Fig 1.
Deficiency and also transgenic overexpression of Timp-3 generate defects in trabecular bone.
Trabecular bone phenotype of WTB6 (black), Timp-3 KO (grey), WTF1 (dark red), Timp-3 Tg (light red) tibia at 8 weeks of age. (A) Representative 3D Micro-CT thickness colour-coded images of tibial trabecular bone. Ex vivo high-resolution analyses of distal proximal metaphysical tibia to determine (B) trabecular bone volume/total volume, (C) trabecular number, (D) trabecular thickness and (G) bone surface/bone volume. (H) Representative Alcian blue haematoxylin and orange G/eosin stained sections with outline of trabeculae showing the region of interest used for measurements. These sections were analysed using Osteoid Histo to provide (I) trabecular thickness and (J) trabecular bone surface/bone volume. Box-plots represent means ± SEM. Group sizes for Micro-CT (B-G) were n = 5 for WT littermates and n = 6 for Timp-3 KO and Tg mice. Group sizes for histological data (I-L) were n = 4 for all groups. Two-sample t-test was used to compare means between KO and WTB6, and between Tg and WTF1. Normality of variance assumption was violated for total area of KO group (E) and homogeneity for trabecular thickness of WT vs. KO group (I) (p > 0.05), thus, for these groups Kruskal-Wallis test was performed. Statistical comparisons: * denotes p ≤ 0.05.
Table 1.
Mean value of morphometric parameters from the 2D and 3D analysis representing trabecular and cortical mass and architecture of WTB6, Timp-3 KO, WTF1 and Timp-3 Tg mice at 8 weeks of age.
Fig 2.
Timp-3 deficiency and transgenic chondrocyte-specific overexpression do not alter thickness of different zones in growth plate.
Representative images of toluidine blue stained sections from WTB6 (A and E), KO (B and F), WTF1 (C and G) and Tg (D and H) mice showing overall structure of growth plate measured from resting zone to primary spongiosa. (I) Different zones of growth plate were marked and measured. No significant differences in size overall size of growth plate (J), percent proliferative zone (K), percent hypertrophic zone (L) and percent resting zone (M) between KO and their WT controls as well as Tg and their respective WT were observed. Two-sample t-test was used to compare means between KO and WTB6, and between Tg and WTF1. Normality and homogeneity of variance assumptions were not violated in any experimental group (p ≥ 0.05). Group sizes were n = 4. Data are mean ± SEM.
Fig 3.
Timp-3 deficiency and transgenic chondrocyte-specific overexpression produce gross changes in cortical bone.
(A) Representative 3D Micro-CT colour-coded images of tibial cortical bone thickness. (B) Comparable cross-sectional reconstructed 2D images along the length of the tibia. (C) Bone cross sectional area (CSA) and (D) mean cortical thickness of WTB6 (black), Timp-3 KO (grey), WTF1 (dark red) and Timp-3 Tg (light red) tibia at 8 weeks of age. Whole bone analyses of cortical bone between 15–75% of total tibial length, excluding proximal and distal metaphyseal bone. Line graphs represent means ± SEM. Group sizes were n = 5 for WT littermates and n = 6 for KO and Tg mice. Two-sample t-test was used to compare means between KO and WTB6, and between Tg and WTF1. Graphical heat map summarises statistical differences at specific matched locations along the tibial length, representation of the overall effect of genotype and post-hoc analysis are also shown. Red p≤0.000–0.001, yellow p≤0.001–0.01, green p≤0.01–0.05 and blue p ≥ 0.05.
Fig 4.
Timp-3 deficiency and transgenic chondrocyte-specific overexpression produce gross changes in tibial geometry.
(A) Minimum and (B) maximum second moments of area (Imin and Imax respectively), (C) ellipticity and J (D; resistance to torsion) of WTB6 (black), Timp-3 KO (grey), WTF1 (dark red) and Timp-3 Tg (light red) tibia at 8 weeks of age. Whole bone analyses of cortical bone between 15–75% of total tibial length, excluding proximal and distal metaphyseal bone. Two-sample t-test was used to compare means between KO and WTB6, and between Tg and WTF1. Line graphs represent means ± SEM. Group sizes were n = 5 for WT littermates and n = 6 for KO and Tg mice. Graphical heat map summarises statistical differences at specific matched locations along the tibial length, representative of overall effect of genotype and post-hoc analysis are also shown. Red p≤0.000–0.001, yellow p≤0.001–0.01, green p≤0.01–0.05 and blue p ≥ 0.05.
Fig 5.
Timp-3 deficiency and transgenic chondrocyte-specific overexpression increase trabecular TRAP staining.
Representative images of TRAP stained sections from WTB6 (A-D), KO (E-H), WTF1 (I-L) and Tg (M-P) mice demonstrating TRAP activity in growth plate, trabecular and cortical bone. Staining show that TRAP activity is significantly higher in KO and Tg sections compared with their WT groups. (Q) TRAP activity was quantified to produce osteoclast surface/bone surface and osteoclast number/bone surface. Two-sample t-test was used to compare means between KO and WTB6, and between Tg and WTF1. Normality of variance assumption were not violated for any experimental group. Homogeneity of variance osteoclast surface/bone surface between KO and WTB6 was violated (p >0.05) and thus for these groups Kruskal-Wallis test was performed. Group sizes were n = 4. Data are mean ± SEM. Statistical comparisons: * denotes p ≤ 0.05.
Fig 6.
Timp-3 deficiency and overexpression produce architectural changes in the skull.
Craniometric measurements of WTB6, Timp-3 KO, WTF1 and Timp-3 Tg skull at 8 weeks of age. (A) Representative 3D Micro-CT colour-coded images of skull thickness. (B) Schematic of the structures of the mouse skull depicting landmarks used to obtain measurements in the cranial; 1–2: nasal length, 2–3: frontal length, 3–4: parietal length, 1–5: cranial length, 6–6: bitemporal distance, (C) 7–8: posterior mandible height, 7–9: condilar axis, 7–11: effective mandible length, 8–11: mandible plain, 9–10: mandible axis, 11–12: inferior incisor axis, 13–13: (D) anterior mandible height, 14–14: basisphenoid length, and 15–15: presphenoid length in which the ‘shell’ regions of the bone that correspond to plane of sectioning are coloured in red. (E) Cranial length, mandible plain, presphenoid and basisphenoid length. Box-plots represent means ± SEM. Group sizes were n = 5. Statistical comparisons: * denotes p ≤ 0.05.
Table 2.
Craniometric measurements representing skull parameters of WTB6, KO, WTF1 and Tg mice at 8 weeks of age.