Effects of hydroxybisphosphonates-conjugated sitafloxacin on fracture healing and skeletal growth in mice

Implant-associated osteomyelitis is extremely difficult to treat due to bacterial colonization of the osteocyte-lacuno canalicular network, where bacteria are inaccessible from immune clearance and antibiotic treatment. The pharmacokinetic limitations of drug delivery to this compartment of bone render this infection resistant to standard-of-care antibiotics. Bone-targeted bisphosphonate-conjugated antibiotics have been developed to overcome this issue, and hydroxybisphosphonate-conjugated sitafloxacin (HBCS) has emerged as a leading therapeutic candidate based on safety and efficacy results in mice and sheep. However, potential concerns about its adverse effects on skeletal growth and bone healing remain. To address this, we assessed HBCS effects vs placebo, zoledronic acid (ZA) positive control known to inhibit osteoclasts and increase fracture callus and growth plate bone volume, and hydroxy-phenyl-ethane-hydroxy-bisphosphonate (HPHBP), the bisphosphonate component of HBCS, using a murine closed femur fracture model, and on intramembranous and endochondral ossification in the proximal tibia of 12-wk-old mice. Longitudinal X-rays and micro-CT confirmed that no treatments inhibited fracture healing, although ZA and HBCS increased callus bone volume fraction at 3 wk. Histology results suggested that this was likely due to effects on osteoclasts at 2 and 3 wk but confirmed that all soft callus was remodeled by 3 wk. HBCS did not show the increases in polar moment of inertia or maximum torque observed in the ZA group at 4 wk. Studies on unfractured fluorochrome labeled tibias demonstrated that no treatments affected mineral apposition and bone formation rates. However, HPHBP and HBCS increased growth plate bone volume over total volume (BV/TV) concomitant with a decrease in osteoclasts, while ZA further increased total bone volume and BV/TV with a dramatic increase in osteoclasts. Collectively, these data demonstrate that while HBCS decreases osteoclast numbers in vivo, it does not inhibit fracture healing or normal bone growth in mice.