Development of a rapid matrix digestion technique for ultrastructural analysis of elastic fibers in the intervertebral disc

Collagen and elastic fibers are two major fibrous constituents of the annulus fibrosus (AF) in the disc that contribute to its mechanical and viscoelastic properties. It was thought that elastic fibers play no substantial role in the function and properties of the disc as these fibers were irregularly distributed. Studies that have revealed highly organized elastic fibers with different regional orientation and distribution, while being strongly crosslinked with matrix, suggesting their contribution to disc structure-function properties. These studies that were performed by light microscopic analysis of histologically prepared samples, have not been able to reveal the fine-scale architectural details of the elastic fiber network. Since elastic fibers are intermingled with other fibrous components of the disc and mostly obscured by the extracellular matrix, it is difficult to demonstrate their ultra-structural organization using scanning electron microscopy (SEM). Therefore the aim of this study was to develop a rapid matrix digestion technique for ultrastructural analysis of the disc elastic fibers. This study provides a new method for fundamental visualization of elastic fibers and their architecture in the disc. Through the ultra-structural analysis, the relationship between structure and function, as well as the role of elastic fibers on AF mechanical properties can be studied. This method may be used to develop a three-dimensional map of elastic fibers distribution within the disc, which would provide valuable information for designing tissue engineered scaffolds for AF repair and replacement.