Zoledronic acid suppresses callus remodeling but enhances callus strength in an osteoporotic rat model of fracture healing  Y. Hao, X. Wang, L. Wang, Y. Lu, Z. Mao, S. Ge, K. Dai  Bone  Volume 81, Pages 702-711 (December 2015) DOI: 10.1016/j.bone.2015.09.018 Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 1 X-ray analysis. At 6weeks, the fracture line could still be seen in all the groups. Twelve-week radiographs showed that the cortical plates of the CNT group were united, and the external calluses of the ZOL-treated groups were much larger. Bone 2015 81, 702-711DOI: (10.1016/j.bone.2015.09.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 2 A: Representative images of fracture calluses from μCT cross-sectional scans and 3D reconstructions. At 6weeks, external callus was clearly visible in each group, and internal callus began to appear along the medullary cavity. At 12weeks, the external callus in the CNT group calcified to lamellar bone after remodeling, while the delayed administration groups (ZOLW1 and ZOLW2) showed even larger external callus than CNT and ZOLD1. B: 3D histomorphometry by μCT. CNT group: normal saline control treatment; ZOLD1, ZOLW1, and ZOLW2 groups: zoledronic acid (ZOL) 0.1mg/kg injected intravenously at 1day (D1), 1week (W1), or 2weeks after fracture (W2). Values represent the mean±standard deviation (SD). Each group consisted of 6 rats. *P<0.05 compared to CNT; **P<0.01 compared to CNT. #P<0.05 compared to ZOLD1; ##P<0.01 compared to ZOLD1. ^ P<0.05 compared to ZOLW2; ^^ P<0.01 compared to ZOLW2. Bone 2015 81, 702-711DOI: (10.1016/j.bone.2015.09.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 3 Images of H&E-stained sections. Decalcified bone histology at 6 and 12weeks after surgery (100× or 200× magnification). C=cartilage cells; F=fibrous callus; WB=primary osseous callus or woven bone; LB=lamellar bone; Col = collagen fibers; M=muscle. At 6weeks, the CNT group consisted predominantly of cartilage or fibrocartilage, and was in the conversion stage of primary callus from fibrocartilage formation to bone formation. Meanwhile, the treated groups exhibited larger amounts of newly formed woven bone. At 12weeks post-fracture, the CNT group showed an appearance typical of lamellar bone. In the ZOLD1 group, the majority of woven bone had been converted into calcified lamellar bone. However, the callus in the ZOLW2 group consisted mainly of woven bone undergoing active remodeling. Bone 2015 81, 702-711DOI: (10.1016/j.bone.2015.09.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 4 Safranin O/light green-stained sections of decalcified bone, showing histology at 6 and 12weeks post-surgery (200× magnification). Cartilaginous tissue was stained red or reddish orange while osseous tissue stained green or cyan. At 6weeks, fibrocartilage callus was predominant in the CNT group, while the quantity of woven bone in the ZOLD1 was greater than that in the ZOLW1 or ZOLW2 groups. At 12weeks, lamellar bone dominated the CNT group, while primary lamellar bone was observed in the ZOLD1 group, and no mature osseous callus was observed in the ZOLW1 or ZOLW2 groups. Bone 2015 81, 702-711DOI: (10.1016/j.bone.2015.09.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 5 Bone histology of Van Gieson-stained undecalcified sections at 6 and 12weeks post-surgery (200× magnification). Collagenous fibers were stained green or cyan, muscle fibers blue or purple, osseous tissue red, and osteoid greenish-yellow. At 6weeks, fibrocartilage callus in the CNT group stained bluish-green, indicating collagen, while in the ZOL-treated groups, hard calluses began to form and stained red. At 12weeks, callus in the ZOLD1 group exhibited abundant areas of wide, thick, completely mineralized woven bone, while trabeculae in the ZOLW1 and ZOLW2 groups were discontinuous, with no evidence of remodeling. Bone 2015 81, 702-711DOI: (10.1016/j.bone.2015.09.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Fig. 6 Nanoindentation tests. CNT group: normal saline control treatment; ZOLD1, ZOLW1, and ZOLW2 groups: treatment with zoledronic acid (ZOL) 0.1mg/kg, injected intravenously 1day (D1), 1week (W1), or 2weeks after fracture (W2). Values represent the mean±SD. Each group consisted of 6 rats. *P<0.05 compared to CNT; **P<0.01 compared to CNT. #P<0.05 compared to ZOLD1; ##P<0.01 compared to ZOLD1. ^ P<0.05 compared to ZOLW2; ^^ P<0.01 compared to ZOLW2. Bone 2015 81, 702-711DOI: (10.1016/j.bone.2015.09.018) Copyright © 2015 Elsevier Inc. Terms and Conditions