Three-dimensional characterization of osteoclast bone-resorbing activity in the resorption lacunae.
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.
Soysa
Niroshani Surangika
NS
Section of Pharmacology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Japan. niroshani.hpha@tmd.ac.jp
Alles
Neil
N
Aoki
Kazuhiro
K
Ohya
Keiichi
K
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
D015415:Biomarkers; D007527:Isoenzymes; C467063:NBD peptide, mouse; D016328:NF-kappa B; D010455:Peptides; D002762:Cholecalciferol; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase