Evaluation of osteoclastic resorption activity using calcium phosphate coating combined with labeled polyanion

Tatsuya Miyazaki, Satoshi Miyauchi, Takahisa Anada, Hideki Imaizumi, Osamu Suzuki

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)


Osteoclasts are involved in bone resorption, and its activation is considered one of the causes of osteoporosis. The pit assay is the principal method for evaluating osteoclast function by measuring hydroxyapatite resorption in vitro. However, the pit assay requires time and trained techniques, including the pit image analysis, and there is no other easy method for evaluating bone resorption. In this study, we developed a novel approach to quantify the bone resorption activity using a calcium phosphate (CaP) coating labeled with fluorescent polyanion. Fluoresceinamine-labeled chondroitin polysulfate or Hoechst 33258-labeled deoxyribonucleic acid was used for CaP labeling. When macrophage cell line RAW264 was cultured on the labeled CaP under the stimulation with the receptor activator of the NF-κB ligand (RANKL), RAW264 cells differentiated into osteoclastic cells and the fluorescence intensity of the culture supernatant and pit area increased in a time- and dose-dependent manner. Furthermore, drugs for osteoporosis treatment, such as pamidronate and β-estradiol, inhibited fluorescein release by the cells stimulated with RANKL. A positive correlation between the fluorescence intensity and pit area was observed (r = 0.917). These results indicated that this new method using fluorescent polyanion-labeled CaP is a standardized useful assay system for the evaluation of bone resorption activity.

Original languageEnglish
Pages (from-to)7-12
Number of pages6
JournalAnalytical Biochemistry
Issue number1
Publication statusPublished - Mar 1 2011
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Evaluation of osteoclastic resorption activity using calcium phosphate coating combined with labeled polyanion'. Together they form a unique fingerprint.

Cite this