Osteoclast responses to lipopolysaccharide, parathyroid hormone and bisphosphonates in neonatal murine calvaria analyzed by laser scanning confocal microscopy.
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 microg/ml), parathyroid hormone (PTH; 10(-8) M), and bisphosphonates (BPs; 1-25 microM clodronate or 0.1-2.5 microM risedronate) in cultured neonatal calvaria. Following treatment with LPS or PTH (<48 hr), osteopontin (OPN) and the alphavbeta3 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 beta3 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.
Suzuki
Keiko
K
Department of Pharmacology, School of Dentistry, Showa University, Shinagawa, Tokyo, Japan. suzukik@dent.showa-u.ac.jp
Takeyama
Sadaaki
S
Kikuchi
Takashi
T
Yamada
Shoji
S
Sodek
Jaro
J
Shinoda
Hisashi
H
D000135:Acid Phosphatase / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D015519:Bone Density; D001862:Bone Resorption / Q000378:metabolism; D004002:Clodronic Acid / Q000494:pharmacology; D024042:Collagen Type I / Q000378:metabolism; D004164:Diphosphonates / Q000494:pharmacology*; D012968:Etidronic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes; D039661:Integrin beta3 / Q000378:metabolism; D058574:Integrin-Binding Sialoprotein; D007527:Isoenzymes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D009924:Organ Culture Techniques; D010010:Osteoclasts / Q000187:drug effects* / Q000378:metabolism; D053495:Osteopontin; D010281:Parathyroid Hormone / Q000494:pharmacology*; D000068296:Risedronic Acid; D012795:Sialoglycoproteins / Q000378:metabolism; D012886:Skull / Q000166:cytology / Q000187:drug effects* / Q000254:growth & development; D000071681:Tartrate-Resistant Acid Phosphatase
D000199:Actins; D024042:Collagen Type I; D004164:Diphosphonates; D005456:Fluorescent Dyes; C546872:Ibsp protein, mouse; D039661:Integrin beta3; D058574:Integrin-Binding Sialoprotein; D007527:Isoenzymes; D008070:Lipopolysaccharides; D010281:Parathyroid Hormone; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D004002:Clodronic Acid; D053495:Osteopontin; D000469:Alkaline Phosphatase; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D000068296:Risedronic Acid; D012968:Etidronic Acid