Extracellular vesicles (EVs), which include exosomes and ectosomes/microvesicles, have emerged as important intercellular regulators. EVs can interact with surface receptors of target cells and can transport luminal components, including messenger RNAs (mRNAs), microRNAs, and enzymes, to the cytosol of the target cell. Here, we show that hematopoietic cells grown in culture shed exosome-like EVs as they differentiate from preosteoclasts into osteoclasts. These EVs were between 25 and 120 nm (mean, 40 nm) in diameter determined by transmission electron microscopy. The exosome-associated markers CD63 and EpCAM were enriched in the isolated EVs while markers of Golgi and endoplasmic reticulum were not detected. Treatment of isolated hematopoietic cells with EVs did not affect their receptor activator of nuclear factor κB-ligand (RANKL)-stimulated differentiation into osteoclasts. However, EVs from osteoclast precursors promoted 1,25-dihydroxyvitamin D3-dependent osteoclast formation in whole mouse marrow cultures, and EVs from osteoclast-enriched cultures inhibited osteoclastogenesis in the same cultures. These data suggested that osteoclast-derived EVs are paracrine regulators of osteoclastogenesis. EVs from mature osteoclasts contained receptor activator of nuclear factor κB (RANK). Immunogold labeling showed RANK was enriched in 1 in every 32 EVs isolated from osteoclast-enriched cultures. Depletion of RANK-rich EVs relieved the ability of osteoclast-derived EVs to inhibit osteoclast formation in 1,25-dihydroxyvitamin D3-stimulated marrow cultures. In summary, we show for the first time that EVs released by osteoclasts are novel regulators of osteoclastogenesis. Our data suggest that RANK in EVs may be mechanistically linked to the inhibition of osteoclast formation. RANK present in EVs may function by competitively inhibiting the stimulation of RANK on osteoclast surfaces by RANKL similar to osteoprotegerin. RANK-rich EVs may also take advantage of the RANK/RANKL interaction to target RANK-rich EVs to RANKL-bearing cells for the delivery of other regulatory molecules.

Objective: To collect data regarding Canadian laypersons' perceptions of smile esthetics and compare these data to US data in order to evaluate cultural differences. Materials and Methods: Using Adobe Photoshop 7, a digital image of a posed smile of a sexually ambiguous lower face was prepared so that hard and soft tissue could be manipulated to alter buccal corridor (BC), gingival display (GD), occlusal cant (OC), maxillary midline to face discrepancy (MMFD), and lateral central gingival discrepancy (LCGD). Adult Canadian laypersons (n 5 103) completed an interactive computer-based survey of 29 randomized images to compare smile preferences for these variables. The custom survey was developed to display fluid, continuously appearing modifiable smile variables using MATLAB R2008 for presentation. These data were compared with previously published data for US laypersons. Statistical inference was determined using Wilcoxon rank sum tests. Results: Canadian laypersons were more sensitive in detecting deviations from ideal and had a narrower range of acceptability thresholds for BC, GD, OC, MMFD, and LCGD. Ideal esthetic values were significantly different only for BC.Conclusions: It appears that cultural differences do exist related to smile characteristics. Clinically significant differences in the preference of the smile characteristics were found between Canadian and US laypersons. Canadian laypersons, on average, were more discriminating to deviations from ideal and had a narrower range of acceptability. (Angle Orthod. 2011;81:198-205.)

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