Volume 49, Issue 6, Pages 1178-1185 (December 2011) Increased callus mass and enhanced strength during fracture healing in mice lacking the sclerostin gene  Chaoyang Li, Michael S. Ominsky, Hong-Lin Tan, Mauricio Barrero, Qing-Tian Niu, Franklin J. Asuncion, Edward Lee, Min Liu, William S. Simonet, Chris Paszty, Hua Zhu Ke  Bone  Volume 49, Issue 6, Pages 1178-1185 (December 2011) DOI: 10.1016/j.bone.2011.08.012 Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 1 Representative radiographs of right femurs. The radiographic time course is shown for the mouse in each group with the median ex vivo pQCT vBMD at fracture line (Fig. 2D). A genotypic difference in fracture healing is apparent. Sclerostin knockout (SOST-KO) mice exhibited a larger and denser callus than WT mice at 14days post fracture and thereafter. Both genotypes showed almost full radiographic healing at 28days post fracture. BL: baseline, immediately after fracture; D: days. Bone 2011 49, 1178-1185DOI: (10.1016/j.bone.2011.08.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 2 Mineral content and density, and area analyses of fracture callus at day 45. Calluses remained larger and denser in sclerostin knockout (KO) than wild type (WT) mice after 45days. KO mice showed greater BMD, BMC and area than WT mice by both DXA at the mid-shaft 30% of the fractured femur (A, B and C) and pQCT at the fracture line (D, E and F). The μCT cross-sectional images at fracture line, 1mm proximal and distal from the fracture line of the fractured callus (G) showed larger, denser cross sections in KO mice than WT mice. Bone 2011 49, 1178-1185DOI: (10.1016/j.bone.2011.08.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 3 Goldner-trichrome (A) and toluidine blue (B) stained longitudinal sections of fracture callus. At 14days, overall fracture callus size in the two groups was the same, but callus in sclerostin knockout (KO) mice contained more bone (arrow in A; D) and less cartilage tissue (arrow in B; E) than WT mice; there was no obvious visible difference in the degree of hypertrophic chondrocytes between the WT and KO callus (B lower panel). By 28days, these differences in bone and cartilage composition had disappeared; both KO and WT fractures appeared fully bridged (4 cortices connected, double-arrows in A lower panel photos). There was no genotypic difference in either total callus area (C) or fibrous tissue area (F) at either time point. Values are mean±SEM, N=6–7 WT, N=9–10 KO; *p<0.05 compared with WT. Bone 2011 49, 1178-1185DOI: (10.1016/j.bone.2011.08.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 4 Goldner-trichrome stained (14days) and fluorescent light unstained sections (28days) of fracture callus. (A) Goldner-trichrome stained sections revealed higher surface extent of osteoid (arrows) in the 14day fracture callus in sclerostin knockout (KO) mice compared to WT. KO mice had increased bone formation rate (BFR/BS) at 14 and 28days post fracture. (B) Under fluorescent light, unstained sections from 28day WT callus contain more diffuse label (thin arrow) and less discrete double label than do KO fractures that exhibit more linear double calcein-label surface (arrows) and little or no diffuse label. (C and D) KO mice had significantly greater osteoid surface (OS/BS) and bone formation rate (BFR/BS) than WT at both days 14 and 28 (C and D). Values are mean±SEM, N=6–7 WT, N=9–10 KO; *p<0.05 compared with WT. Bone 2011 49, 1178-1185DOI: (10.1016/j.bone.2011.08.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 5 Bone bending strength endpoints of fractured bones at 45days post fracture. Sclerostin knockout (KO) mice showed significantly greater peak load (A) and energy to peak load (C) than wild type (WT) mice. There was a strong correlation between BMD (assessed by pQCT at the fracture line) and peak load for the fractured femora across both groups (r2=0.69) (D). Values are mean±SEM, N=8 WT, N=14 KO; *p<0.05 compared with WT. Bone 2011 49, 1178-1185DOI: (10.1016/j.bone.2011.08.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Fig. 6 Contralateral femur density and bending strength at 45days post fracture. Sclerostin knockout (KO) mice had greater BMD, BMC and area than wild type (WT) mice at the contralateral midshaft as measured by both DXA (central 30%) (A, B and C) and pQCT (single slice at mid-shaft) (D, E and F). Biomechanical testing (three-point bending) demonstrated that KO mice had higher peak load, stiffness and energy to failure than WT mice (G, H, and I). Values are mean±SEM, N=8 WT, N=14 KO; *p<0.05 compared with WT. Bone 2011 49, 1178-1185DOI: (10.1016/j.bone.2011.08.012) Copyright © 2011 Elsevier Inc. Terms and Conditions