Osteoclast Quantification in Distal Femur Metaphysis

Following microCT scanning, femurs were analyzed for osteoclast abundance in the distal metaphyseal trabecular bone using histomorphometry, as done previously (Putnam et al., 2019 (link)). Femurs were decalcified in 20% EDTA for 3 days, paraffin embedded, and sectioned on a Leica RM2255 microtome (Leica Biosystems, Buffalo Grove, IL, USA) at a thickness of 4 μm. Sections were stained for tartrate-resistant acid phosphatase (TRAP) with hematoxylin counterstain. TRAP-stained slides were imaged with a Leica SCN400 Slide Scanner (Leica Biosystems) in brightfield at 20X or on a Cytation 5 Imaging System [BioTek Instruments, Winooski, VT, USA] in brightfield at 10X and analyzed with Bioquant software (Bioquant Image Analysis Corporation, Nashville, TN, USA). Bioquant software was used to measure osteoclast number, osteoclast surface, and bone surface in the trabeculae of the distal metaphysis of the femur proximal to the growth plate on the TRAP-stained slides. According to ASBMR standards (Dempster et al., 2013 (link)), osteoclast number, osteoclast surface, and bone surface were measured to calculate osteoclast number per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS).