In vivo cellular repopulation of tubular elastin scaffolds mediated by basic fibroblast growth factor

Abstract

In vivo tissue engineering has been explored as a method to repopulate scaffolds with autologous cells to create a functional, living, and non-immunogenic tissue substitute. In this study, we describe an approach to in vivo cellular repopulation of a tissue-derived tubular elastin scaffold. Pure elastin scaffolds were prepared from porcine carotid arteries (elastin tubes). Elastin tubes were filled with agarose gel containing basic fibroblast growth factor (bFGF) to allow sustained release of growth factor. These tubes were implanted in subdermal pouches in adult rats. The elastin tubes with growth factor had significantly more cell infiltration at 28 days than those without growth factor. Immunohistochemical staining indicated that most of these cells were fibroblasts, of which a few were activated fibroblasts (myofibroblasts). Microvasculature was also observed within the scaffolds. Macrophage infiltration was seen at 7 days, which diminished by 28 days of implantation. None of the elastin tubes with bFGF calcified. These results demonstrated that the sustained release of bFGF brings about repopulation of elastin scaffolds in vivo while inhibiting calcification. Results showing myofibroblast infiltration and vascularization are encouraging since such an in vivo implantation technique could be used for autologous cell repopulation of elastin scaffolds for vascular graft applications.

Figure 1

In vitro release of bFGF from agarose gels (A) shows 70% release in 7 days. In vivo release of bFGF (shown as triangles) matches well with in vitro results. The total release in vitro was ~92% in 10 days. Fig 1(B) shows the gradual decrease of bFGF in the gel and a corresponding increase in the surrounding scaffold showing the sustained release through the gel and into the scaffold.

Figure 2

Macroscopic and histological features of explanted EL scaffolds (A) shows the thin capsule over the explants (B) shows the structural integrity of the explants. 7d EL-Gel (C), 7d EL-Gel-FGF (D) and 28d EL-Gel-FGF (E) representative images. Host cells could only infiltrate scaffolds by abluminal side. There is visibly reduced cell infiltration in EL-Gel group. Gomori’s trichrome staining (F) shows new collagen fibers between layers of elastin only in the 28d EL-Gel-FGF group. The red lines indicate the scaffold-capsule interface. H&E stain (cell nuclei are dark blue and matrix is pink), Gomori’s trichrome stain (cell nuclei are dark blue, collagen is blue – green and elastin is pink. Original magnifications (C –E) 200 X and (F) 400 X.

Figure 3

Immunohistochemical characterization of infiltrating cells in EL-Gel-FGF scaffold. A) immunostained for fibroblasts at 28 day, B) immunostained for smooth muscle α-actin at 28 day, C) immunostaining for macrophages at 7 day, D) immunostaining for macrophages at 28 day. The red lines indicate the scaffold-capsule interface. Insets show negative stains. Original magnifications 400 X.

Figure 4

Quantification of DNA within explanted scaffolds. At 28 days EL-Gel-FGF scaffolds had significantly more DNA (p<0.05)

Figure 5

Uniaxial tensile testing of fresh artery and scaffold rings. By 28 days, EL scaffolds were stiffer and less elastic.

Figure 6

Degradation of EL scaffolds in vivo. A) Gel image showing MMPs 2 and 9 in EL-Gel scaffolds 7day (Lane 1), EL-Gel scaffolds 28day (Lane 2), EL-Gel-FGF scaffolds 7day (Lane 3) and EL-Gel-FGF scaffolds 28day (Lane 4). B) VVG stain for elastic fibers. Arrows indicate degraded fibers on abluminal side (EL-Gel-FGF) at 28 days. Degradation was observed in both EL-Gel and EL-Gel-FGF samples at 28 days. Original magnification 200 X.