Figure 4.

Impact of antibiotic (ABX) treatment on osteoblastogenesis. Twelve-week–old male vehicle (VEH)– and ABX-treated mice were euthanized; specimens were harvested for analysis. A–D: Histomorphometric analyses of osteoblast cellular end points were performed in the trabecular bone secondary spongiosa of toluidine blue–stained proximal tibia sections. A: Representative images of toluidine blue–stained secondary spongiosa. B: Osteoblast number per bone perimeter (N.Ob/B.Pm). C: Osteoblast perimeter per bone perimeter (Ob.Pm/B.Pm). D: Osteoid per bone perimeter (O.Pm/B.Pm). E–G: Dynamic histomorphometric analysis of bone formation indexes in proximal tibia trabecular bone; calcein was administered 5 and 2 days before sacrifice. E: Representative images of calcein-labeled secondary spongiosa. F: Mineral apposition rate (MAR). G: Bone formation rate (BFR). H and I: Real-time quantitative RT-PCR (RT-qPCR) analysis of mRNA in femur bone marrow and calvaria. Relative quantification of mRNA was performed via the comparative C_T method (ΔΔC_T); Gapdh was used as an internal control. Data are expressed as fold difference relative to VEH. H: RT-qPCR analysis of Sp7 (osterix) mRNA in femur bone marrow and calvaria. I: RT-qPCR analysis of Bglap [osteocalcin (OCN)] mRNA in femur bone marrow and calvaria. J: Serum was isolated from whole blood; enzyme-linked immunosorbent assay analysis of intact OCN levels. Unpaired t-test was used. Data are expressed as means ± SEM. n = 4 per group (A–G); n = 4 to 5 per group (H and I); n = 5 per group (J). Original magnifications: ×200 (A); ×400 (E).