Osteoclasts are the only cell type capable of resorption of mineralised matrix such as bone or dentine. Resorbing osteoclasts form distinct membrane domains: the functional secretory, the basolateral and the ruffled border (RB) domains. The RB allows acidification of the resorption lacuna, exocytosis of osteolytic enzymes and uptake of degraded bone material, processes that require directed vesicular transport. Few studies have tried to classify the vesicles near the RB into secretory or uptake pathways and given their size (100500 nm) such studies are not possible by light microscopy alone. Using electron tomography, we characterised vesicles adjacent to the RB. Rabbit osteoclasts resorbing dentine discs were processed for transmission electron microscopy (TEM) using standard fixation and embedding techniques. 200 nm thick sections were imaged in a JEOL JEM-1400Plus TEM. Tilt series (±60 degrees) around the structure of interest were acquired with a 2D image captured at each degree. 3D tomograms were generated and regions of interest rendered using Amira software. These renderings allowed better interpretation of 2D TEM images of osteoclastic vesicles and some new observations were made. i) Single membrane-bound vesicles with electron dense content and distinct halos, previously thought to be secretory lysosomes, were found to be tangential sections of collagen fibrils encased in RB membrane. ii) Single membrane-bound vesicles with moderate electron dense content located near the RB were often associated with extracellular collagen fibril tips. They appeared to contain small amounts of degraded collagen and therefore may be part of the uptake pathway. iii) Large, double membrane-bound vesicles (autophagosomes) were frequently seen near the ruffled border. iv) Vesicles (0.11.5 μm) without notable content were released from the apical membrane. Our observations illustrate the need for 3D imaging to avoid erroneous interpretation of 2D TEM and begin to describe the vesicles involved in osteoclastic resorption.
14 May 2016 - 17 May 2016