Quantitative analyses of matrices, osteoblasts, and osteoclasts during bone remodeling using an in vitro system.

Abstract

Bone remodeling plays a central role in the maintenance of bone homeostasis. Our group has established an in vitro system by which the cellular events during bone remodeling can be observed longitudinally. This study used this system to quantitatively analyze osteoblasts, osteoclasts, and matrices to elucidate their temporal changes and correlations. Osteoblasts from EGFP mice were cultured to form calcified nodules, followed by co-culture with bone marrow macrophages from Tnfrsf11aCre/+ x Ai14 mice for 3weeks (resorption phase). Then cells were cultured with osteoblast differentiation medium for 3weeks (formation phase). The same sites were observed weekly using 2-photon microscopy. Matrices were detected using second harmonic generation. Parameters related to matrices, osteoblasts, and osteoclasts were quantified and statistically analyzed. Resorption and replenishment of the matrix were observed at the same sites by 2photon microscopy. Gross quantification revealed that matrix and osteoblast parameters decreased in the resorption phase and increased in the formation phase, while osteoclast parameters showed the opposite pattern. When one field of view was divided into 16 regions of interest (ROIs) and correlations between parameters were analyzed in each ROI, decreased and increased matrix volumes were moderately correlated. Parameters of matricesand osteoblasts, and those of matrices andosteoclasts exhibited moderate correlations, while those of osteoblasts and osteoclasts were only weakly correlated. Several correlations between cells and matrix during remodeling were demonstrated quantitatively. This system may be a powerful tool for the research of bone remodeling.