Sigma 387 a

The mandibles were decalcified in 10% EDTA for 4 weeks. Gradient dehydration was performed for embedding in paraffin followed by perpendicular sectioning to the long axis of the molar roots as previously described [36] (link). Serial sections of 5 µm were cut and mounted on polylysine-coated slides and then performed for H&E staining, Masson trichrome staining (Sigma #HT15; Sigma-Aldrich, St. Louis, USA.) and tartrate-resistant acid phosphatase (TRAP) staining (Sigma #387A; Sigma-Aldrich, St. Louis, USA.) in accordance with the manufacturer's protocol. For histomorphometry, six individual sections were selected from three different locations, which were situated in the middle, coronal and apical levels of the defect (with 400 µm apart from the central). The histometric measurements were determined by processing the images, which were captured with an Olympus DP72 microscope, in Adobe Photoshop CS5 (Adobe Systems, Inc.). New mineralized tissue was identified by Masson trichrome staining and the defect fill was defined as the ratio of area of new mineralized tissue within bony envelope and the total defect area. The number of TRAP-positive cells was performed in a 500 µm square of the defect area.

All in vitro experiments were performed at least 3 times. Primary BMMs were prepared as described^{52 (link)} with slight modifications. Marrow was extracted from femora and tibiae of mice with α minimum essential medium (α-MEM) and cultured in α-MEM containing 10% inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (α-10 medium) with 1:10 of mMCSF producing cell line, CMG 14-12 condition media on petri-plastic dishes. Cells were incubated at 37 °C in 5% CO₂ for 3 days and then washed with phosphate-buffered saline (PBS) and lifted with 1× trypsin/EDTA in PBS. A total of 1.2 × 10⁴ BMMs were cultured in 500 μL α-MEM containing 10% heat-inactivated fetal bovine serum with glutathione-S transferase–RANKL and 30 ng/mL of mouse recombinant macrophage colony-stimulating factor (M-CSF) in 48-well tissue culture plates, some containing sterile bovine bone slices. Cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) activity after 5 days in culture, using a commercial kit (Sigma 387-A; Sigma-Aldrich, St. Louis, MO). The images were captured using a Nikon Eclipse E400 (Melville, NY) upright microscope.

Primary BMMs were prepared as described previously with slight modification. Marrow was extracted from femora and tibias of 8‐ to 10‐week‐old mice with α‐MEM and cultured in α‐MEM containing 10% inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (α‐10 medium) with 1:10 CMG condition media on petri plastic dishes. Cells were incubated at 37°C in 5% CO₂ for 3 days and then washed with PBS and lifted with 1× trypsin/EDTA (Invitrogen, Carlsbad, CA, USA) in PBS. A total of 1.2 × 10⁴ cells were cultured in 500 μL α‐MEM containing 10% heat‐inactivated FBS with 100 ng/mL GST‐RANKL and 30 ng/mL of mouse recombinant M‐CSF in 48‐well tissue culture plates, some containing sterile bone slices. Cells were fixed and stained for tartrate‐resistant acid phosphatase (TRAP) activity after 5 days in culture, using a commercial kit (Sigma 387‐A).