RAW264.7 cells, a mouse macrophage cell line, were used as osteoclast precursor cells and maintained in α modified essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. For osteoclast induction, cells were plated in a 96-well plate at a density of 4×103 cells/well and stimulated with 100 ng/ml RANKL for 4 days. For the inhibition study, cells were pre-incubated in α-MEM supplemented with vehicle or with various concentrations of anthocyanin-rich extracts and anthocyanidins, 1 h before the addition of RANKL. To confirm multinucleated osteoclast formation, the cultured cells were fixed in 10% formalin for 3 minutes, and then stained with an osteoclast marker enzyme, tartrate-resistant acid phosphatase (TRAP). Effects of anthocyanins and anthocyanidins on osteoclast formation were evaluated by morphological observations and the intensity of TRAP staining was measured at 520 nm using a spectrophotometer (SpectraMax M5; Molecular Devices, Sunnyvale, CA, USA).
Osteoblasts were isolated from newborn calvariae of C57BL/6J mice, as described previously with slight modifications [19] (link). Briefly, calvariae were minced and sequentially digested with collagenase solution at 37°C. Cells retrieved from the osteogenic cell fractions were separately cultured in α-MEM supplemented with 10% FBS and antibiotics. After 24 h, cells were pooled and grown in multi-well plates in the same medium containing 50 µg/ml of ascorbic acid (AA), 10 µM dexamethasone (Dex) and 10 mM β-glycerophosphate (β-GP) with or without anthocyanin-rich extracts. After two weeks culture, cells were stained with von Kossa’ s staining to determine the matrix mineralization, as described previously [19] (link).
Osteoblasts were isolated from newborn calvariae of C57BL/6J mice, as described previously with slight modifications [19] (link). Briefly, calvariae were minced and sequentially digested with collagenase solution at 37°C. Cells retrieved from the osteogenic cell fractions were separately cultured in α-MEM supplemented with 10% FBS and antibiotics. After 24 h, cells were pooled and grown in multi-well plates in the same medium containing 50 µg/ml of ascorbic acid (AA), 10 µM dexamethasone (Dex) and 10 mM β-glycerophosphate (β-GP) with or without anthocyanin-rich extracts. After two weeks culture, cells were stained with von Kossa’ s staining to determine the matrix mineralization, as described previously [19] (link).
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