title: Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone.
abstract: Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and is therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments. KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21 days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.
authors.affiliation: Department of Radiology, Leiden University Medical Center, The Netherlands.
authors.forename: Martiene J C
authors.initials: MJ
authors.lastname: Moester
authors.affiliation: Department of Orthopedic Surgery, Leiden University Medical Center, The Netherlands.
authors.forename: Monique A E
authors.initials: MA
authors.lastname: Schoeman
authors.affiliation: Department of Radiology, Leiden University Medical Center, The Netherlands; Percuros BV, Leiden, The Netherlands.
authors.forename: Ineke B
authors.initials: IB
authors.lastname: Oudshoorn
authors.affiliation: Department of Radiology, Leiden University Medical Center, The Netherlands.
authors.forename: Mara M
authors.initials: MM
authors.lastname: van Beusekom
authors.affiliation: Department of Radiology, Leiden University Medical Center, The Netherlands; Percuros BV, Leiden, The Netherlands.
authors.forename: Isabel M
authors.initials: IM
authors.lastname: Mol
authors.affiliation: Department of Radiology, Leiden University Medical Center, The Netherlands.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Kaijzel
authors.affiliation: Department of Radiology, Leiden University Medical Center, The Netherlands.
authors.forename: Clemens W G M
authors.initials: CW
authors.lastname: Löwik
authors.affiliation: Department of Radiology, Leiden University Medical Center, The Netherlands; Percuros BV, Leiden, The Netherlands. Electronic address: k.e.de_rooij@lumc.nl.
authors.forename: Karien E
authors.initials: KE
authors.lastname: de Rooij
mesh_terms: D000818:Animals; D000880:Anthraquinones; D002113:Calcification, Physiologic / Q000502:physiology*; D002454:Cell Differentiation; D002460:Cell Line; D005456:Fluorescent Dyes; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D010006:Osteoblasts / Q000502:physiology; D010012:Osteogenesis / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000880:Anthraquinones; D005456:Fluorescent Dyes; C004468:Alizarin Red S
keywords: Alizarin Red S; Fluorescence; Mineralization; Osteoblast; Quantification; Staining