Metabolomic signatures distinguish extracellular vesicles from osteoclasts and odontoclasts.

AIMS: Pathological dental root resorption and alveolar bone loss are often detected only after irreversible damage. Biomarkers in the gingival crevicular fluid or saliva could provide a means for early detection; however, such biomarkers have proven elusive. We hypothesize that a multiomic approach might yield reliable diagnostic signatures for root resorption and alveolar bone loss. Previously, we showed that extracellular vesicles (EVs) from osteoclasts and odontoclasts differ in their protein composition. In this study, we investigated the metabolome of EVs from osteoclasts, odontoclasts and clasts (non-resorbing clastic cells). MATERIALS AND METHODS: Mouse haematopoietic precursors were cultured on dentine, bone or plastic, in the presence of recombinant RANKL and CSF-1 to trigger differentiation along the clastic line. On Day 7, the cells were fixed and the differentiation state and resorptive status of the clastic cells were confirmed. EVs were isolated from the conditioned media on Day 7 and characterized by nanoparticle tracking and electron microscopy to ensure quality. Global metabolomic profiling was performed using a Thermo Q-Exactive Orbitrap mass spectrometer with a Dionex UHPLC and autosampler. RESULTS: We identified 978 metabolites in clastic EVs. Of those, 79 are potential biomarkers with Variable Interdependent Parameters scores of 2 or greater. Known metabolites cytidine, isocytosine, thymine, succinate and citrulline were found at statistically higher levels in EVs from odontoclasts compared with osteoclasts. CONCLUSION: We conclude that numerous metabolites found in odontoclast EVs differ from those in osteoclast EVs, and thus represent potential biomarkers for root resorption and periodontal tissue destruction.

Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts.

Aim: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. Methods: The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. Results: Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast's cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. Conclusion: RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.