. Scale bar = 50 μm. vascular endothelial cadherin staining of endothelial cells on the surface of electrospun silk (F) and ePTFE (G). Scale bar = 25 μm. (H) Quantification of fluorescent fibrinogen on silk, polycaprolactone (PCL), and ePTFE scaffolds. Representative images of the scaffolds demonstrating fibrin network formation in green. (I) Macroscopic images of the whole blood assay on silk, PCL, and ePTFE scaffolds. Representative scanning electron micrograph images of these same scaffolds revealing red blood cells (black arrows) and fibrin deposition (white arrows) on the different scaffolds. Scale bar = 25 μm.

Figure 3

Figure 3

In Vivo Experimental Design in a Rat Model of Abdominal Aortic Replacement (A) Schematic of in vivo implantation procedure in rat. (B) Silk and (C) expanded polytetrafluoroethylene (ePTFE) control grafts at implantation and at the explantation time points of 6 and 24 weeks. (D) Kaplan-Meier curve depicting graft survival of silk (blue) and ePTFE (red). n = 21 and 22 for silk and ePTFE grafts, respectively.

Figure 4

Figure 4

Scanning Electron Microscopy of Silk and ePTFE Grafts at Explantation Scanning electron micrograph images of explanted silk (A) and expanded polytetrafluoroethylene (ePTFE) (C) grafts at 3, 6, 12, and 24 weeks. Scale bar = 500 μm. Representative high magnification images (black boxes in A and C) of silk (B) and ePTFE (D). Scale bar = 50 μm.

Figure 5

Figure 5

Immunohistologic Staining for vWF+ Cells in Silk Grafts at Explantation Representative micrograph images of von Willebrand factor (vWF) immunohistochemistry staining on cross sections of silk grafts at 3 (A), 6 (B), 12 (C), and 24 (D) weeks. vWF staining is red, and 4′,6-diamidino-2-phenylindole staining is blue. Scale bar = 200 μm. (E) Quantification of vWF coverage on the lumen of the graft depicting vWF positivity as early as 3 weeks and complete coverage by 12 weeks. N = 4 animals per time point with 3 images analyzed per region per graft. The red dashed line indicates the average vWF+ staining for each time point. Statistical analysis was performed on these values. D → P indicates selected equidistant regions within the graft, from distal to proximal.

Figure 6

Figure 6

Histologic Assessment of Neointima Formation and SMC Phenotype in Silk Grafts (A) Representative micrograph images of hematoxylin and eosin (H&E) staining of silk grafts at all time points. Scale bar = 200 μm. (B) Representative fluorescent micrograph image of proliferating cell nuclear antigen (PCNA)/smooth muscle cell actin (SMC-α) double stain at all explantation time points. Scale bar = 200 μm. (C) Quantification of neointima, represented as a percentage of total lumen size for each graft. N = 4 animals per time point with 3 images analyzed per region per graft. (D) Quantification of positive PCNA (red) and SMC-α (green) staining, represented as a percentage of the total positive staining in each graft section. N = 4 animals per time point with 3 images analyzed per region per graft. The dashed lines indicate the average neointima values (C) and average PCNA and SMC-α actin staining (D) for each time point. Statistical analysis was performed on these values. D → P indicates selected equidistant regions within the graft, from distal to proximal.

Figure 7

Figure 7

Granulation Tissue Response and Immune Cell Accumulation in the Adventitia of Silk Grafts (A) Representative micrograph images of hematoxylin and eosin (H&E) staining of the adventitia surrounding the silk grafts at all time points. Scale bar = 100 μm. (B) Representative fluorescent micrograph images of CD68 immunostaining at all time points. Scale bar = 100 μm. (C) Quantification of granulation tissue area, represented as a percentage of total lumen size for each graft. N = 4 animals per time point with 3 images analyzed per region per graft. (D) Quantification of positive CD68 staining, indicating macrophage presence, in the granulation tissue area, represented as a percentage of total granulation tissue for each section. N = 4 animals per time point with 3 images analyzed per region per graft. CD68 staining is red, and 4′,6-diamidino-2-phenylindole staining is blue. Scale bar = 100 μm. The red dashed lines indicate the average granulation tissue thickness values (C) and average CD68 staining (D) for each time point. Statistical analysis was performed on these values. D → P indicates selected equidistant regions within the graft, from distal to proximal.

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