Zoledronic Acid affects the formation and remodeling of cartilage and bone during tibial fracture repair
To examine the long-term effects of ZA on healing of non-stabilized tibial fractures, we injected ZA intravenously 4 weeks before fracture and on the day of fracture. We chose a high dose of ZA as used in cancer patients and delivered two doses in order to maximize the incorporation of ZA within bone prior to injury 
. Histological analyses showed a delay in fracture repair in ZA-treated mice (). There was less cartilage during the soft callus phase of repair (day 7 to 10 post-fracture) in ZA-treated compared to PBS-treated mice as shown by Safranin-O staining (). Trichrome staining did not reveal an apparent delay in bone deposition within the callus until day 14 (data not shown). However, there was more woven bone in the callus of ZA treated mice by day 28, indicating a delay in the restoration of the bone marrow cavity (). Histomorphometric analyses showed that total callus volume was smaller in ZA-treated samples compared to controls at day 7. Total callus volume gradually increased from day 7 to 14 and was reduced by day 28 post-fracture in control samples, while it remained high by day 28 in ZA-treated samples indicating a delay in callus formation and remodeling (, top). Histomorphometric analyses confirmed that there was significantly less cartilage in the callus of ZA-treated samples at days 7 and 10 post-fracture (, middle). Cartilage volume peaked by day 10 and decreased by day 14 in PBS-treated samples, while cartilage volume remained high by day 14 compared to day 10 in ZA-treated samples (, middle). These results indicate that ZA delays both cartilage deposition and remodeling during tibial fracture repair. Bone volume was significantly higher by day 10 post-fracture and remained high by day 28 in ZA-treated samples compared to controls implying an increase in bone deposition during the soft callus phase of repair followed by a delay in bone remodeling (, bottom).
Zoledronic Acid delays cartilage and bone remodeling during non-stabilized mandibular fracture repair
To compare the effects of ZA on tibial and mandibular fracture repair, we developed a non-stabilized mandibular fracture model. Fractures were created from the top of the coronoid process to the gonial angle of mandible bone (). Histological analyses showed that mandibular fractures healed via endochondral ossification with a lesser degree of cartilage deposition compared to non-stabilized tibial fractures (). Callus size was smaller in mandibular compared to tibial fractures. As observed in tibial fractures, mandible fracture calluses were filled with woven bone by day 14 post-fracture, and gradually resorbed and bridged by day 21. Bone remodeling was well underway by day 28 (, left). Histomorphometric analyses confirmed these histological analyses. Total callus volume peaked at day 10 and gradually reduced by day 28 post-fracture in PBS- and ZA-treated samples, but the total callus volume was significantly greater in ZA-treated samples compared to the controls at days 14 and 21 post-fracture indicating that there was a delay in callus remodeling (, top). Cartilage formed by day 7, peaked at day 10 and decreased by day 14, but cartilage volume remained significantly high in ZA-treated calluses compared to PBS-treated calluses. Cartilage was completely resorbed by day 21 in both ZA- and PBS-treated calluses (, middle). Bone volume gradually increased from day 7 to day 14 and decreased by day 28 in PBS-treated calluses, but remained high from day 14 to day 28 in ZA-treated calluses (, bottom). These data indicate a delay in cartilage and bone remodeling in the repair of non-stabilized mandible fractures treated with ZA.
Zoledronic acid delays cartilage hypertrophy during tibial fracture healing
Since cartilage formation was delayed in tibial but not in mandibular fracture healing, we performed immunohistochemistry to examine chondrocyte differentiation following ZA and PBS treatment. In tibial fracture calluses, immunostaining for collagen type II was detected in chondrocytes of both PBS and ZA-treated mice by day 5 post-injury (). However, collagen type X immunostaining was observed in PBS but not in ZA-treated calluses (). By day 10, collagen type X was expressed in both groups ( and data not shown), therefore cartilage hypertrophy was delayed during tibial fracture repair as a result of ZA treatment. In mandibular fracture calluses, immunostaining for collagen type II was markedly decreased compared to tibial fractures (), which was in concordance with decreased cartilage formation reported via histology and histomorphometry. Immunostaining for collagen type X was seen in the calluses of PBS and ZA-treated mice at days 5 and 10 post-fracture ( and data not shown).
Effects of Zoledronate on collagen types II and X expression during tibial and mandibular fracture repair.
Zoledronic acid reduces osteoclastogenesis during mandibular fracture healing
Callus formation and remodeling involves the deposition and breakdown of extracellular matrix by the coordinated action of osteoblasts, chondrocytes and osteoclasts. Osteoclasts are a well-known target of bisphosphonates action. Therefore, we investigated the effects of ZA on osteoclast numbers in tibial and mandibular fractures. There were no significant differences in the number of TRAP-positive cells per callus area between ZA-treated and control samples in tibial fractures, but there were less TRAP-positive cells per callus area in ZA-treated samples compared to the controls in mandibular fractures at both days 7 and 14 post-fracture (). To assess whether the decrease in the number of TRAP-positive cells was due to apoptosis, we performed double staining for TUNEL and TRAP at day 5 post-fracture. Overall there was minimal cell death in the callus of both tibial and mandibular fractures, and we did not observe significant differences between ZA-treated and control groups (data not shown). Dead cells were found at the fracture site, within the bone marrow and in the cortex, while TRAP-positive cells were found primarily around new bone adjacent to the periosteum and within the fracture callus ( and data not shown). Double staining and quantitative analyses showed that TRAP-positive cells were not TUNEL-positive, suggesting that ZA treatment did not induce osteoclast cell death ( and data not shown). Therefore, the decrease in the number of TRAP-positive cells were not due to increase cell death in mandibular fractures.
Effects of zoledronate on osteoclastogenesis during tibial and mandibular fracture repair.
Effects of zoledronate on apoptosis during tibial and mandibular fracture repair.
Zoledronic acid reduces angiogenesis in the tibial fracture callus
Skeletal tissue deposition and remodeling is tightly linked with revascularization of the fracture site. To examine whether ZA affects vascularization, we quantified blood vessel length and surface densities in ZA- and PBS-treated calluses. Immunohistochemistry of PECAM () and stereological analyses revealed that blood vessel length density () and surface density () were significantly reduced in ZA-treated calluses compared to controls in tibial fractures but not in mandibular fractures at day 5 post-fracture. Our results also revealed that in the absence of ZA treatment, blood vessel length density and surface density were significantly higher in tibial fractures compared to mandibular fractures ().
Effects of zoledronate on angiogenesis during tibial and mandibular fracture repair.