New bone formation and microstructure assessed by combination of confocal laser scanning microscopy and differential interference contrast microscopy.

Bone is a mineralized connective tissue that is continuously and microstructurally remodeled. Altered bone formation and microstructure arise in pathological bone conditions such as osteoporosis, osteonecrosis, fracture repair, and Paget disease of bone. A proper and objective assessment of bone formation and microstructure will provide insight into the understanding of bone pathogenesis and remodeling. Here, new bone formation ex vitro and its microstructure were evaluated in in vivo multiple sequential polychrome-labeled samples using confocal laser scanning microscopy (CLSM), which generated clearer and more reliable images of thick bone sections than conventional fluorescence microscopy (CFM). Intriguingly, fine details of the bone microstructural features, including the mineralization fronts, quiescent versus active osteons, and Volkmann's channel, were elucidated using CLSM, which defines the relationship between morphological changes and function, when combined with differential interference contrast microscopy. Furthermore, CLSM provided objective evaluations of bone formation, such as the ratio of labeled areas of new bone formation in a rabbit model when compared with CFM. Altogether, new bone formation and its microstructure can be evaluated more adequately using a combination of CLSM and DIC microscopies.