[20] Role of 1α,25-dihydroxyvitamin D3 in osteoclast differentiation and function

This chapter describes the methods for examining osteoclast development and function. Cytochemical staining for tartrate-resistant acid phosphatase (TRAP) is widely used for identifying osteoclasts in vivo and in vitro. Osteoclasts are shown to possess abundant calcitonin receptors. Expression of calcitonin receptors is one of the most reliable markers for identifying osteoclasts. For autoradiography of 125I-labeled calcitonin, cultures are performed on plastic coverslips placed in 24-well culture plates. Cocultures of primary osteoblastic cells with bone marrow cells or spleen cells produce more osteoclasts than bone marrow cultures do. Osteoclasts formed on plastic culture dishes cannot be detached by treatment with either trypsin-EDTA or bacterial collagenase. To obtain functionally active osteoclasts formed in cocultures, a collagen gel culture is developed. Osteoclasts adhere to the bone surface through specialized discrete structures called podosomes in the clear zone, which consist mainly of dots containing F-actin. Therefore, the ringed structure of podosomes (actin ring) formed in osteoclasts is a characteristic of polarized osteoclasts. The actin rings are visualized by staining F-actin with rhodamine-conjugated phalloidin.