Strontium potently inhibits mineralisation in bone-forming primary rat osteoblast cultures and reduces numbers of osteoclasts in mouse marrow cultures

Abstract

Summary: The basic mechanisms by which strontium ranelate acts on bone are still unclear. We show that an important action of strontium salts is to block calcification in cultures of osteoblasts, the bone-forming cells. These results suggest that strontium treatment could have previously overlooked effects on bone.

Introduction: The basic mechanisms of action of strontium ranelate (SrR) on bone have remained unclear. We studied the direct actions of Sr(2+) salts in functional cultures of osteoblasts and osteoclasts.

Methods: Cultures of primary osteoblasts from rat calvariae and osteoclast-forming mouse marrow cells were treated continuously with either SrR or strontium chloride (SrCl2).

Results: Abundant, discretely mineralised 'trabecular' bone structures formed in control osteoblast cultures after 14 days. SrR at 0.01, 0.1 and 1 mM inhibited mineralisation to 59, 98 and 100 % (all p < 0.001) of control values, respectively. SrCl2 at the same concentrations caused similar inhibitions. Osteoblast cell numbers and alkaline phosphatase activity were unaltered. SrR dose-dependently reduced the formation of multinucleated osteoclasts from marrow mononuclear cells cultured on dentine for 8 days in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL), with a 50 % inhibition occurring at 1 mM; SrCl2 was slightly less effective, eliciting a maximal 30 % inhibition. Corresponding decreases in total resorption pit formation were observed, suggesting Sr(2+) salts affect osteoclast formation rather than resorptive activity.

Conclusion: Our findings are consistent with the documented physicochemical inhibitory action of Sr(2+) on mineralisation but contrast with reports that Sr(2+) increases osteoblast activity and number in vitro. Our results suggest that rather than acting as an agent that 'uncouples' bone formation and resorption, Sr(2+) acts as a global inhibitor of bone cell function, with particularly marked effects on mineralisation. The potential effects of long-term Sr(2+) on secondary mineralisation in bone deserve investigation.

Fig. 1

Strontium ranelate (SrR) blocks bone matrix mineralisation in 14-day primary cultures of rat osteoblasts. Upper row images are reflected light scans of unstained cell wells; mineralised bone nodules appear as white features; scale bar = 0.5 cm. Lower row images are corresponding phase contrast (transmitted light) micrographs of unstained cell layers; mineralised bone nodules appear as black features, and unmineralised collagenous matrix is white-grey; scale bar = 0.5 mm

Fig. 2

a Dose-dependent inhibition of mineralised bone formation in 14-day rat osteoblast cultures by strontium salts. Strontium ranelate (SrR) caused near-total blockade at concentrations ≥0.1 mM; strontium chloride (SrCl₂) completely blocked mineralisation at a concentration of 1 mM. b Strontium salts caused a corresponding increase in the retention of ionised calcium in the culture medium of bone-forming osteoblasts; the measured concentration of ionised Ca²⁺ in the culture medium at day 3 (i.e. before onset of osteoblast differentiation and mineral deposition) was 1.03 mM. Culture medium pH remained within 0.01 unit of the control value. pH was corrected for CO₂ concentration. Data are means ± SEM for six replicate determinations; ***p < 0.001, significantly different from control

Fig. 3

a Effect of strontium salts on alkaline phosphatase (ALP) activity in 14-day rat osteoblast cultures: modest inhibition by SrCl₂. b Strontium salts were also associated with moderate decreases in cell viability, assessed as lactate dehydrogenase activity, although cell numbers were not affected. Data are means ± SEM for six replicate determinations; *p < 0.05; **p < 0.01; ***p < 0.001, significantly different from control

Fig. 4

Strontium ranelate causes a moderate inhibition of numbers of multinucleated osteoclasts observed in mouse marrow mononuclear cells cultured for 8 days on ivory discs with M-CSF and RANKL. TRAP-stained preparation; resorption pits are visible as tan areas associated with osteoclasts. Scale bar = 100 μm

Fig. 5

a Strontium salts cause a moderate inhibition of resorption pit formation by osteoclasts generated in 8-day cultures of mouse marrow cells on ivory discs. b Strontium salts were associated with corresponding decreases in numbers of multinucleated, TRAP-stained osteoclasts. c The resorptive activity of individual osteoclasts was not affected by strontium salts. Data are means ± SEM for eight replicate determinations; *p < 0.05; ***p < 0.001, significantly different from control