Quantification of temporal changes in 3D osteoclastic resorption pit using confocal laser scanning microscopy

Abstract

Osteoclasts, cells responsible for bone resorption, generate 3D resorption pits by attaching to the bone surface and excavating the underneath bone matrix. Changes in trabecular bone microstructure due to these excavations may be detrimental to overall bone strength. However, most studies on bone resorption analyze osteoclastic activity using 2D staining techniques which do not correctly show the effects of a 3D resorption pit. Therefore, in this study, our goal was to (1) understand how osteoclastic bone resorption pits are generated temporally by focusing on the changes in resorptive area and resorptive depth and (2) understand how changes in the number of osteoclasts, increases in pit area and depth individually contribute to the eventual increases in overall resorption pit volume at each time period. We accurately measured the changes in resorption pit volume, depth, and area by using 3D confocal laser scanning microscopy and counted tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and pit number by using conventional 2D staining techniques at Day 7, 14, and 21 after osteoclasts were seeded on dentine slices (Day 0). Resorption pit numbers significantly increased by Day 7 and then decreased by Day 14 and the level was maintained until Day 21. The numbers of osteoclast per resorption pit are similar at Day 7 and Day 14 but the number significantly decreases at Day 21. Pit depth and pit volume both increased between Day 7 and Day 14 but only pit volume continued to increase between Day 14 and Day 21. These results indicate that up to Day 7, the dispersed osteoclasts generate many individual pits on various spots of the dentine surface. Between Day 7 and Day 14, several osteoclasts in the process of fusion appear to target a single pit and then the larger fused osteoclasts with more resorptive efficiency excavate resorption pits deeper resulting in the increase in pit volume. Between Day 14 and Day 21, osteoclastic bone-resorbing activities appear to be significantly decreased due to osteoclast degradation.

An Ammonia-Induced Calcium Phosphate Nanostructure: A Potential Assay for Studying Osteoporosis and Bone Metastasis

“…Comparing the AiM-CaP coating with the bone slice in terms of resorption in vitro , the osteoclasts left shallow resorption pits on the AiM-CaP insert, but they left the irregular shape of resorption pits with various depths on the bone slice. On the bone slice, the depth of resorption pits varied from 2 to 70 μm. − To quantify the resorption activity accurately on the bone slice, a three-dimensional measurement method was necessary, and it increased the difficulties of quantification . Additionally, cell lacunae in bone may also cause difficulties in differentiating the newly formed resorption pits from the pre-existing cavities .…”

“…Developing a reliable in vitro assay that simulates osteoclastic resorption of bones would be a first step to take before proceeding to any animal or human studies. − Traditionally, slices made from bones and dentin have been well accepted for in vitro studies on osteoclastic resorption. ,, Both the number of osteoclasts and the area of the resorption pits can be measured and used as indicators for osteoclast activities. ,,− However, the organization of structural elements including porosity and mineral content in dentin and cortical bone varies between species and even within different locations on the same sample, preventing one from obtaining probes with consistent quality …”