Figure 6
Effect of combination of osteocytes and M-CSF/RANKL-treated bone marrow (BM) cells from Ccn2-deficient mice at 7 months-old on osteoclastogenesis. (A) Illustration of co-culture system of collagen gel-embedded WT or KO osteocyte-enriched population and BM cells treated with M-CSF and RANKL. (B) WT and KO mice at 1 month-old were injected with tamoxifen; and after 6 months, femurs were collected. After the bone marrow had been removed, BM cells were isolated from it and cultured for 1 day. Floating cells were re-plated and treated with M-CSF (50 ng/ml) for 2 days. The osteocyte-enriched population was isolated from the remaining bone tissues and cultured for 3 days. Then, the WT or KO osteocyte-enriched cell population of cells at a density of 2.5 × 104/well were embedded into collagen gel in 24-well plates; and the M-CSF-treated adherent BM cells (1.5 × 105 cells/ml) were inoculated onto the gel and treated with RANKL (100 ng/ml) for 10 days, with media containing RANKL being refreshed every 3 days. Then, cell lysate was prepared; and Western blot analysis was performed by using anti-NFATc1, CTSK and β-actin antibodies. (C) The amounts of NFATc1 and CTSK were determined densitometrically and were normalized to that of β-actin. Left graph shows the relative density of NFATc1 in the combination of WT osteocytes with WT BM cells (WT/WT; n = 6), WT osteocytes with KO BM cells (WT/KO; n = 5), KO osteocytes with WT BM cells (KO/WT; n = 6) and KO osteocytes with KO BM cells (KO/KO; n = 3). Right graph shows the relative density of CTSK in WT/WT (n = 5), WT/KO (n = 4), KO/WT (n = 5) and KO/KO (n = 3). In these graphs, the ordinate indicates the relative ratio with respect to the data of WT/WT (ratio = 1.0), and bars represent mean and standard deviation. The data were analyzed by Bonferroni’s test, and p < 0.05 (*) was considered significant. (D) Immunoreactivity for CCN2 was detected in osteocytes (arrows) and osteoblasts (arrowheads) of wild type murine cortical bone (7 months-old; b). Serial section was stained with hematoxylin-eosin (H-E; a), or without primary antibody as a negative control (c). The scale bar represents 100 μm. BM represents bone marrow.
