Pmc ak04f cos

Osteoclast differentiation and activity were determined by TRAP staining and a bone resorption pit assay, respectively. TRAP staining was performed using a TRAP staining kit (PMC-AK04F-COS; Cosmo Bio Co., Ltd, Tokyo, Japan) following the manufacturer’s instructions. TRAP-positive multinucleated cells with more than three nuclei were counted under a microscope using ImageJ software (NIH, Bethesda, MD). The bone resorption pit assay was performed using dentin discs (IDS AE-8050; Immunodiagnostic Systems, Tyne & Wear, UK). Cells were differentiated to osteoclasts on the discs over a 4-day period, after which the discs were stained with 1% toluidine blue solution and the resorption pit area was quantified using ImageJ software.

Bone histomorphometric analyses were performed on undecalcified long bones and vertebrae in accordance with previously described methods (71 (link)). Briefly, the bone was fixed with 4% paraformaldehyde, followed by dehydration in an ethanol series, and subsequently embedded in methyl methacrylate resin. The BV/TV ratio was measured by von Kossa staining. Osteoblast and osteoclast parameters were analyzed by toluidine blue staining (FUJIFILM Wako Pure Chemical 1B481) and TRAP staining (Cosmo Bio PMC-AK04F-COS), respectively. Analyses were performed using the OsteoMeasure analysis system (OsteoMetrics), according to standard protocols. Trabecular architecture was assessed in long bones using a μCT system (Comscan) at 90 kV and 45 μA, and the BV/TV ratio was measured using TRI/3D-BON software (RATOC) (25 (link)). Calcein was injected twice into mice with an interval of 3 days, and then, mice were sacrificed 2 days after the last injection.

An Alkaline Phosphatase (ALP) Staining Kit (PMC-AK20, Cosmo Bio) was used for detecting osteoblastic activity. ALP staining in each field was quantified and presented as a percentage of the ALP active region to the total area. The TRAP Staining Kit (PMC-AK04F-COS, Cosmo Bio) was used for detecting osteoclasts. TRAP+ multinucleated cells (≥ three nuclei) were photographed, and the number of positively stained cells per area were counted. A Masson’s trichrome staining kit (ab150686, Abcam) was used for detecting new bone formation. This staining technique stains mature bone and connective tissue blue, while staining immature new bone (osteoid) and collagen red.²⁷ (link) The histological images were obtained using a BZ-9000 Bio Revo all-in-one microscope.

Bone marrow cells were plated at 1 × 10⁵ cells/well in 96-well plates and cultured in α-MEM containing osteoclastogenic factors for 8 days. Cells were fixed with 10% neutral buffer formalin and incubated with TRAP chromogenic substrate (catalog no. PMC-AK04F-COS; Cosmo Bio, Tokyo, Japan) at 37 °C for 60 min. TRAP-positive multinucleated osteoclasts containing ≥3 nuclei were visualized and manually counted under an inverted microscope (model IX83ZDC; Olympus) as previously described^{56 (link),57 (link)}. The number of cells was represented as TRAP-positive cells per well.

Four weeks after surgery, bone marrow was collected from six euthanized rats in each group to isolate BMMs and determine osteoclast formation, as described [20 (link)]. Briefly, bone marrow cells were flushed from rat tibias using α-minimum essential media (MEM, 01-042-1ACS; BioInd, Shanghai, China) containing 2% fetal bovine serum (FBS, 10099141; Thermo Fisher Inc.), erythrocytes were removed with lysis buffer (R1010; Solarbio Co Ltd.), and the remaining cells were resuspended in α-MEM containing 10% FBS. After an overnight incubation, unattached cells (osteoclast precursors) were incubated in α-MEM containing 10% FBS, 30 ng/mL of macrophage-colony stimulating factor (M-CSF, 216-MC-025; R&D Systems, Minneapolis, MN, USA), and 20 ng/mL RANKL (390-TN-010; R&D Systems) for 3 days, followed by α-MEM containing 10% FBS, 30 ng/mL of M-CSF, and 100 ng/mL of RANKL for 3 days. The cells were fixed, and then stained for TRAP using kits (PMC-AK04F-COS; Cosmo Bio Co., Ltd., Tokyo, Japan), as described by the manufacturer. Multinucleated cells (> 3 nuclei) that were TRAP positive were identified as mature osteoclasts and counted using the DP71 light microscope.

Histological analysis was performed using a modified method for H&E and TRAP stains, as previously described. To prepare paraffin wax-embedded sections, the mice were sacrificed at six weeks (n = 6 mice/group). The isolated femur was fixed in 4% paraformaldehyde solution for 24 h and then immersed in 10% EDTA decalcifying solution for one week. After complete decalcification, paraffin was embedded and sectioned longitudinally (4-μm thickness). Before tartrate-resistant acid phosphatase (TRAP) staining, the sections were stained with H&E to detect structural changes. TRAP activity was demonstrated using an acid phosphate kit (Cat. No. PMC-AK04F-COS, Cosmo Bio Co., Tokyo, Japan). TRAP staining was performed in accordance with the manufacturer’s protocol. The sections were counter-stained with methyl green. TRAP-staining positive multinucleated cells containing at least three nuclei were counted as osteoclast cells. The number and area of TRAP-staining positive multinucleated cells were counted in a randomized order by the same researcher.