Dinamic and functional analysis of osteoclast activity using video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy

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Quantitative evaluation of the ability of bone resorption activity in live osteoclast-like cells (OCLs) has not reported yet. To analyze the ability of bone resorption in the same alive cells for long periods, we observed OCLs maintaining normal culture condition, and recorded by video: microscopy. The coverslips were placed in the incubator equipped inverted microscope (Biostation IM, Nikon, Japan), and OCLs were observed with phase contrast microscope. We observed the morphological change of OCLs and measured calcium phosphate free area made by OCLs affected by elcatonin using incubator facilitated video-enhanced microscopy. OCLs, which were obtained from co-culture of ddy-mouse osteoblastic cells and bone marrow cells, were cultured on calcium phosphate (CP)-coated quartz coverslips to observe sequential morphological change and to measure the increase of CP-free area. The CP-free area increase constantly in the control group, while that didn't increase after the addition of agent in the elcatonin group. In this study we could evaluate the resorbed areas increased on the same CP-coated coverslips, even with the migrated osteoclast. Furthermore, we observed the morphological change of OCLs after elcatonin addition. The nuclei were observed above the CP-free area and were gathered each other before chemical addition. After addition of elcatonin, however, the vacuoles spread from CP-free area, and the nuclei were dispersed. These results suggest that elcatonin could not only weaken the bone resorption ability but also change the polarization of nuclei. This study showed that analysis of the resorbed areas under the osteoclast-like cell body using this method enables the quantitative evaluation of the bone resorption activity to the several therapeutic agents like bisphosphonates in vitro.