Zoledronic Acid Deteriorates Soft and Hard Tissue Healing of Murine Tooth Extraction Sockets in a Dose-Dependent Manner.

The pathophysiology, histopathology, and immunopathology of bisphosphonate-related osteonecrosis of the jaw (BRONJ) Stage 0 remain unclear. The aim of this study was to investigate the effects of high-dose bisphosphonates on tooth extraction socket healing by creating a murine model of BRONJ Stage 0-like lesions using 8-week-old female C57BL/6J mice. Zoledronic acid (Zol) was administered subcutaneously twice a week for 7 weeks at doses of 0.1 mg/kg/week (moderate dose; Zol-M), 0.5 mg/kg/week (high dose; Zol-H1), and 1.0 mg/kg/week (higher dose; Zol-H2). Saline was used as a control (VC). Both maxillary first molars were extracted 3 weeks after drug treatment. Maxillae, long bones, and sera were collected 4 weeks post-extraction (n = 7 mice/group). Microcomputed tomography, histological, immunohistochemical, and ELISA analyses were performed. A ceiling effect for Zol was noted at the Zol-H1 dose. Osseous healing of extraction sites was significantly impaired with increased necrotic bone and the number of empty lacunae in a Zol dose-dependent manner. Zol significantly decreased epithelial thickness, due to a decrease in thickness of the stratum spinosum, in both Zol-H1 and Zol-H2. Both Zol-H1 and Zol-H2 significantly suppressed the distribution of F4/80+ macrophages in the connective tissue of tooth extraction sockets, although gross healing appeared to be normal. Intriguingly, both Zol-H1 and Zol-H2 significantly increased the numbers of TRAP+ mononuclear cells and detached osteoclasts in the connective tissue and bone marrow of extraction sites compared to VC and Zol-M, correlated with serum TRAcP5b levels. The created murine model of BRONJ Stage 0-like lesions becoming more severe in a dose-dependent manner may help to understand the pathophysiology and histopathology of BRONJ Stage 0 in humans.

Zoledronate/Anti-VEGF Neutralizing Antibody Combination Administration Increases Osteal Macrophages in a Murine Model of MRONJ Stage 0-like Lesions.

The pathophysiology, pathogenesis, histopathology, and immunopathology of medication-related osteonecrosis of the jaw (MRONJ) Stage 0 remain unclear, although 50% of MRONJ Stage 0 cases could progress to higher stages. The aim of this study was to investigate the effects of zoledronate (Zol) and anti-vascular endothelial cell growth factor A (VEGFA) neutralizing antibody (Vab) administration on polarization shifting of macrophage subsets in tooth extraction sockets by creating a murine model of MRONJ Stage 0-like lesions. Eight-week-old, female C57BL/6J mice were randomly divided into 4 groups: Zol, Vab, Zol/Vab combination, and vehicle control (VC). Subcutaneous Zol and intraperitoneal Vab administration were performed for 5 weeks with extraction of both maxillary first molars 3 weeks after drug administration. Euthanasia was conducted 2 weeks after tooth extraction. Maxillae, tibiae, femora, tongues, and sera were collected. Structural, histological, immunohistochemical, and biochemical analyses were comprehensively performed. Tooth extraction sites appeared to be completely healed in all groups. However, osseous healing and soft tissue healing of tooth extraction sites were quite different. The Zol/Vab combination significantly induced abnormal epithelial healing, and delayed connective tissue healing due to decreased rete ridge length and thickness of the stratum granulosum and due to decreased collagen production, respectively. Moreover, Zol/Vab significantly increased necrotic bone area with increased numbers of empty lacunae compared with Vab and VC. Most interestingly, Zol/Vab significantly increased the number of CD169+ osteal macrophages (osteomacs) in the bone marrow and decreased F4/80+ macrophages, with a slightly increased ratio of F4/80+CD38+ M1 macrophages compared to VC. These findings are the first to provide new evidence of the involvement of osteal macrophages in the immunopathology of MRONJ Stage 0-like lesions.

Depletion of macrophages deteriorates bisphosphonate-related osteonecrosis of the jaw-like lesions in mice.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a potentially intractable disease with no definitive pathophysiology and no treatment and prevention strategies. This study aimed to investigate whether time-selective depletion of macrophages worsens BRONJ-like lesions in mice. A murine model of high-prevalence BRONJ-like lesions in combination with zoledronate/chemotherapeutic drug administration and tooth extraction was created according to the methods of our previous studies. Daily intra-oral submucosal administration of clodronate-loaded liposomes, which temporarily depletes systemic macrophages, was performed immediately after tooth extraction. Spleens, femora, tibiae, and maxillae were dissected 2 weeks after extraction to evaluate BRONJ-like lesions and systemic conditions by micro-computed tomography analysis, histomorphometric and immunofluorescent analyses, and serum chemistry with ELISA. Depletion of macrophages significantly decreased the numbers of local and systemic macrophages and osteoclasts on the bone surface, which markedly worsened osseous healing, with increased necrotic bone and empty lacunae in the existing alveolar bone and newly formed bone in the extraction sockets, and soft tissue healing, with decreased collagen production and increased infiltration of polymorphonuclear cells. Interestingly, the depletion of macrophages significantly shifted macrophage polarization to M1 macrophages through an increase in F4/80+CD38+ M1 macrophages and a decrease in F4/80+CD163+ M2 macrophages, with decreases in the total number of F4/80+ macrophages. These data demonstrated that severe inhibition of osteoclasts in bone tissue and polarization shifting of macrophages in soft tissue are essential factors associated with BRONJ.

Gene expression analysis of fresh extraction wounds prior to onset of bisphosphonate-related osteonecrosis of the jaw-like lesions in mice: A preliminary animal study.

Purpose The aim of the present study was to investigate the effects of chemotherapeutic/bisphosphonate combination therapy with tooth extraction on gene expression patterns of fresh extraction wounds during initial stages prior to their diagnosis as bisphosphonate-related osteonecrosis of the jaw (BRONJ)-like lesions in mice.Methods Female C57BL/6J mice were used. To create a high-prevalence BRONJ mouse model, combination therapy with the chemotherapy drug cyclophosphamide (CY) and zoledronic acid (ZA) was performed (CY/ZA). Both maxillary first molars were extracted 3 weeks after drug therapy. Saline was used as the control (VC). Soft tissues near the fresh extraction wounds were dissected at 72 h postextraction to investigate the gene expression patterns. Maxillae and long bones at 2 and 4 weeks postextraction were also analyzed.Results CY/ZA significantly increased the relative expression levels of IL-6 and decreased those of IL-10 and IGF-1 when compared with those in VC. Moreover, CY/ZA significantly reduced the relative expression levels of CCR-7, cxcl12, cxcr4, and CD105 when compared with those in VC, whereas the level of F4/80 was significantly increased by CY/ZA. Furthermore, CY/ZA significantly decreased the relative expression levels of VEGFA, VEGFB, and VEGFC at 72 h postextraction compared with those in VC.Conclusions Considering that the present study lacked adequate in vitro models, CY/ZA markedly changed the gene expression patterns associated with wound healing from the initial stages prior to onset of BRONJ-like lesions, which may help us to understand the pathophysiology of BRONJ in humans.