Ossification is a developmental process where bone is created in appositional layers through the gradual calcification of the native extracellular matrix (ECM). This evolution is orchestrated by biological processes that allow for mesenchymal condensation of stem cells and their subsequent differentiation into bone-forming cells. While bone mechanobiology has been studied in skeletal development and fracture healing, the interplay between mechanics and biology during the transition of calcifying osteoid to mature bone remains poorly understood. Our objective is to replicate the critical changes in the ECM during bone formation to study the step-by-step process of bone regeneration in vitro, as current models are limited. Ultimately, we hope to advance the fundamental understanding of the mechanobiology behind bone formation, contributing to future research on regenerative therapies.