Real-Time Activity Bioassay of Single Osteoclasts Using a Silicon Nanocrystal-lmpregnated Artificial Matrix

摘要

The lack of an in vitro real-time osteoclast (OC) activity assay has hampered mechanistic studies of bone resorption. Such an assay is developed, employing a hydroxy apatite matrix impregnated with alkyl-capped silicon nanocrystals, which is capable of monitoring the time-course of resorption by single osteoclasts. Resorption of the matrix by OC releases the nanocrystals, which are internalized by the cell and detected as an increase in OC luminescence. This particular choice of nanocrystals is motivated by their bright pH-independent luminescence, proportional to concentration, and by their rapid uptake without cytotoxicity. In this in vitro assay, OCs are inhibited by calcitonin (CT) and methyl-β-cyclodextrin (MCD), and stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) in the expected manner. The kinetics of the assay exhibit a lag phase representing cell attachment and commencement of resorption processes, followed by a growth of cell luminescence intensity, and the whole time-course is satisfactorily described by the logistic equation.