Figure 1.

Study design for in vivo experiment to induce BONJ-like lesion. (A) Experiment 1 (Exp 1). To examine the effects of Pam and Fn on the development BONJ-like lesions, we divided the mice into 4 groups (n = 10/group) as described in the “Materials & Methods” section: (Group 1) saline (control), (Group 2) Fn, (Group 3) Pam alone, and (Group 4) Pam+Fn. To Groups 3 and 4, Pam (1 mg/kg/wk in saline) was subcutaneously (s.c.) injected weekly for 4 wks prior to tooth extraction (-28, -21, -14, and -7 days). Group 1 (saline) and Group 2 (Fn) received control saline injection (s.c.) instead of Pam, following the same injection schedule as Pam (-28, -21, -14, and -7 days). On Day 0, the first right maxillary molar was extracted under anesthesia (ketamine/xylazine) without causing fracture in alveolar bone or tooth (B, C). After tooth extraction, each animal in Group 2 (Fn) or Group 4 (Pam+Fn) was inoculated orally with live Fn (10⁹ CFU/50 µL PBS with 5% carboxymethyl cellulose/animal) every other day until termination of the experiment scheduled at Day 15. Experiment 2 (Exp 2). For examination of the effects of antibiotics on the BONJ-like lesions induced by Pam+Fn, both Group A (n = 10; Pam+Fn) and Group B (n = 10; Pam+Fn) received Pam injection (1 mg/kg/wk in saline s.c.) weekly for 4 wks prior to tooth extraction (-28, -21, -14, and -7 days). After tooth extraction, both groups received oral inoculation with live Fn (10⁹ CFU) every other day until termination of the experiment scheduled at Day 15. After tooth extraction until termination (Day 0 to Day 15), Group A animals were supplied with control regular drinking water, while Group B received drinking water containing an antibiotic cocktail, consisting of ampicillin (1 g/L), metronidazole (1 g/L), vancomycin (0.5 g/L), and neomycin sulfate (1 g/L). (B) Image of extracted maxillary first molar is shown. (C) Image of extraction socket is shown. Soft tissue was removed so that structure of alveolar bone can be visualized. Bar = 1 mm. (D) Histological image (H&E staining) of first right maxillary molar of control mice receiving no treatment is shown. The specific location and area where gingival epithelium, connective tissue, and bone were evaluated in Fig. 3 as well as Appendix Fig. 4 are indicated by “X” (gingival epithelium and connective tissue) and “Y” (bone), respectively.