Quantification of Bone Formation on Calcium Phosphate Ceramic Thin Film, in Vitro by Tetracycline Labelling

Abstract Introduction We have previously described a quantitative in vitro method for the assessment of bone matrix deposition, employing the fluorescent antibiotic tetracycline1. In a present study we demonstrate the relationship of bone matrix formation by osteoblasts as a function of cell seeding density, and time, on calcium phosphate ceramic thin film substrates. Materials and Methods Substrata. Commercially available 3x1 inch quartz Osteologic™ chamber slides (Millenium Biologix Inc., Kingston, Ont.) previously shown to support bone growth, were used as substrates for osteoblastic cell growth2. These chamber slides are coated with a sintered sub-micron calcium phosphate ceramic thin film, and are fitted with a removable 16 well superstructure. Rat Bone Marrow Cell Culture. Bone marrow cells from young adult male Wistar rat (115-120g) femora were cultured according to a previously described protocol. Each well of each Osteologic™ chamber slide (14/16 wells; 2 wells contained media, but without cells, to provide background for tetracycline fluorescence) was inoculated with a 250 μl of cell suspension.