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Osteoclast Responses to Lipopolysaccharide, Parathyroid Hormone and Bisphosphonates in Neonatal Murine Calvaria Analyzed by Laser Scanning Confocal Microscopy

Keiko Suzuki, Sadaaki Takeyama, Takashi Kikuchi, Shoji Yamada, Jaro Sodek and Hisashi Shinoda

Department of Pharmacology, School of Dentistry, Showa University, Tokyo, Japan (KS,SY); Division of Pharmacology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, Sendai, Japan (ST,TK,HS); and CIHR Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada (JS)

Correspondence to: Keiko Suzuki, PhD, Department of Pharmacology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan. E-mail: suzukik{at}dent.showa-u.ac.jp

Because the development and activity of osteoclasts in bone remodeling is critically dependent on cell–cell and cell–matrix interactions, we used laser confocal microscopy to study the response of osteoclasts to lipopolysaccharide (LPS; 10 µg/ml), parathyroid hormone (PTH; 10^–8 M), and bisphosphonates (BPs; 1–25 µM clodronate or 0.1–2.5 µM risedronate) in cultured neonatal calvaria. Following treatment with LPS or PTH (<48 hr), osteopontin (OPN) and the vß3 integrin were found colocalized with the actin ring in the sealing zone of actively resorbing osteoclasts. In contrast, non-resorbing osteoclasts in BP-treated cultures showed morphological abnormalities, including retraction of pseudopods and vacuolization of cytoplasm. In the combined presence of LPS and BP, bone-resorbing osteoclasts were smaller and the sealing zone diffuse, reflecting reduced actin, OPN, and ß3 integrin staining. Depth analyses of calvaria showed that the area of resorbed bone was filled with proliferating osteoblastic cells that stained for alkaline phosphatase, collagen type I, and bone sialoprotein, regardless of the presence of BPs. These studies show that confocal microscopy of neonatal calvaria in culture can be used to assess the cytological relationships between osteoclasts and osteoblastic cells in response to agents that regulate bone remodeling in situ, avoiding systemic effects that can compromise in vivo studies and artifacts associated with studies of isolated osteoclasts. (J Histochem Cytochem 53:1525–1537, 2005)

Key Words: calvarial cells • bone remodeling • bisphosphonates • osteopontin • vß3 integrin • confocal microscopy

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