Three-Dimensional Structural Characterization of Tissue Engineered and Native Ovine Pulmonary Valves

Abstract

OBJECTIVES: Efforts in tissue-engineered heart valves (TEHV) have shown increasingly equivalent mechanical/structural properties compared to native valves, though a literature gap exists regarding detailed structural information. This work was performed to provide such data of implanted TEHV, the native pulmonary valve (PV), and pre-implant scaffold to better understand developing TEHV. METHODS: Dynamically-cultured in vivo samples (“pre-implant”) and ovine TEHV PV in vitro samples (“explant”) were produced based on previous techniques; ovine PVs were excised. Samples were stained with picrosirius red and resinmounted. Using extended-volume scanning laser confocal microscopy (EV-SLCM), 1.5 x 1.5 x 0.4 mm full-thickness samples were imaged at 1 pixel/μm in 1 μm Z-direction steps. Custom software was used to process and visualize samples. Collagen, cell nuclei, and scaffold volume fractions were quantified; scaffold fiber trajectory and length were tracked using custom software. RESULTS: In a scaffold representative volume (90 μm thick), 104 fibers were tracked with a mean fiber length of 137.94 μm 55.4 μm (Fig.1). A comparison between pre-implant and explant samples showed collagen volume fraction increasing from 76.6% to 85.9%, with nuclei and scaffold decreasing from 2.8% to 0.5% and from 5.9% to 0.8%, respectively. With the native collagen volume fraction measured at 70%, pre-implant and explant samples showed an increase in collagen. CONCLUSIONS: This work captured important differences between in vivo/in vitro TEHV constituents; it is the first known work to utilize EV-SLCM on TEHV. A comparison to the native valve showed structural differences that could impact longterm functionality and improve design.