Animals and surgical procedures were handled according to guidelines of the UCLA Chancellor’s Animal Research Committee. Thirty-five 4-month old C57BL/6J male mice (Jackson Laboratories, Bar Harbor, ME, USA) received intraperitoneal (IP) injections of veh (endotoxin free water) or 200 µg/kg zoledronic acid (BP group) three times per week for 1 week prior to periapical lesion induction. The dose of 200 µg/kg is approximately 3 fold higher than the oncologic zoledronic acid dose of 66 µg/kg [32 (link)], and in the range of experimental BP doses used in other animal models [27 (link), 29 (link)]. We chose to use this higher dose to increase the incidence of ONJ in our animals, since there appears to be a dose-dependent effect of BPs on ONJ incidence in humans [33 (link), 34 (link)].
Mice were anesthetized with isoflurane and mounted on a jaw retraction board. Pulpal exposure of the left first and second mandibular molars was performed utilizing a size 1/4 round bur, avoiding furcal perforation (Fig 1A, taken at sacrifice) as described [35 (link), 36 (link)]. Exposed teeth were left open to the oral environment. Vehicle or ZA IP injections continued for an additional 7 weeks, following the same protocol of 200 µg/kg ZA at three times per week. We elected to inject the animals three times per week for the eight weeks of the experiment, to mimic the monthly injections in humans given the estimation that 17 days of a rodent life correspond to one human year [37 (link)]. Thus, the animals received a total of 24 ZA injections, which corresponds to 2 years of treatment for a cancer patient [38 (link)]. The mean time to onset of ONJ in patients treated with ZA is approximately 18 months [39 (link)].
At the end of the experiment, animals were sacrificed, mandibles were removed, placed in 4% paraformaldehyde for 48 hours and stored in 70% ethanol. Seventeen veh and 18 BP treated animals were utilized.