Three-dimensional characterization of osteoclast bone-resorbing activity in the resorption lacunae

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  • Niroshani Surangika Soysa
    Section of Pharmacology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Japan
  • Neil Alles
    Section of Pharmacology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Japan Global COE program, Tokyo Medical and Dental University, Japan.
  • Aoki Kazuhiro
    Section of Pharmacology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Japan
  • Ohya Keiichi
    Section of Pharmacology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Japan

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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-κB 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.

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