Osteoblast isolation and cultures.
All animal studies were approved by the Animal Research Council at Okayama University, Okayama, Japan. Animal experiments were carried out according to the NIH guidelines (Guide for the Care and Use of Laboratory Animals). Neonatal calvariae-derived osteoblasts from C57BL/6 J mice (CLEA Japan Inc., Osaka, Japan) were harvested and cultured as described previously (Fig. 1b).3 Briefly, neonatal mouse pups were euthanized by decapitation, and the heads were placed in a petri dish with PBS. The skin of each head was cut away, and the calvariae were cut and washed with PBS in a petri dish. The calvariae were incubated in 4 ml of digestion solution supplemented with collagenase (0.64 mg/ml) and trypsin (0.05%) at 37 °C in a shaking water bath. During the incubation, the calvariae were shaken by hand for a few seconds. After incubation for 20 min, 700 µl FBS was added to the cell suspension to inhibit trypsin activity. The calvariae were washed with 3 ml DMEM without FBS and shaken well and then the supernatant was transferred to a tube containing the cell suspension. This is population number 1. The calvariae were transferred to a new digestion solution to repeat the previous steps in order to obtain population number 2. The entire procedure was repeated four times to obtain populations 1–4. Pooled populations 3 and 4 are enriched with cells exhibiting biochemical characteristics of differentiated osteoblasts such as high ALP activity and osteopontin expression. In this study, 2 µL of benzonase (E1014, Sigma) was added to 4 ml of digestion solution before adding FBS in the 3rd or 4th session of calvarial digestion, and isolated osteoblasts with or without Benzonase were analyzed. Osteoblast differentiation was induced by culturing cells in an osteogenic medium (α-MEM containing 10% FBS, 100 µg/ml ascorbic acid and 10 mM β-glycerophosphate) for 21 days as described previously.11, 13 Alizarin red staining of mineralization was accomplished by cell fixation in 4% formaldehyde for 30 minutes followed by staining with 0.1% Alizarin Red-S solution (pH 4.8) for 20 minutes. Alizarin red dye was extracted with 10% formic acid, and the absorbance at 415 nm was determined with a microplate reader as described previously.14
Histomorphometry.
The in vivo isolated cells were analyzed from sections of undecalcified neonatal calvariae that had been digested with digestion solution in the presence or absence of Benzonase, embedded in paraffin, and stained with H&E or DNA Feulgen staining, which specifically stains deoxyribonucleoprotein. We examined the number of cells and the amount of viscous substance surrounding bones by H&E staining. The number of cells was automatically estimated by counting the hematoxylin-stained nuclei using “Analyze Particles” function in ImageJ/Fiji and normalized by the reference area. Before analysis of the substance area and cell number, the images were pre-processed by white balance correction and "subtract background”. Then they were cropped according to ROI selection.
Reagents and antibodies.
Unless stated otherwise, all chemicals were purchased from Sigma. Antibodies were obtained from the following sources: anti-ABL, anti-TAZ (BD Pharmingen), anti-RUNX2 (MBL International) and anti-Actin (Santa Cruz Biotechnologies). Halt™ Protease and Phosphatase Inhibitor Cocktail was from Thermo Fisher Scientific.
RNA extraction and quantitative real-time PCR analysis.
Total cellular RNA was extracted using RNeasy Plus Mini Kit (QIAGEN). A High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Science) was used for reverse transcription, and qPCR was performed on a Step One Plus Real-Time PCR System (Applied Biosystems) according to the manufacturer’s protocol. The sequences of primers are as follows: mouse Rpl19 (forward primer, 5’-CTG AAG GTC AAA GGG AAT GTG-3’; reverse primer, 5’-GGA CAG AGT CTT GAT GAT CTC-3’), mouse Alp (forward primer, 5’-GCT GAT CAT TCC CAC GTT TTC-3’; reverse primer, 5’-CTG GGC CTG GTA GTT GTT GT-3’), mouse Osteocalcin (forward primer, 5’-CTG ACA AAG CCT TCA TGT CCA A-3’; reverse primer, 5’-GCG CCG GAG TCT GTT CAC TA-3’) and mouse Runx2 (forward primer, 5’-GCT ATT AAA GTG ACA GTG GAC G-3’; reverse primer, 5’-CAC GTC AGT GAT GGC AGG TAG C-3’). The relative expression of each mRNA was calculated by the ΔCt method.
Western blot analysis.
Cells were lysed with RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP40, 0.1% SDS, 0.25% sodium deoxycholate, 1 mM EDTA) supplemented with protease and phosphatase inhibitors. Lysates were cleared by centrifugation for 10 minutes at 14,000 rpm and 4 oC. For Western blotting, protein in whole cell lysates was resolved by SDS-PAGE and transferred to a PVDF membrane (Immobilon; Millipore). Membranes were blocked in 5% BSA or 5% nonfat dried milk in PBST (PBS + 0.1% Tween-20) as described previously.15 The images are representative of three independent experiments.
Cell growth assays.
Primary murine osteoblasts were plated in regular growth medium and placed into the INCUCYTE™ Kinetic Imaging System (Essen Bioscience) to monitor cell growth and percent cell confluence.
Statistics.
All results are shown as means ± SEM of data from at least three separate experiments. The data were subjected to the unpaired t-test with JMP® 7 (SAS Institute Inc, USA) to determine differences. P values < 0.05 were accepted as statistically significant.