Wharton's Jelly-Mesenchymal Stem Cell-Engineered Conduit for Pulmonary Artery Reconstruction in Growing Piglets

Abstract

Surgical treatment of congenital heart defects affecting the right ventricular outflow tract often requires complex reconstruction and multiple reoperations. With a randomized controlled trial, we compared a novel tissue-engineered small intestine submucosa-based graft for pulmonary artery reconstruction (seeded with mesenchymal stem cells derived from Wharton's Jelly) with conventional small intestine submucosa in growing piglets. Six months after implantation, seeded grafts showed integration with host tissues at cellular level and exhibited growth potential on transthoracic echocardiography and cardiovascular magnetic resonance. Our seeded graft is a promising biomaterial for pulmonary artery reconstruction in pediatric patients with right ventricular outflow tract abnormalities.

Keywords: CMR, cardiovascular magnetic resonance; FISH, fluorescent in situ hybridization; MPA, main pulmonary artery; RVOT, right ventricular outflow tract; SIS, small intestinal submucosa; SMA, smooth muscle actin; TTE, transthoracic echocardiography; WJ-MSC, Wharton’s Jelly–mesenchymal stem cells; growing swine model; right ventricular outflow tract reconstruction; small intestinal submucosa; tissue engineering.

Graphical abstract

Figure 1

Graft Preparation and in Vivo Implantation (A) Circumferential graft created from patch of small intestinal submucosa (running longitudinal suture visible). (B) Graft after implantation on main pulmonary artery.

Figure 2

Doppler Analysis (A,B) Color Doppler images of the main pulmonary artery at baseline and 6 months following graft implantation in seeded and unseeded animals. Box plots summarizing Doppler measurements on MPA (C) and RVOT (D) at baseline and at 6-month follow-up, in unseeded and seeded animals (n = 4, ± SD). MPA = main pulmonary artery; RVOT = right ventricular outflow tract.

Figure 3

Cardiac Magnetic Resonance Analysis (A) Images of the RVOT/MPA at baseline and 6 months following graft implantation in seeded and unseeded animals. (B) Cardiac magnetic resonance long-axis view of the RVOT/MPA after implantation, showing lack of growth in an unseeded animal (yellow asterisk), and better growth and remodeling with no graft stenosis in a similar picture of a seeded graft. (C,D) Box plots summarizing MPA area and flow measurements on cardiovascular magnetic resonance at baseline and at 6-month follow-up, in unseeded and seeded animals. MPA area growth was significant in seeded graft, but not in unseeded grafts (n = 4, ± SD). Abbreviations as in Figure 2.

Figure 4

Histological Findings on Explanted MPAs and Grafts (A) Representative image of the explanted acellular and cell-engineered conduit. (B) Von Kossa staining showing no calcification in the grafts. (C) Hematoxylin and eosin staining showing extensive nucleation throughout the structure of seeded and unseeded grafts. (D) Van Gieson staining of collagen (pink) and elastin (purple) content of the grafts. (E) Immunohistochemistry showing elastin expression (brown staining) in the unseeded and seeded grafts. (F) Box plots showing elastin concentration, as quantified by histology and immunohistochemistry, in unseeded/seeded grafts and in the native MPA (n = 4, ± SD). Zoomed pictures in B, C, D and E indicated by dashed lines. Scale bars = 1,000 μm, 50 μm higher magnification. Abbreviation as in Figure 2.

Figure 5

Immunohistochemistry of Explanted Native MPAs and Grafts Representative images showing longitudinal sections of the grafts, far from the anastomotic sites. A highly organized layer of smooth muscle cells (smooth muscle actin [SMA]–positive, red) and a newly formed layer of endothelial cells (isolectin-positive, green), similar to the native MPA (C), were more pronounced in the seeded graft (B), compared to the unseeded graft (A). 4′,6-diamidino-2-phenylindole was used to mark nuclei (blue). The isolectin-positive cell concentration was similar between native MPA and seeded graft, whereas the slightly different staining pattern is most likely artifactual. Panoramic pictures (small) and corresponding zoomed pictures (large). (D) Box plot showing the different SMA concentrations in unseeded/seeded grafts and in native MPA (n = 4, ± SD). Scale bars = 20 μm, 10 μm higher magnification. Abbreviation as in Figure 2.

Figure 6

Y Chromosome Fluorescent In Situ Hybridization of the Explanted Grafts and Non-Target Tissues The male seeded cells (Y chromosome-positive, red) were detected on the MPA before implantation and 1 week and 2 months postoperatively. The presence of seeded cells decreased with time in vivo. No seeded cells were observed in the pulmonary artery away from the grafting site or in any of the non-target tissues after 2 months. The right ventricle of a male pig was used as a positive control. 4′,6-diamidino-2-phenylindole was used to mark nuclei (blue). Scale bars = 50 μm. Abbreviation as in Figure 2.