Fig. 6: Increased extracellular Ca²⁺ in osteoclast-activated bone resorption conditioned medium affect MSCs phenotype.

a Preparation of osteoclastic bone resorption conditioned medium. Osteoclastic precursors isolated from mouse bone marrow were cultured on mouse calvairal bone slices in the presence of MCSF and RANKL to induce osteoclast differentiation (OC), or with only MCSF as a control (Pre). To monitor morphological change of precursors and determine osteoclast differentiation, precursors were cultured under the same condition without bone slices (a, b’). After 16 days of incubation, cells were stained for TRAP activity. Precursors and bone slice only showed TRAP-negative (a, a’, c, c’, d, d’), and active osteoclast revealed TRAP-positive (b, b’, e, e’). Image, x100 magnification. b Analysis of bone surface topology by using scanning electronic microscopy (SEM). SEM image showed a rough bone surface with several dimples by the osteoclast-activated bone resorption (e, e’, h). Bone slice surface in the presence of media alone was clean without any cells and dents (c, c’, f). Image, x300. The 1000x magnification images represent dotted rectangle boxes. c After collecting the bone resorption conditioned medium as described in Materials and methods section, the extracellular Ca²⁺ concentration was analyzed. BCCM bone control-conditioned medium, BPCM precursor with bone-conditioned medium, BRCM osteoclast-activated bone resorption-conditioned medium. d BRCM induces secretion of OPN in BM-MSCs. BM-MSCs were treated with the indicated bone resorption-conditioned media (4, 8, 12, and 16th day) for 48 h. Culture medium was collected and used for ELISA to measure secreted OPN levels. e BRCM increases cell proliferation of BM-MSCs. Cells were incubated in the indicated conditioned medium, and cell proliferation was measured by BrdU assay after 48 h treatment. f BRCM-treated culture medium (CM) enhances the migration of BM-MSCs. BRCM-treated CM from the 16th days was loaded in the lower chamber. To neutralize secreted OPN, the indicated CMs were pre-incubated with individual neutralizing antibodies for 1 h and used as the medium for the lower chamber. Migration of BM-MSCs for 6 h to the lower chamber were visualized with staining (left). Number of migrated cells per field of view (FV, x50 magnification) is expressed (right). g Working model illustrating the role of ionized calcium in bone remodeling surface. **p < 0.01; ***p < 0.001 vs. BCCM. ^##p < 0.01; ^###p < 0.001 vs. BPCM. ***p < 0.001 vs. first bar