A novel resorption assay for osteoclast functionality based on an osteoblast-derived native extracellular matrix.
Osteoclasts are large, mobile, bone-resorbing cells and play a critical role in bone remodeling and catabolic bone diseases. The major function of osteoclasts is to hydrolyze inorganic hydroxyapatite and degrade organic bone matrix, mainly collagen. For evaluation of differentiation to fully functional osteoclasts in vitro, a quantitative functional resorption assay is essential. Currently available commercial test systems are either based on the organic or the inorganic part of the bone matrix. The novel resorption assay presented here is based on decellularized osteoblast-derived matrix. SaOS-2 cells were used for the synthesis of a densely mineralized extracellular bone matrix (ECM) in alpha-MEM medium, which strongly accelerates their matrix synthesis. After removal of the SaOS-2 cells, osteoclast precursors are plated on the osteoblast-derived matrix and stained by von Kossa. Subsequently, resorption pits were quantified by densitometry using an imaging program. Using this novel assay, we show that (i) RAW 264.7 cells resorbed the osteoblast-derived matrix continuously from day 6 until day 9 of culture, a process that is dose dependent on the macrophage colony-stimulating factor (M-CSF) concentration, (ii) the resorption performance of RAW 264.7 was dose-dependently inhibited by IFN-gamma, and (iii) the assay is working with primary human and mouse osteoclast precursors as well. In conclusion, this quantitative, functional, easy-to-use, inexpensive assay will advance analysis of osteoclast biology.
Lutter
Anne-Helen
AH
Medical Faculty Carl Gustav Carus, Institute of Physiological Chemistry, Dresden University of Technology, Fiedlerstrasse 42, 01307 Dresden, Germany. ute.hempel@tu-dresden.de
Hempel
Ute
U
Wolf-Brandstetter
Cornelia
C
Garbe
Annette I
AI
Goettsch
Claudia
C
Hofbauer
Lorenz C
LC
Jessberger
Rolf
R
Dieter
Peter
P
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010710:Phosphates / Q000378:metabolism; D013194:Staining and Labeling
D016207:Cytokines; D010710:Phosphates; D002118:Calcium