Development of a BMU-on-a-chip model based on spatiotemporal regulation of cellular interactions in the bone remodeling cycle

Bone remodeling is essential for maintaining bone homeostasis throughout life by replacing old bone with new tissue. This dynamic process occurs continuously within basic multicellular unit (BMU) through well-coordinated interactions among osteocytes, osteoblasts, and osteoclasts. However, a precise <i>in vitro</i> model that accurately replicates this mechanism has not yet been developed. In this study, we created a human <i>in vitro</i> BMU-modeling chip platform by tri-culturing cells within a chip unit integrated into a tissue culture well plate, enabling high-throughput three-dimensional (3D) cell culture. To establish the tri-culture, human osteoblasts were isolated from human surgical bone samples and differentiated into osteocytes within collagen gel inside the chip unit. Subsequently, osteoblasts and peripheral blood mononuclear cells (PBMCs) containing osteoclast precursors were added to the chip unit. To simulate each phase of the bone remodeling cycle, we optimized the tri-culture process by adjusting the timing and using two types of osteoblasts at different stages of differentiation. The completed tri-culture model successfully mimicked the bone formation phase. When receptor activators of nuclear factor kappa-Β (RANKL) and macrophage colony-stimulating factor (M-CSF) were introduced, the cells exhibited characteristics of the reversal phase, where osteogenic and osteoclastogenic environments coexist. Additionally, using more differentiated osteoblasts within the tri-culture platform induced osteoclast differentiation, resembling the bone resorption phase. Overall, our model effectively replicates each phase of the bone remodeling cycle in BMUs, both spatially and temporally. This advancement not only facilitates the study of the intricate mechanisms of bone remodeling and cellular function but also aids drug development by providing a robust bone model for testing target drugs.