Deconstructing the Tissue Engineered Vascular Graft: Evaluating Scaffold Pre-Wetting, Conditioned Media Incubation, and Determining the Optimal Mononuclear Cell Source

Abstract

Stenosis limits widespread use of tissue-engineered vascular grafts (TEVGs), and bone marrow mononuclear cell (BM-MNC) seeding attenuates this complication. Yet seeding is a multistep process, and the singular effects of each component are unknown. We investigated which components of the clinical seeding protocol confer graft patency and sought to identify the optimal MNC source. Scaffolds composed of polyglycolic acid and ε-caprolactone/ι-lactic acid underwent conditioned media (CM) incubation (n = 25) and syngeneic BM-MNC (n = 9) or peripheral blood (PB)-MNC (n = 20) seeding. TEVGs were implanted for 2 weeks in the mouse IVC. CM incubation and PB-MNC seeding did not increase graft patency compared to control scaffolds prewet with PBS (n = 10), while BM-MNC seeding reduced stenosis by suppressing inflammation and smooth muscle cell, myofibroblast, and pericyte proliferation. IL-1β, IL-6, and TNFα were elevated in the seeded BM-MNC supernatant. Further, BM-MNC seeding reduced platelet activation in a dose-dependent manner, possibly contributing to TEVG patency.

Keywords: IL-1β; IL-6; TNFα; biodegradable scaffold; bone marrow; mononuclear cell; mouse model; peripheral blood; platelet; seeding; stenosis; tissue engineered vascular graft.

Figure 1

Lumen diameter measurements (A) and representative H&E photomicrographs of patent (B–E) and occluded (F–I) TEVGs after 2-week implantation from the PBS (B, F), conditioned media (C, G), BM-MNC seeded (D, H), and PB-MNC seeded (E, I) groups. TEVGs with lumen diameters <50% of the original lumen diameter at implant (dashed line, 0.41 mm) were considered critically stenotic. The PBS, conditioned media, and PB-MNC seeded groups all had significantly smaller lumen diameters than the BM-MNC seeded group (p = 0.0291, 0.0093, and 0.0455, respectively). No other significant differences in lumen diameter between groups were identified. Images were acquired at 5× magnification.

Figure 2

Immunohistochemical evaluation of F4/80⁺ macrophages (A) and representative photomicrographs of patent (B–E) and occluded (F–I) grafts from the PBS (B, F), conditioned media (C, G), BM-MNC seeded (D, H), and PB-MNC seeded (E, I) groups. **p ≤ 0.005, ***p ≤ 0.0005, ****p ≤ 0.0001. Images were acquired at 63× magnification.

Figure 3

Immunohistochemical evaluation of αSMA⁺ area fraction (A) and representative photomicrographs of patent (B-E) and occluded (F–I) grafts from the PBS (B, F), BM-MNC seeded (C, G) conditioned media (D, H), and PB-MNC seeded (E, I groups). *p ≤ 0.05, **p ≤ 0.005. Images were acquired at 63× magnification.

Figure 4

In vitro cytokine analysis (A) from the media in which grafts were immersed after overnight incubation. Seeded BM-MNCs secreted significantly more IL-1β, IL-6, and TNFα than seeded PB-MNCs. ATP secretion from thrombin activated platelets in the presence of TEVGs seeded with varying doses of either BM-MNCs or PB-MNCs demonstrates a dose-responsive decrease in measurable ATP from wells with BM-MNC seeded grafts, and this phenomenon was not identified with seeded PB-MNCs (B, **p ≤ 0.005, ***p ≤ 0.0005, ****p ≤ 0.0001).

Scheme 1

Clinical Protocol for TEVG Assembly