Fig 1.
Bone loss in 13- and 18-mo-old C57BL/6J mice.
Representative microCT images of mouse tibiae, femurs, and L3 from 3-, 13-, and 18-mo-old C57BL/6J mice. Graphs show quantitative volumetric analysis of microCT data. Data are Means ± SD (n = 5–15). *, p<0.05 vs 3 mo as determined with ANOVA.
Fig 2.
Decline in bone architecture, strength, and formation in 13-mo-old C57BL/6J mice.
A) Histological evaluation of mouse bones. Sections stained with ABH/OG were analyzed using histomorphometry and OsteoMeasure software and trabecular Bone to Total Area ratios were calculated; B) Biomechanical torsion test of mouse femurs; C) Bone formation assay. Shown are representative sections and quantitative analyses of mouse bones labeled with Alizarin Red and Calcein for dynamic Mineral Apposition Rate (MAR), Mineralizing Surface/Bone Surface (MS/BS) and Bone Formation Rate (BFR) assay. Scale bar is 5 μm; D) TRAP-positive osteoclast surface relative to bone surface (Oc.S/B.S.) and CTX-I bone resorption/OC activity marker measured using ELISA. Data are Means ± SD (n = 5). *, p<0.05 vs 3 mo as determined with t-test.
Fig 3.
Metabolic changes in bone samples from 13-mo-old C57BL/6J mice.
Small metabolites were extracted from bone shafts of tibia and femurs and analyzed using metabolomic LC-MS. Metabolites are grouped into appropriate metabolic pathways. Data are Means ± SD (n = 3). *, p<0.05 vs 3 mo as determined with t-test.
Fig 4.
Mitochondrial swelling in osteocytes in bones from 13-mo-old C57BL/6J mice.
A) Electron micrographs of osteocytes (OTs) in mouse tibia. Arrowheads indicate mitochondria. Boxed mitochondria are enlarged and shown in the insets; B) Quantitative analysis of mitochondrial morphology. Fifteen cells per sample and 3 mice per group were blindly analyzed by three independent scorers and the number of mitochondria in a swollen conformation was determined. Data are Means ± SD. *, p<0.05 as determined with t-test.
Fig 5.
CypD KO mice do not show bone loss at 13 and 18 months of age.
Representative microCT images of mouse tibiae, femurs, and L3 from 3-, 13-, and 18- mo-old CypD KO mice and graphs showing quantitative volumetric analysis of microCT data. Data are Means ± SD (n = 5–15). *, p<0.05 vs 3 mo as determined with ANOVA.
Fig 6.
CypD deletion protects against the decline in bone architecture, strength, and formation in 13-mo-old mice.
A) Histological evaluation of mouse bones, Trabecular Bone to Total Area ratios; B) Biomechanical torsion test of mouse femurs; C) Bone formation assay. Quantitative analysis of Mineral Apposition Rate (MAR), Mineralizing Surface/Bone Surface (MS/BS) and Bone Formation Rate (BFR). Scale bar is 5 μm; D) Bone resorption assay. TRAP-positive osteoclast surface relative to bone surface (Oc.S/B.S.) and CTX-I bone resorption/OC activity marker measured using ELISA. Data are Means ± SD (n = 5). *, p<0.05 vs 3 mo as determined with t-test.
Fig 7.
CypD deletion protects against metabolic changes in bone samples from 13-mo-old mice.
Metabolomic analysis of mouse bones using MS-LC. Data are Means ± SD (n = 3). *, p<0.05 as determined with t-test.
Fig 8.
CypD deletion protects against osteocyte mitochondrial swelling in bone samples from 13-mo-old mice.
A) Electron micrographs of osteocytes (OTs) in mouse tibia. Arrowheads indicate mitochondria. Boxed mitochondria are enlarged and shown in the insets; B) Quantitative analysis of mitochondrial morphology. Fifteen cells per sample and 3 mice per group were blindly analyzed by three independent scorers and the number of mitochondria in a swollen conformation was determined. Data are Means ± SD. *, p<0.05 as determined with t-test.