Morphodynamic cellular changes prior to and during cell fusion related to osteoclast formation

Osteoclasts (OCs) play an important role in bone remodeling and bone resorption. Furthermore, OCs are considered to initiate the process of osteoinduction by stimulating the osteogenic differentiation of mesenchymal stromal cells (MSCs). OCs are large, multi-nucleated cells that originate from mononuclear precursors via cell fusion. Despite progress in understanding the molecular pathways involved, the dynamic morphological changes that precede and accompany fusion remain poorly defined. This study aims to characterize the spatiotemporal morphodynamics of osteoclast precursor cells (OCPs) during RANKL-stimulated differentiation, and identify structural features associated with fusion potential. Murine macrophages (RAW264.7 cells) were used as an in vitro model to study osteoclastogenesis. OCPs fusion and formation were induced by using 50 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL). Time-lapse holotomographic live-cell recordings were performed immediately after RANKL addition over 72 h using a Nanolive 3D cell explorer-fluo microscope, enabling continuous, label-free visualization of individual cell dynamics. Quantitative morphometric analysis was conducted using refractive index (RI)-based measurements with EVE analysis software to assess parameters including form factor, eccentricity, compactness, and cell extent. Finally, OCPs underwent progressive morphological remodeling prior to fusion, exhibiting increased elongation and shape irregularity. Multiple fusion phases were captured, including mono-mononuclear, mono-multinuclear, and multi-multinuclear fusion events. Notably, tunneling nanotubes-like membrane tethers (TNTs) frequently appeared before and during fusion and were associated with mediate directional movement and intercellular contact. Mononuclear cells displaying larger and more irregular morphology were more likely to participate in TNT-mediated fusion with multinucleated osteoclasts. Our findings demonstrate the predictive value of cell morphology in osteoclastogenesis and suggest that TNTs facilitate fusion coordination. This dynamic, single-cell-level perspective offers new insights into the morphodynamic characteristics of osteoclastogenesis and supports the notion that modulation of cell morphology may serve as a potential strategy to regulate osteoclast differentiation and function.