Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes

Abstract

ObjectivesThe aim of this study was the biological application of focused ion beam-scanning electron microscopy (FIB-SEM) to obtain serial sectional images of skeletal tissues that showed the ultrastructure of 1) cartilaginous extracellular fibrils and 2) osteoblastic cytoplasmic processes. MethodsSeven-week-old female wild-type mice were fixed with half-Karnovsky solution and then OsO4, and tibiae were extracted for block staining prior to observation under transmission electron microscope (TEM) and FIB-SEM. ResultsTEM showed the fine fibrillar but somewhat amorphous ultrastructure of the intercolumnar septa in the growth plate cartilage. In contrast, FIB-SEM revealed bundles of stout fibrils at regular intervals paralleling the septa's longitudinal axis, as well as vesicular structures embedded in the cartilaginous matrix of the proliferative zone. In the primary trabeculae, both TEM and FIB-SEM showed several osteoblastic cytoplasmic processes on the osteoid, in greater numbers than those seen in the bone matrix. FIB-SEM revealed the agglomeration of cytoplasmic processes beneath osteoblasts that formed a tubular continuum extending from those cells. Based on these findings, we postulated that osteoblasts not only extend their cytoplasmic processes to the bone matrix, but also stack these cell processes on the osteoid of the primary trabeculae. ConclusionTaken together, these data suggest that FIB-SEM imaging of serial bone sections may facilitate new insights on the ultrastructure of cartilage and bone tissues.