Frontal cryosectioning: An improved protocol for sectioning large areas of fibrous scaffolds

Abstract

Fibrous tissue engineering scaffolds, such as those produced by electrospinning, cannot achieve their clinical potential until deep cell-scaffold interactions are understood. Even the most advanced imaging techniques are limited to capturing data at depths of 100 μm due to light scatter associated with the fibers that compose these scaffolds. Conventional cross-sectional analysis provides information on relatively small volumes of space and frontal sections are difficult to generate. Current understanding of cellular penetration into fibrous scaffolds is limited predominantly to the scaffold surface. Although some information is available from cross-sections, sections vary in quality, can distort spatial scaffold properties, and offer virtually no spatial cues as to what scaffold properties instigate specific cellular responses. Without the definitive ability to understand how cells interact with the architecture of an entire scaffold it is difficult to justify scaffold modifications or in-depth cell penetration analyses until appropriate techniques are developed. To address this limitation we have developed a cryosectioning protocol that makes it possible to obtain serial frontal sections from electrospun scaffolds. Microscopic images assembled into montage images from serial sections were then used to create three-dimensional (3D) models of cellular infiltration throughout the entire scaffold.