Figure 1.
Normal trabecular bone mass, but increased cortical bone strength in fetuin-A deficient mice.
(A) Von Kossa/van Gieson-stained undecalcified sections of the spine from 4 months old wildtype and fetuin-A deficient mice. (B) Histomorphometric quantification of the trabecular bone volume (BV/TV, bone volume per tissue volume) and the trabecular number (Tb.N.). (C) Histomorphometric quantification of the osteoblast number (N.Ob./B.Pm, number of osteoblasts per bone perimeter) and the osteoclast number (N.Oc./B.Pm, number of osteoclasts per bone perimeter). (D) Contact radiographs of the hindlegs from 4 months old Ahsg+/+ and Ahsg−/− mice. The femoral length is given below. (E) Cross-sectional µCT scanning of the femora. (F) Quantification of the cortical thickness and the force to failure in three-point-bending assays. All values represent mean±SD (n = 8 per group). Asterisks indicate statistically significant differences (p<0.05).
Figure 2.
Microstructural and micro-scale mechanical properties of Ahsg+/+ and Ahsg−/− cortical bone.
(A). Light microscopy of osteocyte lacunae in Ahsg+/+ and Ahsg−/− samples, respectively (scale bar: 20 µm) (B). Laser scanning confocal microscopy using Rhodamine-B as a contrasting agent showing osteocytic and canalicular networks in Ahsg+/+ and Ahsg−/− samples, light areas are intensely stained with Rhodamine-B, scale bar: 20 µm. (C) Backscatter scanning electron microscopy revealing the microstructure at the surface and no significant differences in density, scale bar: 10 µm. (D) Nanoindentation (n = 80) measurements of the indentation moduli and hardness of Ahsg+/+ and Ahsg−/− bone samples (E) Micro-tensile (n = 20) measurements of tensile strength and elastic moduli in wildtype and fetuin-A deficient bone samples (F) Representative Ahsg+/+ and Ahsg−/− fracture surfaces showing evidence of brittle failure, scale bar: 20 µm, inset: higher magnification of the fracture surface, scale bar: 2 µm.
Figure 3.
Mineral and organic components in Ahsg+/+ and Ahsg−/− femoral cortical bone.
(A) A typical Raman spectrum of representative Ahsg+/+ and Ahsg−/− samples with peaks at 910–990 cm−1 and 1600–1700 cm−1 representing PO43− (mineral) and Amide I (organic matrix) groups, respectively. inset: Normalized intensity measurements at polarization angles of −45, 0, 45, 90 were made to address the orientation artifacts of Raman intensity of type I collagen for both Ahsg+/+ and Ahsg−/−. The dashed lines are fits which estimate parameters characteristic of sample orientation and mineralization. The solid lines indicate mean intensity values of mineralization in the samples. (B) The mineral content normalized with the organic matrix can be observed by Raman ratios between Ahsg+/+ and Ahsg−/− samples. Further complementing these observations, measurements of the mineral component were made with X-ray attenuation, absorption, as well as small-angle X-ray scattering. (C) Comparing fibrillar versus tissue strains in Ahsg+/+ (green circles) and Ahsg−/− (red squares) bone. Samples were measured with in-situ synchrotron small angle X-ray scattering to determine the amount of strain contributed by the collagenous fibrils within the Ahsg+/+ and Ahsg−/− samples. Dashed lines represent orientation guides.
Figure 4.
Growth plate morphology and mineralization in Ahsg+/+ and Ahsg−/− mice.
(A) Wildtype mice had dark blue stained cartilage cores within metaphyseal trabeculae of the growth plates, whereas Ahsg−/− had completed the cartilage-to-bone transition without the remains of cartilage cores within the trabeculae. Moreover, Ahsg −/− mice showed pronounced discontinuities in the chondrocyte column organization in comparison to the wildtype mice (Toluidine blue staining). (B) Ahsg −/− mice frequently showed thickened calcified bridge formations across their growth plates, which was confirmed by backscattered electron microscopy. The orange and black areas correspond to mineralized and non-mineralized tissue, respectively. (C) The mineral content in Ahsg−/− mice was significantly increased in both tibial and femoral growth plates in comparison to wildtype mice as judged by quantitative backscattered electron imaging. (D) In contrast, the mineralization (mean Ca Wt%) of the femoral and tibial cortices was similar in Ahsg−/− and Ahsg+/+ mice.