In vitro mineralization of fetal rat parietal bones in defined serum-free medium: effect of beta-glycerol phosphate.

We have developed a bone organ culture system that mineralizes in vitro. Fetal rat parietal bones (20 days old) were cultured in a chemically defined serum-free medium containing physiological 3 mM phosphate. During 5 days in culture, calcium content increased from 26 to 55 micrograms and dry weight increased from 137 to 194 micrograms. After 2 days in vivo, the calcium content of the parietal bone showed a comparable increase to 49 micrograms and dry weight increased to 183 micrograms. During culture, the mineralized bone area in thick sections increased from 11 to 23%, which paralleled the doubling in calcium content. Fluorescent calcein labeling during the 5 day culture period demonstrated that calcification occurs in an ordered pattern. Protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP). The percentage collagen synthesis decreased from 17.5% at 0 time to 5.0% at 2 days and then increased to 9.4% at 5 days of culture. Varying the inorganic phosphate concentration in the medium or adding beta-glycerol phosphate was found to affect mineralization. After 5 days in culture, bones treated with 1 mM phosphate exhibited a large region of unmineralized osteoid with only a 23% increase in calcium content compared with 112% in control (3 mM phosphate) bones and a 28% increase in dry weight compared with a 40% increase in control. Treatment for 5 days with 6 mM phosphate or 1, 3, or 10 mM beta-glycerol phosphate had no significant effect on dry weight compared to control bones. However, bone calcium content increased significantly from 55 +/- 5 micrograms in control cultures to 105 +/- 7 with 6 mM phosphate, 74 +/- 6 with 3 mM beta-glycerol phosphate, and 75 +/- 5 micrograms with 10 mM beta-glycerol phosphate. Calcified area measured by histomorphometry was also significantly greater than in control bones, but this was mainly due to ectopic calcification in the periosteum, representing from 23 to 74% of the total increase in calcified matrix in bones cultured with 6 mM phosphate or 1-10 mM beta-glycerol phosphate. Ultrastructural analysis demonstrated that ectopic calcification was associated with cell death and debris. Therefore, calcification with beta-glycerol phosphate and high concentrations of inorganic phosphate differed from mineralization in vivo or in bones cultured with a physiologically concentration of phosphate.