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 journalArticle

14 Citations (Scopus)

Abstract

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
Volume410
Issue number1
DOIs
Publication statusPublished - Mar 1 2011
Externally publishedYes

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Bone Resorption
Bone
Assays
Coatings
pamidronate
Osteoclasts
Osteoporosis
Fluorescence
Chondroitin
Bisbenzimidazole
Macrophages
Durapatite
Fluorescein
Labeling
Image analysis
Estradiol
Chemical activation
Cells
Ligands
Cell Line

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Evaluation of osteoclastic resorption activity using calcium phosphate coating combined with labeled polyanion. / Miyazaki, Tatsuya; Miyauchi, Satoshi; Anada, Takahisa; Imaizumi, Hideki; Suzuki, Osamu.

In: Analytical Biochemistry, Vol. 410, No. 1, 01.03.2011, p. 7-12.

Research output: Contribution to journalArticle

Miyazaki, Tatsuya ; Miyauchi, Satoshi ; Anada, Takahisa ; Imaizumi, Hideki ; Suzuki, Osamu. / Evaluation of osteoclastic resorption activity using calcium phosphate coating combined with labeled polyanion. In: Analytical Biochemistry. 2011 ; Vol. 410, No. 1. pp. 7-12.
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