Osteoclasts' extracellular vesicles induce osteogenesis of human periodontal ligament cells via AKT activation.

OBJECTIVES: Our study has shown an enhanced effect on osteogenesis of periodontal ligament (PDL) cells upon treatment with osteoclasts' conditioned medium. However, characterization of osteoclasts' extracellular vesicles (OC-EVs), their osteogenic enhancement, and the underlying mechanism within PDL cells have been unexplored. DESIGNS: Before PDL cell treatment and staining with alkaline phosphatase, Alizarin Red, or von Kossa, OC-EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis with their sizes demonstrated at 100-200 nm, and immunoblotting for expressions of OC-EVs' markers. Expressions of receptor activator of nuclear factor-kappa B (RANK) on OC-EVs' membrane and RANK ligand (RANKL) on PDL cells' membrane were determined by flow cytometry and immunofluorescence, respectively. Involvement of the RANK-RANKL and AKT signaling in OC-EVs-induced osteogenesis of PDL cells was studied by pretreatment with osteoprotegerin, RANKL, or MK-2206, an AKT inhibitor. RESULTS: RANK expression was found at 5.02% of total OC-EVs. Treatment of PDL cells with OC-EVs significantly increased osteogenic differentiation, mineralization, and gene expressions (P < 0.05). Pretreatment of osteoclasts with GW4869, a blocker of EVs' secretion, abolished the expression of RANK on OC-EVs and significantly reduced the enhanced osteogenesis of PDL cells (P < 0.05). Moreover, pretreatment with osteoprotegerin or RANKL before treatment with OC-EVs significantly diminished the enhanced osteogenesis (P < 0.05), without any inhibition on enhanced phosphorylated AKT expression by OC-EVs. Pretreatment with MK-2206 could significantly decrease differentiation and mineralization of OC-EVs-treated PDL cells (P < 0.05). CONCLUSION: OC-EVs can induce osteogenesis of PDL cells via RANK-RANKL signaling and AKT activation as parallel contributions.