Methodological assessment of acid-etching for visualizing the osteocyte lacunar-canalicular networks using scanning electron microscopy

Abstract

Osteocytes are the most abundant of the bone cells. Each osteocyte is contained within its own lacuna and connected to adjacent osteocytes via fillipodial processes, which form an intricate network of canaliculi within the matrix. Studying this intricate network of cells and their processes is difficult, because it exists embedded within a densely mineralized matrix. Scanning electron microscopy (SEM) has been shown to be a useful tool for visualizing this cellular network, yet the techniques involved for preparing specimens has not been systematically explored. The goal of this study was to investigate how variations in acid-etching, both etching media and etching duration, affect SEM-based visualization of the osteocyte lacunar-canalicular network. Bone samples were embedded in plastic and then acid etched in either 9% (10, 20, 40, and 60 s durations) or 37% (5, 10, and 15 s) phosphoric acid. Specimens were imaged using SEM, and qualitative evaluation of the lacunar-canalicular network was undertaken. Our findings show acid etchingwith a 9% phosphoric acid solution for 20 s provided the most favorable visualization of the osteocyte lacunar-canalicular network.