Figure 1.
Analyses of message and proteins in CHAD−/− and WT mice.
A: PCR and agarose gel electrophoresis of mouse tail samples. There is a faint, barely visible, reactivity at the position of the wild type allele (320 bp), but this is not observed in the CHAD−/− mice. It probably represents some weak reactivity of the wild type allele by the primers for the deleted allele B: Protein stained gel and Western blot of cartilages and liver as a control for non-specific reactions. Different cartilages were extracted with 4 M guanidine hydrochloride, proteins precipitated with ethanol and electrophoresed on 4–16% SDS-PAGE. Left picture represents a Coomassie stained gel and the right picture represents Western blots with the anti-CHAD antibody. The lanes represent extracts of 1. Trachea (−/−); 2. Nasal cartilage (−/−); 3. Knee cartilage (−/−); 4. Trachea (+/+); 5. Nasal cartilage (+/+); 6. Knee cartilage (+/+); 7. Liver (+/+); 8. Recombinant CHAD; −/− represents CHAD−/− and +/+ wild type mice.
Figure 2.
Micrographs of the epiphyseal plate in CHAD −/− and WT mice.
Light micrographs of CHAD−/− (2a) and WT (2b) mice at 3 weeks of age. A small but significant increase in the height of the growth plate mostly confined to the proliferative zone was confirmed by histomorphometry (table 3). Epon-embedded tissue, toluidine blue staining (x 20).
Table 1.
Histomorphometric analyses of the epiphyseal growth plate in 3 and 6 weeks old CHAD−/− mice and age-matched WT mice.
Table 2.
Changes in the proteome of cartilage from mice with the CHAD gene inactivated.
Table 3.
Micro-CT cortical/trabecular bone parameters in 4 month old mice.
Figure 3.
BSP mRNA expressing cells in the distal femur of CHAD−/− and WT mice at 6 weeks of age.
In situ hybridization showed intense signal in multiple osteoblastic cells (arrows) in the metaphysis (M) of WT mice (3b), while CHAD−/− mice (3a) showed very sparse signal in cells in the corresponding area (p = 0.01). Also chondrocytes in the epiphyseal growth plate (EGF) showed signal, although no quantitative difference in number of cells were detected between the groups at this site (x 20). Negative control with sense probe was without signal (3c).
Table 4.
Ultrastructural distribution of OPN and BSP in bone of CHAD−/− and WT mice.
Figure 4.
Mechanical properties of bone.
4a: Femoral neck failure load (Newtons) for 6 weeks, 4 months and 8 months old wild type (wt) and CHAD−/− male and female mice. The difference between CHAD−/− and wt in 4 months old male mice is significant (p<0.01). 4b: Tibial shaft failure load (Newtons) for 6 weeks, 4 months and 8 months old wild type (wt) and CHAD−/− male and female mice. The difference between CHAD−/− and wt in 4 months old mice is significant for both genders (p<0.001).