An In Vitro Model System Based on Calcium- and Phosphate Ion-Induced hMSC Spheroid Mineralization

A challenge in regenerative medicine is recapitulating the complex three-dimensional microenvironment of native organ or tissue, such as bone. Recreating the three-dimensional microenvironment of bone in the culture dish would allow the study of musculoskeletal disorders and the creation generation of building blocks for bone regeneration. In this study, we propose the use of a microwell-based platform to create spheroidal aggregates of human mesenchymal stromal cells and apply ionic calcium and phosphate supplementation to achieve rapid mineralization. These mineralized spheroids promote an osteogenic gene signature in cells through the influence of the spheroids biophysical environment and inorganic signaling, and require less calcium or phosphate to achieve mineralization compared to a monolayer culture. In addition, we found that mineralized spheroids represent a physiological model suitable for small molecule perturbations and the study of extracellular vesicle calcification. Furthermore, we demonstrate that understanding the molecular pathways promoted by the biophysical environment of spheroids allows mimicking these pathways in traditional monolayer culture, enabling similar rapid mineralization events. In sum, this study demonstrates the generation and application of physiological mineralized cell systems for multiple regenerative medicine goals.