Bone mimetic resorption plates: A reliable method to quantify osteoclast resorption in vitro

• A novel in vitro resorption assay to measure osteoclast activity • A quick and easy protocol for producing osteoclast resorption plates. • A customized ImageJ macro for batch analysis of resorption. Osteoclasts resorb bone and excessive osteoclast activity can lead to osteoporosis and increased risk of fractures. Methods are needed to measure osteoclast bone resorption activity in vitro to test potential therapeutics and to better understand osteoclast biology. Classical in vitro osteoclast activity assays use dentin wafers as a resorption substrate. As dentin is increasingly scarce, we formulated an alternative “bone mimetic” tissue culture plate (resorption assay plate) to reproducibly quantify osteoclast resorptive activity. The protocol requires an initial coating of untreated tissue cultures plates with bovine serum albumin (BSA) followed by serial treatment with calcium and other salt solutions to precipitate a mineral substratum on the plate surface. In cultures without cells or in cultures of RAW264.7 or mouse bone marrow monocytes cells (BMMs), no discernable resorption of the resorption assay plate substratum was observed after 14 days. However, when RAW264.7 or mouse BMMs were differentiated into osteoclasts, substantial resorption of the resorption assay plate substratum was observed after 10 days in RAW264.7-derived osteoclast cultures and after 14 days in BMM-derived osteoclast cultures. Treatment of osteoclast cultures with zoledronic acid, a bisphosphonate, significantly inhibited resorption. An ImageJ macro was developed to quantify resorption activity by identifying, counting, and measuring areas of resorption assay plate resorption from brightfield images. This protocol is a practical and consistent method to analyze osteoclast activity based on resorption of the resorption assay plate substratum.