Targeted imaging of matrix metalloproteinase activity in the evaluation of remodeling tissue-engineered vascular grafts implanted in a growing lamb model

Abstract

Objectives: The clinical translation of tissue-engineered vascular grafts has been demonstrated in children. The remodeling of biodegradable, cell-seeded scaffolds to functional neovessels has been partially attributed to matrix metalloproteinases. Noninvasive assessment of matrix metalloproteinase activity can indicate graft remodeling and elucidate the progression of neovessel formation. Therefore, matrix metalloproteinase activity was evaluated in grafts implanted in lambs using in vivo and ex vivo hybrid imaging. Graft growth and remodeling was quantified using in vivo x-ray computed tomography angiography.

Methods: Cell-seeded and unseeded scaffolds were implanted in 5 lambs as inferior vena cava interposition grafts. At 2 and 6 months after implantation, in vivo angiography was used to assess graft morphology. In vivo and ex vivo single photon emission tomography/computed tomography imaging was performed with a radiolabeled compound targeting matrix metalloproteinase activity at 6 months. The neotissue was examined at 6 months using qualitative histologic and immunohistochemical staining and quantitative biochemical analysis.

Results: The seeded grafts demonstrated significant luminal and longitudinal growth from 2 to 6 months. In vivo imaging revealed subjectively greater matrix metalloproteinase activity in grafts versus native tissue. Ex vivo imaging confirmed a quantitative increase in matrix metalloproteinase activity and demonstrated greater activity in unseeded versus seeded grafts. The glycosaminoglycan content was increased in seeded grafts versus unseeded grafts, without significant differences in collagen content.

Conclusions: Matrix metalloproteinase activity remained elevated in tissue-engineered grafts 6 months after implantation and could indicate remodeling. Optimization of in vivo imaging to noninvasively evaluate matrix metalloproteinase activity could assist in the serial assessment of vascular graft remodeling.

Figure 1

Graft scaffold (A) and scanning electron micrograph (B) of polymer scaffold prior to surgical implantation. C) Schematic representation of surgical implantation of inferior vena cava interposition graft. Intra-operative photograph prior to (D) and immediately following (E) surgical implantation of an unseeded scaffold. Staples denote proximal and distal anastomoses.

Figure 2

In vivo CT angiography at 2 and 6 months following implantation of an unseeded (A) and seeded (B) scaffold demonstrates graft patency (lumen indicated by red markers). Serial quantification of CT angiography revealed significant increases in TEVG luminal volume (C) and length (D) in seeded grafts.

Figure 3

Increased ^99mTc-RP805 activity is observed in the blood pool and the region of IVC graft implantation (indicated by yellow marker) in axial (A), sagittal (B), and coronal (C) views.

Figure 4

Ex vivo SPECT/CT imaging and quantification. A) Representative sample of explanted TEVG and native IVC tissue prior to ex vivo SPECT/CT imaging. SPECT/CT imaging demonstrated heterogeneous uptake of ^99mTc-RP805 in the graft, as illustrated by representative cross sections of the vessel wall in both unseeded (B) and seeded (C) grafts. Significantly higher MMP activity is seen in unseeded compared to seeded grafts (D).

Figure 5

H&E, Elastica-Van Gieson, Masson's trichrome, and Alcian blue staining of native IVC (A), unseeded TEVG at 6 months (B), and seeded TEVGs at 6 months (C). Implanted scaffolds completely degraded over 6 months and were replaced with collagen-dominated neotissue (B & C). Elastic fiber formation was comparable in TEVGs, but appeared suboptimal compared to native IVC (A). Alcian blue staining demonstrated higher amount of glycosaminoglycans in the seeded compared to unseeded TEVG.