Growth capacity of a Wharton's Jelly derived mesenchymal stromal cells tissue engineered vascular graft used for main pulmonary artery reconstruction in piglets

Abstract

Background: Surgical treatment of congenital heart defects affecting the right ventricular outflow tract (RVOT) often requires complex reconstruction and multiple reoperations due to structural degeneration and lack of growth of currently available materials. Hence, alternative approaches for RVOT reconstruction, which meet the requirements of biocompatibility and long-term durability of an ideal scaffold, are needed. Through this full scale pre-clinical study, we demonstrated the growth capacity of a Wharton's Jelly derived mesenchymal stromal cells (WJ-MSC) tissue engineered vascular graft used in reconstructing the main pulmonary artery in piglets, providing proof of biocompatibility and efficacy. Methods: Sixteen four-week-old Landrace pigs were randomized to undergo supravalvar Main Pulmonary Artery (MPA) replacement with either unseeded or WJ-MSCs-seeded Small Intestinal Submucosa-derived grafts. Animals were followed up for 6 months by clinical examinations and cardiac imaging. At termination, sections of MPAs were assessed by macroscopic inspection, histology and fluorescent immunohistochemistry. Results: Data collected at 6 months follow up showed no sign of graft thrombosis or calcification. The explanted main pulmonary arteries demonstrated a significantly higher degree of cellular organization and elastin content in the WJ-MSCs seeded grafts compared to the acellular counterparts. Transthoracic echocardiography and cardiovascular magnetic resonance confirmed the superior growth and remodelling of the WJ-MSCs seeded conduit compared to the unseeded. Conclusion: Our findings indicate that the addition of WJ-MSCs to the acellular scaffold can upgrade the material, converting it into a biologically active tissue, with the potential to grow, repair and remodel the RVOT.

Keywords: growing swine model; preclinical efficacy; right ventricular outflow tract reconstruction; small intestinal submucosa; tissue engineering.

FIGURE 1

Visual abstract summarizing the experimental and results sections.

FIGURE 2

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. (C) Representative image of the explanted acellular and cell-engineered conduit.

FIGURE 3

In vivo assessments. (A, B) Representative ultrasound images of the pulmonary arteries (PA) of unseeded and WJ-MSC seeded SIS grafts at 6 months post-surgery. Arrows indicate level of graft insertion (a, aorta; pa, pulmonary artery). (C) Circumference of the WJ-MSC seeded SIS and unseeded grafts at implantation and 6 months post-surgery. (D, E) Representative images of Colour Doppler blood velocities in PA of unseeded and cell-seeded groups. (F) Blood flow peak velocities through the PA of unseeded and WJ-MSC seeded groups pre-surgery and at 6 months of follow-up. (G) MPA flow measurements by cardiovascular magnetic resonance at pre-operation baseline and at 6-month follow-up, in unseeded and seeded animals. (H) Pigs’ weight pre-surgery and at 6 months of follow-up. ANOVA with post-hoc testing were used [n = 8, mean ± (SD), * p < 0.05, ** p < 0.01, *** p < 0.001].

FIGURE 4

(A) Hematoxylin and eosin staining showing extensive nucleation throughout the structure of seeded and unseeded grafts. (B) Von Kossa staining showing no calcification in the grafts. (C) Van Gieson staining of collagen (pink) and elastin (purple) content of the grafts. A higher content of elastin, a major extracellular matrix component of the pulmonary artery, is visible in the seeded graft. (D) Bar plots showing collagen and elastin concentration in unseeded/seeded grafts and in the native MPA [n = 8, mean ± (SD), *p < 0.05, **p < 0.005]. Scale bars = 1,000 μm, 50 µm higher magnification.

FIGURE 5

Representative images showing longitudinal sections of the grafts. 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 WJ-MSC seeded SIS graft (B), compared to the unseeded graft (A). 40,6-diamidino-2-phenylindole was used to mark nuclei (blue). (D) Bar chart showing the different SMA concentrations in unseeded/seeded grafts and in native MPA [n = 8, mean ± (SD), *p < 0.05, **p < 0.005]. Scale bars = 1,000 μm, 50 µm higher magnification (smaller image).

FIGURE 6

Tensile strength and elasticity of the explanted graft. Ultimate Tensile Strength (A) and Young’s modulus (B) were not significantly different between the WJ-MSC seeded and unseeded SIS scaffold, and the native MPA. MPa, megapascal.