The murine monocyte/macrophage cell line RAW264.7 (TIB-71™; American Type Culture Collection, Manassas, VA, USA) was used as osteoclast precursors. RAW264.7 cells have been shown to differentiate into osteoclast-like cells in the presence of RANKL (5). Cells were cultured in Dulbecco's modified Eagle's medium (Wako, Osaka, Japan) containing 10% heat-inactivated fetal bovine serum (FBS; Invitrogen Life Technologies, Carlsbad, CA, USA) and 66.7 μg/ml kanamycin sulfate (Meiji Seika, Tokyo, Japan) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Cells were seeded onto 100-mm standard dishes (BD Biosciences, Franklin Lakes, NJ, USA) and cultured overnight. 12-mm diameter glass cover slips (slips; Fisher Microscope Cover Glass; Thermo Fisher Scientific, Waltham, MA, USA) was placed into the wells of a 24-well culture plate (BD Biosciences). For osteoclast differentiation, RAW264.7 cells were transferred into the 24-well culture plates at 1.0×10⁴ cells/well and were cultured in α-minimum essential medium (α-MEM; Wako) supplemented with 10% heat-inactivated FBS, 2 μM L-alanyl-L-glutamine (Wako), 284 μM L-ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO, USA), 66.7 μg/ml kanamycin sulfate and 50 ng/ml RANKL (Oriental Yeast Co., Ltd., Tokyo, Japan) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Medium containing these reagents was replaced every other day.

Prior to culture, 15 mm-diameter silicone tubes were cut into 2-mm slices and sterilized in an autoclave (Fig. 1A). RAW264.7 cells were cultured on slips which had been placed on sliced silicone tubes in 24-well culture plates for five days, and the slips were reversed in the same wells filled with culture medium after three days for 48 h, or after four days for 24 h (Fig. 1B). The control group was cultured for the same period without reversing the slips.

Acid-soluble collagen solution (Cellmatrix; Nitta Gelatin, Inc., Osaka, Japan), ten-fold concentrated α-MEM (Invitrogen Life Technologies), and reconstruction buffer (2.2 g NaHCO₃ and 4.77 g 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in 100 ml 0.05 N NaOH; Nittal Gelatin, Inc.) were mixed at a volume ratio of 8:1:1, and 10% heat-inactivated FBS, 284 µM l-ascorbic acid 2-phosphate (Sigma-Aldrich) and 2 mM L-alanyl-L-gluta-mine (Wako) were added in an ice-cold bath kept at <4°C. This prepared collagen mixture was added to 24-well culture plates at a volume of 500 µl/well, and was subjected to gelation in a CO₂ incubator at 37°C for 30 min. After the collagen mixture had solidified, it was covered with 50 ng/ml RANKL in culture medium at 1 ml/well.

Prior to the application of compressive forces, RAW264.7 cells were incubated on the slip for three days (Fig. 1C, left). Collagen gel layers were prepared in other 24-well culture plates. Subsequently, osteoclasts on the slips were reversed and placed on top of these collagen gel layers (Fig. 1C, right), and were continuously compressed with layers of 3, 5, 7, 9 or 14 slips (~43 mg/slip) for 24 h, respectively (Fig. 1D). In the control group, the slips with cells attached were reversed onto the collagen gel layer without application of any additional force (Fig. 1C, right).

After cells were compressed for the indicated times, they were fixed in 10% neutral formalin. They were then washed with distilled water and stained with Fast Red Violet LB Salt (Sigma-Aldrich) in TRAP staining solution [acetate buffer (pH 5.0) (Sigma-Aldrich), naphthol AS-MX phosphate (Sigma-Aldrich) as a substrate, red violet LB (Sigma-Aldrich) as a stain, and 50 mM sodium tartrate (Wako)]. TRAP-positive cells with 2–7 nuclei were considered to be small osteoclasts, and those with >8 nuclei were considered to be large osteoclasts (3,4). Osteoclasts were counted under a light microscope (magnification, ×100; Olympus IMT-2; Olympus, Tokyo, Japan) in a 20-mm² rectangle within the circular field.

Osteologic™ was used to characterize and quantify osteoclast-mediated bone resorption in order to determine the amount of osteoclastic differentiation. A disc (φ12-m m) made of Osteologic™ (BD Biosciences) was coated with calcium phosphate. Osteoclasts were cultured for three days on these discs under normal cell culturing conditions as described above, and discs were reversed onto the top of a collagen gel layer in 24-well culture plates on the fourth day for 24 has a control group. Another group of reversed osteoclasts was subjected to the compressive force of seven layered slips. On the fifth day, the control and compressed osteoclasts on the discs were rinsed twice with distilled water. Subsequently, 1 ml bleach solution (6% NaOCl and 5.2% NaCl) was added to each well, followed by pipetting up and down to remove cells and incubation for five minutes at room temperature. Discs were washed with 2 ml distilled water three times and were examined by light microscopy (magnification, ×100; Olympus IMT-2; Olympus) after drying (19,20). The resorption area was measured using Image J software (v. 1.48a; National Institutes of Health, Bethesda, MD, USA).

