Methods For Assaying Osteoblasts And Osteoclasts In Vitro

In general, these activities have been evaluated using an experimental system, which leads to the differentiation of mature osteoclasts, in which bone marrow cells are cultured in the presence of osteoblasts.

Bone remodeling is a continuous process that helps to repair the microdamage to the bone matrix and adjusts the bone architecture to maintain bone strength. In this tightly regulated process, osteoclasts, which are multinucleated cells derived from the myeloid hemopoietic lineage, comprise the principal cell population that is involved in bone resorption, whereas osteoblasts, which originate from multipotent mesenchymal stem cells, carry out bone formation. The overall integrity of bone is controlled by biochemical factors, which include hormones, cyto-kines and other proteins, and mechanical factors. Perturbations of this complex but well-coordinated process result in skeletal abnormalities characterized by increased bone loss or excessive bone formation.

Osteoclast-like multinucleated cells can be differentiated in vitro from cocultures of mouse bone marrow cells and calvarial osteoblastic cells by treatment with the osteotropic factor 1a,25-dihydroxyvitamin D3 (1,25 (OH)2D3) (Miura et al., 2002). It has been shown that RANKL induces osteoclast formation in a culture of bone marrow cells in the presence of macrophage colony-stimulating factor without the need for osteoblasts. A mouse macrophage RAW264 cells are also known to differentiate into osteoclasts in the presence of RANKL (Hsu et al., 1999). When osteoblast/ stromal cells are stimulated by osteotropic factors such as 1,25(OH)2D3, RANKL is expressed and induces the differentiation of osteoclast progenitors by binding to RANK (Jimi et al., 1999).

Osteoblasts were isolated from 3-week-old mice that had been killed by cervical dislocation. The calvariae were digested in 2 ml of an enzyme solution containing 0.2% collagenase (Wako, Osaka, Japan) for 5 min at 37 °C in a shaking water bath. The supernatant was discarded and 2 ml of the enzyme solution was added. After the mixture was shaken at 37 °C for 20 min, the supernatant was carefully collected and transferred to a new tube. This digestion of calvariae by collagenase was repeated three times. The collected supernatant (6 ml) was placed in a centrifuge at 1500 x g for 5 min to collect osteoblastic cells. Cells were resuspended in a-minimal essential medium (a-MEM) (MP Biomedicals, Germany) containing 10% fetal bovine serum (FBS) and cultured to confluence in culture dishes for about 1 week. The cells were then detached from the culture dishes using trypsin-EDTA, suspended in a-MEM containing 10% FBS, and used for the coculture as osteoblastic cells.

Femoral and tibial bone marrow cells were collected from 7-week-old mice that had been killed by cervical dislocation. The tibiae and femora were removed and dissected free of adhering tissues. The bone ends were removed, and the marrow cavities were flushed by slowly injecting media at one end using a 26-gauge needle. The calvariae and bone marrow cells were washed and used in the coculture.

Mouse calvarial cells (1.3 x 105 cells/ml) were cocultured with bone marrow cells (5.0 x 106 cells/ml) in a-MEM containing 10% FBS in 48-well plates (Corning Inc., NY, USA). The culture volume was made up to 250 ml per well with a-MEM supplemented with 10% FBS, in the presence of 10 ng/ml 1,25(OH)2D3 (Biomol, PA, USA), with or without samples. All cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 in air. Half of the medium was changed after coculture for 3 days.

To count multinucleated cells, cultured cells were stained as described below. After cells were cultured, adherent cells were fixed with 10% formaldehyde in phosphate-buffered saline (—) for 20 min. After cells were treated with 95% ethanol for 1 min, the well surface was dried and treated with tartrate-resistant acid phosphatase (TRAP)-staining solution [0.1 M sodium acetate buffer (pH 5.0) containing 50 mM sodium tartrate, 0.1 mg/ ml naphthol AS-MX phosphate (Sigma Chemical Co., St. Louis, USA), and 1 mg/ml fast red violet LB salt (Sigma Chemical Co.)] for 30 min. TRAP-(+) multinucleated cells were then counted under a microscope.

Cell viability was evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Chemical Co.) assay. After culture, cells were treated with 1 mg/ml MTT for 2 h, precipitated dye was solubilized in dimethylsulfoxide, and the absorbance at 570 nm was measured.

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