Spheroids with Artificial Mineral Organelles: Machine-Learning-Assisted Analysis and Control of Mechanical Properties, Fusion, and Ossification

Controlling the balance between structural integration and functional maturation of microtissues remains a central challenge in bone tissue engineering, particularly in the design of composite cell-material constructs. Here, we demonstrate that intracellular loading of preosteoblast cells with submicron vaterite particles serves as a programmable material-based parameter to regulate spheroid behavior. Following controlled pre-loading and assembly into composite spheroids, we systematically quantified particle localization (confocal microscopy, PCC/Manders coefficients, Cellpose-based segmentation), mechanical properties (atomic force microscopy (AFM)), cell viability, osteogenic activity (ALP expression and hydroxyapatite deposition), and spheroid fusion kinetics. Ridge regression mediation analysis with bootstrap-validated pathway weights identifies particle uptake as the dominant control parameter governing a functional trade-off between structural integration and osteogenic maturation: Low loading facilitates a rapid spheroid fusion, whereas high loading enhances osteogenic maturation through direct positive effects on day-7 alkaline phosphatase activity and hydroxyapatite deposition and through a positive mediated pathway via the day-1 fusion descriptor. Importantly, early-stage fusion dynamics act as key mediators linking intracellular material loading to late-stage functional outcomes. These results establish intracellular particle loading as a tunable design variable in composite microtissues and define a practical design space for selecting loading regimes depending on whether rapid construct assembly or enhanced osteogenic maturation is required.