In Vitro Assay to Examine Osteoclast Resorptive Activity Under Estrogen Withdrawal

The bone is a highly dynamic organ that undergoes continuous remodeling through an intricate balance of bone formation and degradation. Hyperactivation of the bone-degrading cells, the osteoclasts (OCs), occurs in disease conditions and hormonal changes in females, resulting in osteoporosis, a disease characterized by altered microarchitecture of the bone tissue, and increased bone fragility. Thus, building robust assays to quantify OC resorptive activity to examine the molecular mechanisms underlying bone degradation is critical. Here, we establish an in vitro model to investigate the effect of estrogen withdrawal on OCs derived from the mouse macrophage RAW 264.7 cell line in a bone biomimetic microenvironment. This simple and robust model can also be adapted to examine the effect of drugs and genetic factors influencing OC resorptive activity in addition to being compatible with fluorescent imaging. Key features • A robust in vitro protocol that allows molecular and functional studies of mature osteoclasts in response to estrogen and its withdrawal. • Generation of inorganic bone-mimetic substrates for culturing and examining osteoclast resorptive behavior. • This quantitative image-based approach is compatible with brightfield and fluorescence microscopy to assess osteoclast resorptive activity.