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.

Histone acetylation is an important epigenetic mechanism that controls expression of certain genes. It includes non-sequence-based changes of chromosomal regional structure that can alter the expression of genes. Acetylation of histones is controlled by the activity of two groups of enzymes: the histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs remove acetyl groups from the histone tail, which alters its charge and thus promotes compaction of DNA in the nucleosome. HDACs render the chromatin structure into a more compact form of heterochromatin, which makes the genes inaccessible for transcription. By altering the transcriptional activity of bone-associated genes, HDACs control both osteogenesis and osteoclastogenesis. This review presents an overview of the function of HDACs in the modulation of bone formation. Special attention is paid to the use of HDAC inhibitors in mineralized tissue regeneration from cells of dental origin.

One of the characteristics of periodontal ligament (PDL) cells is their plasticity. Yet, the underlying mechanisms responsible for this phenomenon are unknown. One possible mechanism might be related to epigenetics, since histone deacetylases (HDACs) have been shown to play a role in osteoblast differentiation. This study was aimed to investigate the role of HDACs in osteogenic differentiation of human PDL (hPDL) cells. HDAC inhibitor trichostatin A (TSA) had no effect on cell viability as was assessed by MTT assay. Osteogenic and adipogenic differentiation was analyzed by gene expression, ALP activity and mineral deposition. Western blotting was used to investigate the effect of TSA on histone acetylation and protein expression. In the presence of the HDAC inhibitor osteogenic differentiation was induced; osteoblast-related gene expression was increased significantly. ALP activity and mineral nodule formation were also enhanced. Inhibition of HDACs did not induce differentiation into the adipocyte lineage. hPDL highly expressed HDACs of both class I (HDAC 1, 2, 3) and class II (HDAC 4, 6). During osteogenic differentiation HDAC 3 expression gradually decreased. This was apparent in the absence and presence of the inhibitor. The level of acetylated Histone H3 was increased during osteogenic differentiation. Inhibition of HDAC activity induced hyperacetylation of Histone H3, therefore, demonstrating Histone H3 as a candidate target molecule for HDAC inhibition. In conclusion, hPDL cells express a distinguished series of HDACs and these enzymes appear to be involved in osteogenic differentiation. This finding suggests a potential application of TSA for bone regeneration therapy by hPDL cells.

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