title: Three-dimensional characterization of osteoclast bone-resorbing activity in the resorption lacunae.
abstract: Confocal laser microscopy is a well-recognized research tool in the fields of biological and material science which enables high-resolution images of samples with minimum requirements for specimen preparation. Here we introduce an innovative technique for the 3-D description and measurement of resorption pits using Super Depth Surface Profile Measurement Microscope based on the principle of confocal microscope. We show one example of culturing for 48 h with an established NF-kappaB inhibitor named NBD-peptide after plating mature osteoclasts on dentine slices with osteoblasts. The activity of osteoclasts is measured by determining the volume of resorbed portion of dentine by osteoclasts in vitro. The 3-D surface profile could be obtained by detecting the position at which the reflected laser intensity from the target becomes the maximum on z-axis. The volume and depth of resorption lacunae by stimulated osteoclasts is significantly increased compared to the un-stimulated group without changing of resorption area. The increase in volume and depth are dose-dependently inhibited by the NBD-peptide. Comparing to the classical method by measuring 2-D area of pits, analysis based on this technique could provide reliable quantitative assessment reflecting the osteoclast activity.
authors.affiliation: Section of Pharmacology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Japan. niroshani.hpha@tmd.ac.jp
authors.forename: Niroshani Surangika
authors.initials: NS
authors.lastname: Soysa
authors.forename: Neil
authors.initials: N
authors.lastname: Alles
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Aoki
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ohya
mesh_terms: D000135:Acid Phosphatase / Q000032:analysis; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001854:Bone Marrow Cells / Q000187:drug effects / Q000473:pathology; D001862:Bone Resorption / Q000473:pathology*; D002478:Cells, Cultured; D002762:Cholecalciferol / Q000494:pharmacology; D018920:Coculture Techniques; D003804:Dentin / Q000187:drug effects / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D021621:Imaging, Three-Dimensional; D007527:Isoenzymes / Q000032:analysis; D051379:Mice; D008813:Mice, Inbred ICR; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D010006:Osteoblasts / Q000187:drug effects / Q000473:pathology; D010010:Osteoclasts / Q000187:drug effects / Q000473:pathology*; D010455:Peptides / Q000008:administration & dosage / Q000494:pharmacology; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: D015415:Biomarkers; D007527:Isoenzymes; C467063:NBD peptide, mouse; D016328:NF-kappa B; D010455:Peptides; D002762:Cholecalciferol; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase