Alendronate Alters the Release of EVs by Raw 264.7 Osteoclasts

Abstract

Alendronate (ALN), a nitrogen-containing bisphosphonate, is widely used to treat bone disorders. While its inhibitory effect on osteoclast activity is well-established, its impact on the release of extracellular vesicles (EVs) is less understood. This study investigated the effect of ALN on the quantity and size distribution of EVs released by osteoclasts cultured on bovine bone slices pretreated with 10-µM ALN, 100-µM ALN, or vehicle. Raw 264.7 cells were differentiated into osteoclasts using RANK-ligand, and EVs were isolated from conditioned media. Tartrate-resistant acid phosphatase (TRAP) staining, phalloidin staining for actin rings, and nanoparticle tracking analysis (NTA) were performed. TRAP staining showed a significant reduction in the number of TRAP-positive multinucleated cells in the 100-µM ALN group, confirming that high-concentration ALN also impairs osteoclast formation. Phalloidin staining showed a significant decrease in actin ring formation in the 100-µM ALN group, confirming ALN’s inhibitory effect on osteoclast activity. NTA revealed a lower total EV concentration in the 100-µM ALN group, with a distinct peak of smaller EVs (<100 nm), suggestive of exosomes. These findings indicate that ALN, especially at higher concentrations, alters the release profile of osteoclast-derived EVs, potentially affecting intercellular communication and bone remodeling beyond its direct inhibition of resorption.