Femur Histomorphometric Analysis

All right femurs were put in a 19% EDTA solution for 14 days, and the solution was changed daily. Subsequently, all samples were sent to the Translational Pathology Core Laboratory (TPCL) at the UCLA Department of Pathology for paraffin embedding. Longitudinal sections of 5 μm thickness were created by microtome. All slides were used for hematoxylin and eosin (H&E), trichrome, tartrate-resistant acid phosphatase (TRAP), and osteocalcin (OCN) staining. All specimens were analyzed under an Olympus BX51 microscope (Olympus Corp.) using cellSens software version 1.6 (Olympus Corp.). Six consecutive images at the distal femur region were acquired for OCN and TRAP analyses, which were completed by three blinded examiners using ImageJ software v1.48 (National Institutes of Health). Parameters of osteocalcin+bone-lining cells per bone perimeter (OCN+cells/Bpm, mm⁻¹) and surface (Ob.S/Bs, %), TRAP+bone-lining cells per bone perimeter (TRAP+cells/Bpm, mm⁻¹), and surface (Oc.S/Bs, %) were used as previously reported.^{12 (link)}