Evaluation of small-diameter silk vascular grafts implanted in dogs

Abstract

Objectives: Vascular replacement is one treatment for cardiovascular disease. However, in blood vessels with a diameter less than 6 mm, the existing artificial vascular grafts may be occluded by thrombus formation or intimal hyperplasia. Thus, new artificial vascular grafts need to be developed. We have developed a small-diameter artificial vascular graft made of silk fibroin. The implantation of such graft has been evaluated mainly in rats. However, only a few reports describe long-term implantation in large animal models. Therefore, modified silk fibroin artificial vascular grafts were implanted in the femoral arteries of dogs, and their patency and remodeling ability were investigated.

Methods: Six beagles weighing 10 to 12 kg were used for the in vivo study. Grafts (4 cm length × 3.5 mm inner diameter) were implanted in the femoral artery of 6 dogs. The patency of the graft was monitored using vascular ultrasound apparatus. At 3 months', 5 months', and 1-year postimplantation, the graft was retrieved and conducted histopathologic examination.

Results: No side effects, such as ischemia, paralysis, and edema of the hind legs, were observed postimplantation. Five of the 6 grafts exhibited a high patency rate, and the lumen was covered with vascular endothelial cells in the central part of the graft 3 months' postimplantation.

Conclusions: Based on these results, artificial silk fibroin vascular grafts implanted in the femoral arteries of dogs exhibit high patency and remodeling ability. Silk fibroin grafts may be clinically applicable as an artificial vascular graft in small-diameter <6 mm.

Keywords: SF, silk fibroin; ePTFE, expanded polytetrafluoroethylene; endothelialization; large animal model; remodeling ability; silk fibroin; small-diameter vascular grafts.

SF artificial vascular grafts were implanted in the femoral arteries of dogs, and their patency and remodeling ability were investigated. Five of the 6 grafts exhibited a high patency rate, and remodeling ability. SF grafts may be clinically applicable as an artificial vascular graft in small-diameter <6 mm. SF, Silk fibroin.

Remodeling to the autologous tissue was observed at 3 months' postimplantation.

Figure 1

Process of preparation of a SF vascular graft coated by SF sponge using glycerin. SF, Silk fibroin; PTFE, polytetrafluoroethylene.

Figure 2

A, A 3.5-mm-inner-diameter and 4-cm-length artificial vascular graft was implanted in the femoral artery of a beagle. Scanning electron microscope pictures of the morphologies of the inner (B) and outer surfaces (C) of the silk fibroin vascular grafts.

Figure 3

Photographs of artificial vascular grafts at the time of implantation. A, Artificial vascular grafts immediately after implantation in the femoral artery of a beagle; B, artificial vascular grafts after blood flow resumed; and C, artificial vascular grafts after hemostasis. A small amount of bleeding was observed in the graft, but it could be stopped by compression for several minutes. Photographs of vascular grafts at D, 3 months; E, 5 months; and F, 1 year postimplantation. The implanted vascular grafts adhered to the surrounding tissue and could be easily separated at all time points.

Figure 4

Vascular ultrasound examination of the implanted artificial vascular grafts. Vascular ultrasound photograph 4 weeks after transplantation. A structure with high echogenicity was confirmed on the lumen surface of the bent artificial vascular graft (A: circled). Blood flow could be confirmed via color Doppler. However, no blood flow was observed at the site of the thrombus (B: arrow). Vascular ultrasound photograph of artificial vascular graft 1 year postimplantation (C). No bending or aneurysm of the artificial vascular graft was observed. D, In addition, the blood flow was confirmed using a color Doppler scan.

Figure 5

Peak systolic velocity and end-diastolic velocity values of the central part of the graft postimplantation. No significant change was observed in all postimplant periods. Peak systolic velocity preimplantation was 101.25 ± 12.31, 1-month postimplantation was 102.51 ± 8.04, 3-month postimplantation was 106.04 ± 5.31, 5-month postimplantation was 92.63 ± 8.88, and 1-year postimplantation was 117.05 ± 3.75. End-diastolic velocity preimplantation was 16.51 ± 4.46, 1-month postimplantation was 18.46 ± 5.164, 3-month postimplantation was 21.28 ± 4.25, 5-month postimplantation was 14.9 ± 4.52, and 1-year postimplantation was 22.2 ± 3.9.

Figure 6

HE and MTC staining 3 months, 5 months, and 1-year postimplantation. HE staining at 3 months' postimplantation (A) and its high-magnification image (D), HE staining at 5 months' postimplantation (B) and its high-magnification image (E), HE staining at 1-year postimplantation (C) and its high-magnification image (F), MTC staining at 3 months' postimplantation (G) and its high-magnification image (J), MTC staining at 5 months' postimplantation (H) and its high-magnification image (K), and MTC staining at 1-year postimplantation (I) and its high-magnification image (L) are presented. Layered structures formed in the lumen of the artificial vascular graft at 3 months' postimplantation, and their thickness increased 5 months and 1-year postimplantation. Collagen fibers were mainly gathered around the outer periphery of the artificial vascular graft 3 months' postimplantation. At 5 months and 1-year postimplantation, the collagen fibers on the luminal surface increased, and the collagen fibers on the outer peripheral surface decreased.

Figure 7

Photographs at 3 months' postimplantation. A, Elastica van Gieson staining, B, α-smooth muscle actin staining, and C, CD31 staining are presented. Elastic fibers, smooth muscle cells, and endothelial cells were observed in the intimal layer of artificial vascular grafts and their surfaces.