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Updated: Jun 12, 2026
A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro
Published on: June 16, 2022
Alterations in osteoclast function and phenotype induced by different inhibitors of bone resorption--implications for
Anita V Neutzsky-Wulff1, Mette G Sørensen, Dino Kocijancic
1Nordic Bioscience A/S, Herlev DK 2730, Denmark.
Background:
Normal osteoclasts resorb bone by secretion of acid and proteases. Recent studies of patients with loss of function mutations affecting either of these processes have indicated a divergence in osteoclastic phenotypes. These difference in osteoclast phenotypes may directly or indirectly have secondary effects on bone remodeling, a process which is of importance for the pathogenesis of both osteoporosis and osteoarthritis. We treated human osteoclasts with different inhibitors and characterized their resulting function.
Methods:
Human CD14 + monocytes were differentiated into mature osteoclasts using RANKL and M-CSF. The osteoclasts were cultured on bone in the presence or absence of various inhibitors: Inhibitors of acidification (bafilomycin A1, diphyllin, ethoxyzolamide), inhibitors of proteolysis (E64, GM6001), or a bisphosphonate (ibandronate). Osteoclast numbers and bone resorption were monitored by measurements of TRACP activity, the release of calcium, CTX-I and ICTP, as well as by counting resorption pits.
Results:
All inhibitors of acidification were equally potent with respect to inhibition of both organic and inorganic resorption. In contrast, inhibition of proteolysis by E64 potently reduced organic resorption, but only modestly suppressed inorganic resorption. GM6001 alone did not greatly affect bone resorption. However, when GM6001 and E64 were combined, a complete abrogation of organic bone resorption was observed, without a great effect on inorganic resorption. Ibandronate abrogated both organic and inorganic resorption at all concentrations tested [0.3-100 microM], however, this treatment dramatically reduced TRACP activity.
Conclusions:
We present evidence highlighting important differences with respect to osteoclast function, when comparing the different types of osteoclast inhibitors. Each class of osteoclast inhibitors will lead to different alterations in osteoclast quality, which secondarily may lead to different bone qualities.
Insights
Different osteoclast inhibitors impact bone resorption uniquely. Acidification inhibitors block both organic and inorganic resorption, while proteolysis inhibitors selectively affect organic components, influencing bone quality differently.
Area of Science:
- Bone Biology
- Cell Biology
- Pharmacology
Background:
- Osteoclasts resorb bone via acid and protease secretion.
- Functional mutations reveal distinct osteoclast phenotypes.
- Altered bone remodeling contributes to osteoporosis and osteoarthritis.
Purpose of the Study:
- To investigate the functional consequences of inhibiting osteoclast acidification and proteolysis.
- To compare the effects of different classes of osteoclast inhibitors on bone resorption.
Main Methods:
- Human monocytes differentiated into osteoclasts.
- Osteoclasts cultured on bone with inhibitors of acidification (bafilomycin A1, diphyllin, ethoxyzolamide), proteolysis (E64, GM6001), or bisphosphonate (ibandronate).
- Bone resorption monitored by TRACP activity, calcium release, CTX-I, ICTP, and resorption pits.
Main Results:
- Acidification inhibitors equally inhibited organic and inorganic resorption.
- Proteolysis inhibition (E64) reduced organic resorption more than inorganic.
- Combined proteolysis inhibitors abrogated organic resorption; ibandronate inhibited both resorption types and TRACP activity.
Conclusions:
- Osteoclast inhibitors exhibit distinct functional effects on bone resorption.
- Different inhibitor classes induce unique alterations in osteoclast activity.
- These alterations may lead to varied secondary effects on bone quality.
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