Mineralization-driven cellular dynamics and tissue remodeling in scaffold-free MC3T3-E1 spheroids

Bone mineralization is a critical process in bone tissue formation and remodeling, yet the underlying mechanisms in three-dimensional (3D) cellular environments remain unclear. In this study, we fabricated scaffold-free spheroids using mouse osteoblast-like MC3T3-E1 cells and evaluated their long-term morphological and mechanical changes to elucidate the mineralization process. The spheroids were cultured for up to 35 days in osteogenic medium, and their size reduction (53.2 % compared to 2-day spheroid), cellular viability, collagen accumulation, and mineral deposition were systematically analyzed. Live/dead staining and nuclear imaging revealed that cell death occurred as early as day 2, predominantly in the spheroid core. Two-photon microscopy showed progressive collagen accumulation, and Alizarin-Red staining confirmed calcium deposition in the inner region by day 35. Mechanical analysis using uniaxial compression test by a microglass plate demonstrated 2.6-fold increase in Young's modulus, indicating increased stiffness associated with mineralization. These findings suggest that mineralization initiates from the spheroid core, potentially facilitated by extracellular matrix accumulation. The study provides new insights into bone-like tissue formation in 3D culture, contributing to the development of bone organoids for regenerative medicine and tissue engineering.