Determining the fate of seeded cells in venous tissue-engineered vascular grafts using serial MRI

Abstract

A major limitation of tissue engineering research is the lack of noninvasive monitoring techniques for observations of dynamic changes in single tissue-engineered constructs. We use cellular magnetic resonance imaging (MRI) to track the fate of cells seeded onto functional tissue-engineered vascular grafts (TEVGs) through serial imaging. After in vitro optimization, murine macrophages were labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles and seeded onto scaffolds that were surgically implanted as inferior vena cava interposition grafts in SCID/bg mice. Serial MRI showed the transverse relaxation times (T(2)) were significantly lower immediately following implantation of USPIO-labeled scaffolds (T(2) = 44 ± 6.8 vs. 71 ± 10.2 ms) but increased rapidly at 2 h to values identical to control implants seeded with unlabeled macrophages (T(2) = 63 ± 12 vs. 63 ± 14 ms). This strongly indicates the rapid loss of seeded cells from the scaffolds, a finding verified using Prussian blue staining for iron containing macrophages on explanted TEVGs. Our results support a novel paradigm where seeded cells are rapidly lost from implanted scaffolds instead of developing into cells of the neovessel, as traditionally thought. Our findings confirm and validate this paradigm shift while demonstrating the first successful application of noninvasive MRI for serial study of cellular-level processes in tissue engineering.

Figure 1.

Evaluation of USPIO cell labeling and scaffold cell seeding. Murine macrophages were stained with Prussian blue to identify intracellular iron. A) Photomicrograph of unlabeled control macrophages (H&E, ×400) B) Photomicrograph of macrophages after USPIO cell labeling (H&E, ×400). C) Photomicrograph of USPIO-labeled macrophages statically seeded onto a biodegradable scaffold (Lee's methylene blue, ×40). D) Representative section of the seeded scaffold in C, showing the PGA scaffold fibers (arrowhead) and the USPIO-labeled macrophages (arrow; Lee's methylene blue, ×400).

Figure 2.

In vitro characterization of USPIO-labeled macrophages and seeded scaffolds. A) T₂-weighted image of a gelatin phantom using an array of concentration gradients of USPIO-labeled macrophages. Labeled cells (n): A, 25 × 10⁶; B, 20 × 10⁶; C, 15 × 10⁶; D, 10 × 10⁶; E, 5 × 10⁶; F, 10⁶; G, 0. B) T₂ mapping of sample in panel A. C) In vitro T₂-weighted image of an unlabeled scaffold, an unseeded scaffold, and a USPIO-labeled scaffold suspended in gelatin. D) T₂ mapping of sample in panel C. E) Relationship between relaxation rate R₂ and labeled cells in gelatin phantoms. The R² exhibits significant correlation with labeled cell concentration (R²=0.97).

Figure 3.

In vivo MR imaging of labeled and unlabeled scaffold implants. A, B) Representative axial T₂-weighted RARE images of mice implanted with labeled (A) and unlabeled (B) seeded scaffolds, illustrating the location of implanted scaffolds (boxes). Kidneys (K) and liver (L) are visible in the images. C, D) Corresponding T₂ maps of the same slices, showing T₂ value differences between the labeled (C) and unlabeled (D) seeded scaffolds.

Figure 4.

In vivo T₂ values for serial MRI of scaffold implants. A, B) T₂ values for scaffold implants and extravascular tissues in mice with USPIO-labeled implants (striped bars) and mice with non-USPIO-labeled implants (open bars) imaged immediately after implantation (A) and 1 wk postimplantation (B). C) To gain further insight into the temporal increase in the T₂ value in the labeled scaffolds, T₂ values were calculated for scaffold implants in one additional mouse with a USPIO-labeled implant (striped bars) and one with a non-USPIO-labeled implant (open bars) at 30-min increments starting immediately after implantation.

Figure 5.

Fate of seeded cells by histological analysis. Scaffold explants were stained with Prussian blue to identify intracellular iron deposits. A) Number of Prussian blue-labeled cells per HPF (shaded bars) and total number of cells per HPF (open bars) were counted immediately after implantation (time 0), 2 h, and 1 wk after implantation. B–D) Representative images of scaffold sections were stained with Prussian blue at time 0 (B), 2 h (C), and 1 wk (D). Arrows indicate macrophages stained with Prussian blue.