Original Full Length ArticleAnalyses of bone modeling and remodeling using in vitro reconstitution system with two-photon microscopy

- We established an in vitro system for reconstituting the bone cell network.
- Cellular phenomena in this system were observed by 2-photon microscopy.
- During the nodule formation, flattening of cuboidal osteoblasts was observed.
- Some evidence that osteoclast pits were replaced with new matrix was presented.

Bone modeling and remodeling are cellular events during which osteoblast lineage cells and osteoclasts interact. During these events, cells undergo drastic changes with time as they become differentiated. Their morphology, topology, and activity are affected by other cells and the extracellular matrices. Since the mechanisms underlying the cellular events of bone metabolism have not been elucidated, there is a need for systems to analyze these cellular networks and their microenvironments spatiotemporally at the cellular level. Here we report a novel in vitro system for reconstituting the bone cell network of osteoclasts, osteoblasts, and osteocytes in the mineralized nodule, allowing for observation of bone modeling and remodeling phenomena by 2-photon microscopy. Using this system, the change in morphology of osteoblasts from cuboidal to flat cells was observed and measured during the formation of mineralized nodules. Furthermore, the recruitment of osteoblasts to resorption pits and their replenishment by newly formed matrices were successfully observed, providing strong evidence for the coupling of bone resorption and bone formation at cellular level. During such remodeling cycle, flat osteoblasts that survived more than 7 weeks were recruited to resorption pits, where they became cuboidal osteoblasts that express osteocalcin. This novel system permitted the elucidation of cellular behavior during bone modeling and remodeling, and can be used to analyze cellular events involved in bone metabolism.

Observed cells and phenomena are depicted in a time course from left to right. White letters within colored rectangles indicate observed phenomena (green: osteoblasts, blue and red: matrix), black letters without highlighting indicate observed cells, and orange boxes indicate differentiation markers detected by immunostaining.261