Trabecular dynamic bone histomorphometry:

All tissues were prepared at the Histology Core of the Indiana Center for Musculoskeletal Health. These parameters were obtained as we have previously reported⁽³³⁾. We analyzed the mice treated with PTH or vehicle for 4wks as opposed to 7wks since our earlier study with global Nmp4^−/− mice indicated that changes in bone formation rate likely occur earlier in treatment, which is consistent with the observed precocious mineralization observed in vitro^(33,35,37). Briefly, mice were administered intraperitoneal injections of calcein green (20 mg/kg; Sigma-Aldrich) and alizarin red (25 mg/kg; Sigma-Aldrich) 6 and 3 days before euthanasia, respectively. Femurs were removed from experimental mice and fixed with 10% buffered formalin followed by transfer to 70% ethanol as described above. The anterior face of the epiphyseal plate of the right femur was cut to expose the marrow cavity. Methyl-methacrylate was used to embed the bones after dehydration with graded alcohols. Specimens were sectioned (4μm) using a Leica RM2255 microtome (Leica Microsystems, Buffalo Grove, IL). Tissue sections were then mounted unstained on microscope slides and scored using the OsteoMeasure High Resolution Digital Video System (OsteoMetrics, Decatur, GA) microscope system. The dynamic bone histomorphometry parameters of mineral apposition rate (MAR, μm/day), bone formation rate (BFR/BS, μm³/μm²/day), and mineralizing surface/bone surface (MS/BS, %) were obtained from a 0.75- to 1-mm² metaphyseal region of interest approximately 200–400μm from the distal femur growth plate.