Cells were compressed for 1, 3, 6, 12 and 24 h in 24-well culture plates. Total RNA was extracted using TRIzol (Invitrogen Life Technologies) according to the manufacturer's instructions. Reverse transcription of 1 µg RNA was performed to obtain cDNA. The PCR reaction mixture contained 50 mM Tri-Hcl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 10 mM dithiothreitol, 0.01% Nonidet^® P-40 and 50% glycerol. The thermocycling protocol was as follows: Annealing at 30°C for 10 min, enzyme reaction at 42°C for 20 min, denaturation at 99°C for 5 min and cooling at 4°C. cDNA was amplified using Rever Tra Ace-α FSK-101 (Toyobo, Osaka Japan). Primers (Applied Biosystems, Thermo Fisher Scientific) for the following genes were used: NFATc1 (Mm00479445_m 1), TRAP (Mm00475698_m1), RANK (Mm00437135_m matrix metalloproteinase-9 (MMP-9; Mm00432271_m1), dendritic cell specific trans membrane protein (DC-STAMP; Mm01168058_m1), osteoclast stimulatory trans membrane protein (OC-STAMP; Mm00512445_m1), integrin-av (Mm00434506_m1), integrin-β3 (Mm00443980_m1), cathepsin-K (cath-K; Mm00484036_m1), chloride channel 7 (ClC-7; Mm00442400_m1), adenosine triphosphatase H⁺ transporting vacuolar proton pump member I (ATP6i; Mm00469395_g1) and GAPDH (Mm99999915_g1). Real-time PCR was performed using the ABI Prism 7300 sequence detection system (Applied Biosystems). Data obtained for each sample were standardized against the expression of GAPDH and were calculated using the 2^−ΔΔCt method (21, 22).

Values are expressed as the mean ± standard deviation. Statistical analysis was performed using Microsoft Excel for Mac 2011, version 14.4.8 (Redmont, WA, USA). Comparisons between two groups were analyzed using the two-tailed unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

In order to determine at what time-point osteoclast differentiation and fusion of RAW264.7 cells were activated, the number of osteoclasts and the number of nuclei per cell were monitored for five days (Fig. 2). RAW264.7 cells were cultured with 50 ng/ml RANKL in 24-well culture plates for five days to induce osteoclastonenesis. No osteoclasts were identified on day two, while the number of osteoclasts was slightly increased on days three and four, and was markedly increased on day five (Fig. 2A). Osteoclasts contained 2–4 nuclei on day three and began to appear with >5 nuclei on day four. On day five, the number of multinuclear cells further increased, and cells with >6 nuclei were increasingly present (Fig. 2B). Only upon osteoclastic differentiation, RQW264.7 cells begin to adhere to the cover slips. Day five was therefore determined to be the ideal time-point for reversing the cover slips in order to apply compressive forces on the cells.

The present study investigated the effects of reversing the slips on osteoclastogenesis on sliced silicone tubes for 24 and 48 h (Fig. 1B). Controls were cultured on slips without reversing and were fixed on day 5. The number of TRAP-positive cells with 2–7 nuclei and those with >8 nuclei was counted. The number of osteoclasts among the reversed cells showed no significant difference when compared with that in the control group at either 24 h or 48 h (Fig. 2C).

Various intensities of compressive force were applied to osteoclasts for 24 h after 4–5 days of culture. RAW264.7 cells on slips were cultured as described above. Cells were reversed onto the collagen gel layer in other plates on the 4th day. Cells were then loaded with layers of 3–14 slips after being reversed. The number of osteoclasts was significantly increased when the cells were compressed with seven slips (Fig. 3A), and the number of osteoclasts with >8 nuclei was significantly elevated (Fig. 3B).

The present study then examined the bone resorption activity of osteoclasts when the cells were subjected to a compressive force of seven slips. RAW264.7 cells were cultured on Osteologic™ discs as described above. Light micrographs of the Osteologic™ discs of the control and compressed (CF) groups were captured (Fig. 3C). The areas in which the calcium phosphate on the discs was completely resorbed were measured. Quantification of the results showed that the resorption areas in the compressed group were significantly larger when compared with those in the control group (Fig. 3D).

The present study examined the effects of compressive forces on mRNA levels of osteoclast-specific genes associated with differentiation, fusion and adherence using RT-qPCR analysis (Fig. 4). The analysis showed that the respective mRNA levels of osteoclast-associated genes, including NFATc-1, TRAP, RANK, cath-K, ClC7, MMP-9, ATP6i, DC-STAMP, OC-STAMP as well as Integrin-αv and -β3 (4), peaked at 3 h in the CF group, whereas the mRNA levels peaked at 24 h in the control group.