Osteoclast isolation and culture
The procedure for the isolation and culture of osteoclasts described earlier by Boyde et al. and by Chambers et al.[17, 18] was slightly modified and has been described in detail previously . Briefly, mechanically harvested osteoclasts from the long bones of 1 or 2-day-old Sprague-Dawley rat pups were allowed to attach to sonicated bovine cortical bone slices (0.5 cm2). After 30 minutes, the non-attached cells were rinsed away, and the remaining cells on the bone slices were cultured in three groups: control, heptanol and Gap 27, described below. Each treatment group had five bone slices and each of the experiments was repeated three times with similar results obtained each time. The control group was cultured in DMEM (Dulbecco's modified Eagle's medium) buffered with 20 mM Hepes and containing 0.84 g of sodium bicarbonate/liter, 2 mM L-glutamine, 100 IU of penicillin/ml, 100 μg of streptomycin/ml and 7% heat-inactivated fetal calf serum (FCS), at +37°C (5% CO2, 95% air), which medium will be later referred to as DMEM-FCS. The heptanol group was cultured in medium with 0.15% ethanol and 3 mM heptanol in DMEM-FCS. We chose not to repeat our earlier experiments with ethanol in this study, since our previous report shows that ethanol has no effect on the parameters studied here . Third group of bone cells, Gap 27 peptide group, was cultured with Gap 27 peptide at concentration 500 μM in DMEM-FCS. In all the three groups cells were cultured for 48 hours, after which the cultures were stopped by fixing the cells and bone slices with freshly made or refrigerated 3% paraformaldehyde (PFA) and 2% sucrose in phosphate-buffered saline (PBS) for 10 minutes at room temperature. Gap 27 peptide (amino acid sequence SRPTEKTIFII) was synthesised by Keck Foundation Biotechnology Resource Laboratory, Yale University School of Medicine, USA; purity was >95%.
Number and activity of osteoclasts
Bone cell cultures were cultured for 48 hours with three different treatments (control, heptanol and Gap 27). After the culture period, bone slices were fixed as described earlier above. The cells were stained for tartrate-resistant acid phosphatase (TRAP), a commonly accepted marker of osteoclasts , using a Sigma TRAP kit (no. 386-A, Sigma Chemicals, St. Louis, MO) according to the manufacturer's instruction. To visualise the nuclei, the cells were incubated with the DNA-binding fluorochrome Hoechst 33258 (1 mg/ml stock diluted 1:800 in PBS, Sigma Chemical Co, St. Louis, MO) for 10 minutes at room temperature. The numbers of mononuclear and multinucleated TRAP-positive cells on each bone slice were counted, using a Nikon Eclipse 600 microscope with either a Nikon Plan Fluor 20x/0.50 or a Nikon Plan Fluor 40x/0.75 objective and a filterset UV-2E/C (DAPI) (Nikon, Tokio, Japan).
The same cultures were also stained for filamentous actin (F-actin), as described before , using tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (T-PHD, Sigma Chemicals, St. Louis, MO) for 20 minutes at +37°C. The cells were studied with the Nikon microscope with a Nikon filterset G-2E/C (TRITC). The percentages of active osteoclasts were determined by counting the number of osteoclasts with actin ring divided by the number of TRAP-positive cells, i.e., osteoclasts.
Area Measurement of Excavated Pits
Osteoclasts were cultured on bone slices for 48 hours in the alternative growth media, after which the resorption pits were stained and visualised as described earlier . Briefly, prior to the staining of the pits, total detachment of the cells from the bone slices was ensured by wiping the cellular surface of the slices with a soft brush. The pits were stained with peroxidase-conjugated wheat germ agglutinin-lectin (WGA; 20 μg/ml, Sigma Chemical Co., St. Louis, MO) for 20 minutes, washed with PBS, and incubated for 15 minutes in diaminobenzidine (0.5 mg/ml) + 0.03% H2O2. Morphometric analysis of the resorption pits was performed with an MCID image analyser, utilising M2 software (Imaging Research Inc., Brock University, Ontario, Canada).
All statistical analyses were performed by first using one-way ANOVA test. After ANOVA indicated the groups were significantly different, Student's t-test for independent samples was performed utilizing computer program Microcal Origin version 4.00 (Microcal Software Inc., Northampton, MA).