Impaired bone resorption and woven bone formation are associated with development of osteonecrosis of the jaw-like lesions by bisphosphonate and anti-receptor activator of NF-κB ligand antibody in mice

Abstract

Drug-induced osteonecrosis of the jaw (ONJ) is a detrimental intraoral lesion that often occurs after dental-related interventions in patients undergoing treatment with bisphosphonates or denosumab, the neutralizing human anti-receptor activator of NF-κB ligand (RANKL) antibody (Ab). The cause of ONJ by these drugs has been speculated to their direct effects on osteoclasts. However, the extent to which osteoclasts contribute to ONJ pathogenesis remains controversial. Herein, by using a tooth-extraction mouse model with i.v. administration of mouse anti-RANKL Ab or the bisphosphonate zoledronate (ZOL), we show that unresorbed bone due to impaired formation or suppressed functions of osteoclasts, respectively, is associated with ONJ development. After tooth extraction, ONJ-like lesions developed 50% in the anti-RANKL Ab-treated mice and 30% in the ZOL-treated mice. Nonviable and unresorbed bone was found more in anti-RANKL Ab-treated mice compared with mice receiving ZOL. All mice receiving anti-RANKL Ab had an undetectable tartrate-resistant acid phosphatase (TRAP) level in the serum and no TRAP-positive osteoclasts at the extracted sockets, whereas ZOL-treated mice had a decreased TRAP level without altering the numbers of TRAP-positive osteoclasts. Interestingly, the absence of newly formed woven bone in the extracted sockets was evident in ONJ-like lesions from both anti-RANKL Ab- and ZOL-treated mice. Our study suggests that the lack of osteoclasts' bone-resorptive functions by these drugs and suppression of woven bone formation after dental trauma may be associated with ONJ development.

Figure 1

Establishment of the osteonecrosis of the jaw (ONJ) mouse models for DRONJ and BRONJ. The schematic timelines for the DRONJ (A) and BRONJ (B) models. C and D: Three weeks after tooth extraction, maxillae were harvested, clinical presentations were imaged (left panels), and μCT scans of the maxillae were taken. The occlusal views (middle panels) and the angled views (right panels) are shown. The black arrows resorbed alveolar ridges (C and D); black arrowheads (C and D), new bone in socket; red arrows (C and D), unresorbed alveolar ridges; red arrowheads (C and D), no bone in socket; white arrowheads (C and D), open wound.

Figure 2

Anti-RANKL Ab treatment induces osteonecrosis of the jaw (ONJ)-like lesions. H&E-stained tissue at tooth-extracted sites from mice i.v. administered with IgG (A) as well as anti-RANKL Ab that induced (B) or did not induce (C) ONJ-like lesions. The numbers of empty lacunae per mm² (D) and the percentage of necrotic bones (E) were quantified. ^∗P < 0.05, ^∗∗P < 0.01. Scale bars: 100 μm (A–C). The black arrow (A) indicates resorbed alveolar ridge; red arrows (B and C), unresorbed ridge; white (A) and red (C) arrowheads, margin between existing lamellar bone and newly formed woven bone. CT, connective tissue; ET, epithelial tissue; LB, lamellar bone; PEH, pseudoepitheliomatous hyperplasia; WB, woven bone.

Figure 3

Zoledronate (ZOL) treatment induces osteonecrosis of the jaw (ONJ)-like lesions. H&E-stained tissue at tooth-extracted sites from mice i.v. administered with Veh (A) or ZOL that induced (B) or did not induce (C) ONJ-like lesions. Quantification of the numbers of empty lacunae per mm² (D) and the percentage of necrotic bones (E). ^∗P < 0.05. Scale bars: 100 μm (A–C). The black arrow (A) indicates the resorbed alveolar ridge; red arrows (B and C), unresorbed ridge; white (A) and red (C) arrowheads, margin between existing lamellar bone and newly formed woven bone CT, connective tissue; ET, epithelial tissue; LB, lamellar bone; PEH, pseudoepitheliomatous hyperplasia; WB, woven bone.

Figure 4

Mature osteoclasts are absent in anti-RANKL Ab-treated, but not in zoledronate (ZOL)-treated, mice. Three weeks after initial i.v. administration and 2 weeks after tooth extraction, the maxillae were harvested and EDTA decalcified. A: TRAP staining was performed at the tooth-extracted sites from mice i.v. administered with IgG or anti-RANKL Ab that induced or did not induce ONJ-like lesions. B: Quantification of the numbers of TRAP⁺ osteoclasts. C and D: Similar experiments were performed in mice treated with Veh and ZOL. ^∗P < 0.05, ^∗∗∗P < 0.001. Scale bar = 100 μm (A and C).

Figure 5

Anti-RANKL Ab or zoledronate (ZOL) treatment diminishes bone remodeling in vivo. Blood was collected from the control IgG- or anti-RANKL Ab-treated mice 4 weeks after initial administration, and the serum levels of TRAP 5b (A) or ALP (B) were evaluated using ELISAs. The serum levels of TRAP 5b (C) and ALP (D) in mice treated with Veh or ZOL. ^∗∗∗P < 0.001.

Figure 6

Newly formed bone in the tooth-extracted sockets is inhibited in osteonecrosis of the jaw (ONJ) lesions. Newly formed bone in the tooth-extracted sockets was quantified using the micro computed tomography scan from the DRONJ (A) or BRONJ (B) mouse model. ^∗∗∗∗P < 0.0001.