title: Quantitative and reliable in vitro method combining scanning electron microscopy and image analysis for the screening of osteotropic modulators.
abstract: The increased generation and up-regulated activity of bone resorbing cells (osteoclasts) play a part in the impairment of bone remodeling in many bone diseases. Numerous drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators) have been proposed to inhibit this increased osteoclastic activity. In this report, we describe a pit resorption assay quantified by scanning electron microscopy coupled with image analysis. Total rabbit bone cells with large numbers of osteoclasts were cultured on dentin slices. The whole surface of the dentin slice was scanned and both the number of resorption pits and the total resorbed surface area were measured. Resorption pits appeared at 48 h and increased gradually up to 96 h. Despite the observation of a strong correlation between the total resorption area and the number of pits, we suggest that area measurement is the most relevant marker for osteoclastic activity. Osteotropic factors stimulating or inhibiting osteoclastic activity were used to test the variations in resorption activity as measured with our method. This reproducible and sensitive quantitative method is a valuable tool for screening for osteoclastic inhibitors and, more generally, for investigating bone modulators.
authors.affiliation: Inserm, UMRS 791, Laboratoire d'ingénierie Ostéo-articulaire et dentaire (LIOAD), Univ Nantes, Faculté de chirurgie dentaire, F-44042 Nantes, France.
authors.forename: Gaël
authors.initials: G
authors.lastname: Grimandi
authors.forename: Assem
authors.initials: A
authors.lastname: Soueidan
authors.forename: Abed Aktam
authors.initials: AA
authors.lastname: Anjrini
authors.forename: Zahi
authors.initials: Z
authors.lastname: Badran
authors.forename: Paul
authors.initials: P
authors.lastname: Pilet
authors.forename: Guy
authors.initials: G
authors.lastname: Daculsi
authors.forename: Corinne
authors.initials: C
authors.lastname: Faucheux
authors.forename: Jean Michel
authors.initials: JM
authors.lastname: Bouler
authors.forename: Jérôme
authors.initials: J
authors.lastname: Guicheux
mesh_terms: D000818:Animals; D050071:Bone Density Conservation Agents / Q000494:pharmacology; D001862:Bone Resorption / Q000378:metabolism / Q000517:prevention & control; D002352:Carrier Proteins / Q000494:pharmacology; D002478:Cells, Cultured; D004164:Diphosphonates / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007093:Imidazoles / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000494:pharmacology; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008856:Microscopy, Fluorescence; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D053245:RANK Ligand; D011817:Rabbits; D013997:Time Factors; D000077211:Zoledronic Acid
chemical_list: D050071:Bone Density Conservation Agents; D002352:Carrier Proteins; D004164:Diphosphonates; D007093:Imidazoles; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D000077211:Zoledronic Acid