A method for visualization and isolation of elastic fibres in annulus fibrosus of the disc.

A simple and cost effective protocol for visualization and isolation of the elastic fibres network in the annulus fibrosus (AF) of the disc is explained, to provide other researchers a method that can be applied in disc ultra-structural analysis, biomechanical assessment of elastic fibre and tissue engineered scaffold fabrication. This protocol is developed based on simultaneous sonication and alkali digestion of tissue that eliminates all matrix constituents except for elastic fibres, which is applicable for different species including human. Thin samples harvested from ovine, bovine, porcine and human, which are commonly used in disc research, were exposed to 0.5 M sodium hydroxide solution along with sonication (25 kHz) in distilled water for defined periods of time at room temperature. Post heat treatment removed collagen fibres via the gelatinization process, for visualization of elastic fibres.