Dynamic, Nondestructive Imaging of a Bioengineered Vascular Graft Endothelium

Abstract

Bioengineering of vascular grafts holds great potential to address the shortcomings associated with autologous and conventional synthetic vascular grafts used for small diameter grafting procedures. Lumen endothelialization of bioengineered vascular grafts is essential to provide an antithrombogenic graft surface to ensure long-term patency after implantation. Conventional methods used to assess endothelialization in vitro typically involve periodic harvesting of the graft for histological sectioning and staining of the lumen. Endpoint testing methods such as these are effective but do not provide real-time information of endothelial cells in their intact microenvironment, rather only a single time point measurement of endothelium development. Therefore, nondestructive methods are needed to provide dynamic information of graft endothelialization and endothelium maturation in vitro. To address this need, we have developed a nondestructive fiber optic based (FOB) imaging method that is capable of dynamic assessment of graft endothelialization without disturbing the graft housed in a bioreactor. In this study we demonstrate the capability of the FOB imaging method to quantify electrospun vascular graft endothelialization, EC detachment, and apoptosis in a nondestructive manner. The electrospun scaffold fiber diameter of the graft lumen was systematically varied and the FOB imaging system was used to noninvasively quantify the affect of topography on graft endothelialization over a 7-day period. Additionally, results demonstrated that the FOB imaging method had a greater imaging penetration depth than that of two-photon microscopy. This imaging method is a powerful tool to optimize vascular grafts and bioreactor conditions in vitro, and can be further adapted to monitor endothelium maturation and response to fluid flow bioreactor preconditioning.

Synthesis and characterization of polycaprolactone urethane hollow fiber membranes as small diameter vascular grafts

“…Cytocompatibility testing of HFMs was performed by direct seeding on the HFMs and by exposing the cells to degradation products, with results consistent with those previously obtained [59,60]. No significant adverse effects were noted in either approach, and well-formed cell layers were formed on the surfaces of HFMs in both static and perfusion conditions.…”

Dynamic Assessment of the Endothelialization of Tissue-Engineered Blood Vessels Using an Optical Coherence Tomography Catheter-Based Fluorescence Imaging System

“…In addition, our imaging system could be potentially used for fluorescent-based immune-staining, which could provide additional spatial information for tracking endothelialization, although this would require sample fixation and destruction. Recently, Whited et al 25 showed dynamic monitoring of endothelium development in a bioengineered vessel using a fiber-optic-based imaging system. However, their imaging technique relies on localized delivery of excitation light using micro-imaging channels embedded within the scaffold, and collection of diffusely scattered fluorescence by optics external to the specimen.…”

Regeneration of the Vascular System

“…Currently, there are different technologies to obtain such information including multiphoton imaging [161]. Another approach is through the use of fiber opticbased imaging [162,163] that provides deeper penetration than any other imaging method and yields information that ranges from cell morphology to functional analysis, all in real time (Figure 3). There is a growing interest in making fluorescent proteins in the far red range that would improve imaging for in vivo application such as in the case of the new infrared fluorescent protein [164].…”