Macroscopic and microscopic biomechanical analysis of mineralized spheroids derived by mesenchymal stem cells

Three-dimensional cultures that simulate bone formation in vitro are promising approaches for elucidating the mineralization process involved in osteogenesis. In this study, we performed a multiscale analysis of spheroids derived from human mesenchymal stem cells. We quantified morphological changes and mechanical conditions from microscale local stiffness to macroscale overall stiffness and viscoelastic/plastic behaviors as mineralization progressed. Specifically, we evaluated how the overall mechanical properties (e.g., Young's modulus), mechanical behaviors (e.g., elastic or viscoelastic behaviors), and local mechanical environments (e.g., Young's modulus and morphological distributions) evolve during the long-term culture of spheroids derived from human mesenchymal stem cells. After 35 days of culture, we observed progression of mineralization within the spheroids, increased stiffness and plastic deformation at the macroscopic level assessed by uniaxial compression tests. A greater heterogeneity in mechanical properties and environments at the microscopic level was observed using atomic force microscopy. These findings indicate changes in mechanical properties and behavior during the in vitro bone formation process, suggesting that the mineralization within spheroids is highly heterogeneous. This study elucidates the complexity of the mineralization process in three-dimensional culture models and, for the first time, evaluates this process from a mechanical perspective, providing new insights for future bone regenerative medicine research utilizing three-dimensional culture models.