Using fluorescent polyanions to assay for osteoclastic calcium-resorption activity

To analyze the bone resorption activity of osteoclast, pit assay is principally performed. The assay measures the calcium phosphate (hydroxyapatite) resorption in vitro, using dentin slices or calcium-phosphate-coated plate. However, it requires trained techniques and time-consuming pit image analysis, and at present there is no alternative easy method for evaluating calciumphosphate resorption. In this chapter, we described a novel approach to quantify the bone resorption activity using a calcium-phosphate labeled with fluorescent polyanion such as chondroitin sulfate or deoxyribonucleic acid. When preosteoclasts were cultured on calcium phosphate coated with fluorescent polyanion under the induction of osteoclast differentiation, a dramatic increase of fluorescence intensity was observed accompanied by the increase of the pit area. Since calcein coating revealed no increase, the long polymer and strong negative charge are thought to be required for tight binding to the calcium-phosphate minerals. The drugs for osteoporosis treatment, such as bisphosphonate inhibited fluorescein release correlated well with the decrease of the pit area. This new method using fluorescent polyanion-labeled calcium phosphate could be a standardized useful assay method evaluating bone resorption activity, which is used as an alternative method for the traditional pit assay.