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It was previously shown that strontium ranelate (SR; S12911-PROTOS, Institut de Recherches Internationales Servier, Courbevoie, France) can modulate bone metabolism in rats and mice. To determine the long-term effects of SR on vertebral bone metabolism in adult mice, the compound or the vehicle was given in the diet to normal male and female mice for 104 weeks at the dose of 200, 600, or 1,800 mg/kg/d corresponding to 0.78, 2.34 or 7.01 mmol Sr(2+)/kg/d. SR dose-dependently increased plasma strontium concentration, as well as exposure to the drug. Histomorphometric analyses of indices of bone volume, bone formation, and resorption were determined in the endosteal vertebral bone. SR significantly increased the trabecular bone volume by 25% and 59% in females treated with SR 600 and 1,800 mg/kg/d, respectively. This was associated with a 27% and 62% increase in mineralized bone volume. Bone volume was also significantly increased by 17% and 38% in male mice treated with SR 200 and 1,800 mg/kg/d, respectively. In parallel, SR increased the osteoblastic surface by 131% in males. In addition to this stimulatory effect on bone formation, a 52% decrease in osteoclastic surface, and a dose-dependent decrease in osteoclastic number (30% to 47%), was observed in female mice. Finally, SR even at the highest dose tested did not alter the osteoid thickness, indicating no deleterious effect on bone mineralization. Altogether, these findings show that SR simultaneously increases bone formation and decreases bone resorption in male or female mice, which results in increased vertebral bone mass in both genders without deleterious effect on bone mineralization.

The hybrid plasmid pK4 containing the early genes of the simian virus SV-40, under the control of the adenovirus type 5 E1a promoter, was introduced into the multipotent embryonal carcinoma (EC) 1003. Expression of the SV-40 oncogenes was observed at the EC cell stage, and this allowed the derivation of immortalized cells corresponding to early stages of differentiation. Among the immortalized mesodermal derivatives obtained, one clone, C1, is committed to the osteogenic pathway. C1 cells have a stable phenotype, synthesize type I collagen, and express alkaline phosphatase activity. Although immortalized and expressing the SV-40 T antigen, the cells continue to be able to differentiate in vivo and in vitro. In vivo, after injection into syngeneic mice, they produce osteosarcomas. In vitro, the cells form nodules and deposit a collagenous matrix that mineralizes, going to hydroxyapatite crystal formation, in the presence of beta-glycerophosphate. This clonal cell line, which originates from an embryonal carcinoma, therefore differentiates into osteogenic cells in vivo and in vitro. This immortalized cell line will be useful in identifying specific molecular markers of the osteogenic pathway, to investigate gene regulation during osteogenesis and to study the ontogeny of osteoblasts.

The mesodermal clone C1 was derived from the multipotent embryonal carcinoma 1003 cell line transformed with the plasmid pK4 carrying SV40 oncogenes under the control of the adenovirus E1A promoter. We have shown that the C1 clone becomes committed to the osteogenic pathway when cultured in aggregates in the presence of mediators of the osteogenic differentiation. To further validate C1 as a model with which to study osteogenesis in vitro the kinetics of its differentiation was studied, focusing on the histology of the aggregates and on the expression of a set of genes corresponding to representative bone matrix proteins. The presence of ascorbic acid and beta- glycerophosphate specifically leads to mineralization in almost 100% of the aggregates. Transcription of the above genes, silent in exponentially growing cells, specifically occurred with the establishment of cell-cell contacts independently of the presence of ascorbic acid and inorganic phosphate. The latter, however, were absolutely required for matrix deposition and mineralization. In their presence, one observed an overall decline in type I collagen and alkaline phosphatase transcripts while osteocalcin and osteopontin transcripts preferentially accumulated in cells lining the mineralizing foci. Concomitantly, type I collagen and osteocalcin became extracellularly deposited. The osteogenic differentiation of C1 occurred while cells were still proliferating. The C1 clone thus behaves as a mesodermal stem cell, becoming committed to the osteogenic pathway upon: firstly, establishment of cellular contacts; and secondly, addition of ascorbate and beta-glycerophosphate. It therefore appears to be a promising in vitro system for deciphering the molecular basis of osteoblast ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)