Calcification of osteoblastlike rat osteosarcoma cells in agarose suspension cultures.

Ros 17/2 clonal rat osteosarcoma cells calcify when cultured in the presence of 10 micrograms/ml beta-glycerol phosphate in an agarose gel. Culture in 1% agarose inhibited cell division while allowing cells to remain metabolically active and viable for over 21 days. Serial photography of the same microscopic field shows a progressive deposition of calcium phosphate during the course of the experiment. The deposition of calcium around cells was confirmed by calcium-specific stains, and by energy dispersive X-ray analysis (EDX) during scanning electron microscopy. Cells with high calcium content analyzed by EDX had Ca:P ratios similar to hydroxyapatite. Total calcium progressively increased in beta-glycerol phosphate-treated cultures whereas the control plates maintained a constant calcium content over 16 days. Alkaline phosphatase activity increased with time in culture whereas cells with beta-glycerol phosphate maintained the alkaline phosphatase values achieved at the time of initial calcification. Alkaline phosphatase staining revealed no correlation between the presence of the enzyme activity and calcification. Radioimmunoassay for the bone-specific vitamin K-dependent protein bone Gla protein showed that beta-glycerol phosphate-treated cells accumulate over sixfold greater amounts of this protein. Our studies show that ROS cells can calcify and accumulate bone-specific matrix components when cultured in a 3-dimensional agarose matrix.