Prevalence of bisphosphonate-related osteonecrosis of the jaw-like lesions is increased in a chemotherapeutic dose-dependent manner in mice

doi:10.1016/j.bone.2018.05.001

Bone

Volume 112, July 2018, Pages 177-186

https://doi.org/10.1016/j.bone.2018.05.001 Get rights and content

Highlights

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Impaired tooth socket healing rarely occurred with cyclophosphamide (CY) monotherapy.
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Bisphosphonate (ZA) monotherapy did not induce BRONJ-like lesions in mice.
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High-dose CY in combination with ZA and tooth extraction induced BRONJ-like lesions in mice.
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BRONJ-like lesions prevalently increased in a CY dose-dependent manner.

Abstract

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) worsens oral health-related quality of life. Most BRONJ occurs in multiple myeloma or metastatic breast cancer patients treated with bisphosphonate/chemotherapeutic combination therapies. Cyclophosphamide (CY), an alkylating chemotherapeutic drug, is used to treat multiple myeloma, although its use has been recently reduced. The aim of this study was to clarify the effects of CY dose on tooth extraction socket healing when CY is used with or without bisphosphonate in mice. Low-dose CY (50 mg/kg; CY-L), moderate-dose CY (100 mg/kg; CY-M), high-dose CY (150 mg/kg; CY-H), and bisphosphonate [Zometa (ZA): 0.05 mg/kg] were administered for 7 weeks. Each dose of CY and ZA in combination was also administered for 7 weeks. Both maxillary first molars were extracted at 3 weeks after the initiation of drug administration. Euthanasia was performed at 4 weeks post-extraction. Gross wound healing, microcomputed tomography analysis, histomorphometry, and immunohistochemistry were used to quantitatively evaluate osseous and soft tissue wound healing of tooth extraction sockets. ZA monotherapy induced no BRONJ-like lesions in mice. CY monotherapy rarely induced open wounds, though delayed osseous wound healing occurred in a CY dose-dependent manner. In contrast, CY/ZA combination therapy prevalently induced BRONJ-like lesions with compromised osseous and soft tissue healing in a CY dose-dependent manner. Interestingly, anti-angiogenesis was noted regardless of CY dose and ZA administration, even though only CY-M/ZA and CY-H/ZA combination therapies induced BRONJ-like lesions. Our findings suggest that high-dose CY may be associated with the development of BRONJ following tooth extraction only when CY is used together with ZA. In addition to anti-angiogenesis, other factors may contribute to the pathoetiology of BRONJ.

Introduction

Nitrogen-containing bisphosphonates bind and inhibit a key enzyme of the intracellular mevalonate pathway, thereby resulting in apoptosis of osteoclasts. In general, oral bisphosphonates are used to treat osteoporosis, whereas intravenous bisphosphonates are utilized to reduce the risks of skeletal-related events such as pathological vertebral fracture and bone pain in oncology or metastasis patients [1]. However, in 2003, bisphosphonate-related osteonecrosis of the jaw (BRONJ) was first reported to occur following tooth extraction in patients taking intravenous bisphosphonates [2]. BRONJ is defined as “exposed bone or bone that can be probed through an intraoral or extraoral fistula in the maxillofacial region that has persisted for more than 8 weeks” [3]. The prevalence of BRONJ is rare, occurring in 0.05% of oral bisphosphonate users following tooth extraction, and 1.6%–14.8% of intravenous bisphosphonate users following tooth extraction [4]. A systematic review reported that BRONJ most frequently occurs in multiple myeloma or metastatic breast cancer patients taking intravenous bisphosphonates [5]. Tooth extraction (61.7%), dental implant treatment (3.9%), dental surgery (7.2%), periodontal disease (5.0%), prosthetic trauma such as ill-fitting removable prostheses (7.4%), and spontaneous cases (14.8%) are triggering factors of BRONJ [5], although all dental therapies including tooth cleaning, root canal filling, and prosthodontic treatment are not contraindicated in patients taking bisphosphonates. BRONJ worsens oral health-related quality of life [6], which results in negative effects on daily life and social activities. However, the exact mechanism of BRONJ has not been clear. Moreover, definitive treatment strategies for BRONJ have not been developed due to its uncertain mechanisms. Therefore, elucidation of the pathoetiology and pathophysiology of BRONJ is required.

Multiple myeloma, which represents 1% of all cancers and approximately 10% of all hematologic malignancies, occurs in 20,000 people in the United States every year [7]. Moreover, the age-adjusted 5-year relative survival rate increased from 39.8% to 53.2% [7]. The increase in survival rate of multiple myeloma patients is due to the development of therapeutic strategies. A current review reported that thalidomide, lenalidomide, bortezomib, carfilzomib, pomalidomide, cyclophosphamide, melphalan, dexamethasone, and prednisone are used to treat multiple myeloma solely or adjunctively [8]. Generally, cyclophosphamide or melphalan are utilized for the treatment of multiple myeloma in combination with intravenous bisphosphonates that reduce the risks of skeletal complications [9]. The use of cyclophosphamide or melphalan in combination with prednisone, thalidomide, bortezomib, or carfilzomib was introduced as the major treatment regimens for multiple myeloma in 2016 [8], whereas at the 2017 American Society of Hematology Annual Meeting, the use of cyclophosphamide and melphalan were reportedly decreased [10]. Different doses of cyclophosphamide are administered to multiple myeloma patients depending on the patient's age and tolerability [9]. However, the effects of different doses of cyclophosphamide on tooth extraction socket healing, with or without bisphosphonate therapy, are not fully understood.

The aims of the present study were 1) to clarify the effects of cyclophosphamide dose on tooth extraction socket healing, and 2) to investigate the effects of each dose of cyclophosphamide and bisphosphonate combination therapy on osseous and soft tissue wound healing of tooth extraction sockets in C57BL6/J mice.

Section snippets

Animals, tooth extraction, and drug therapies

Eight- to twelve-week-old male C57BL/6J mice were used (CLEA Japan Inc., Osaka, Japan). The duration of drug therapies was 7 weeks. Both maxillary first molars were extracted at 3 weeks after the start of drug therapy. All mice were euthanized at 4 weeks post-extraction. Subcutaneous injection of zoledronate (Zometa; Novartis, Stein, Switzerland) was performed at 0.05 mg/kg twice a week for 7 weeks (ZA, n = 7). Intraperitoneal injection of cyclophosphamide (C7397; Sigma-Aldrich, St. Louis, MO,

Effect of CY monotherapy dose on osseous healing of tooth extraction sockets

No open wounds were observed in the VC (0 out of 14 extraction sockets) and CY-L (0 out of 14 extraction sockets) groups at 4 weeks after tooth extraction. However, 7.14% (1 out of 14 extraction sockets) and 14.2% (2 out of 14 extraction sockets) open wounds were noted in CY-M and CY-H groups, respectively, at 4 weeks after tooth extraction (Fig. 2a). Hard tissue healing appeared to be the same between VC and CY-L groups (Fig. 2b). CY monotherapy for 7 weeks did not change osteoclast numbers in

Discussion

In the present study, we showed that CY monotherapy delayed osseous wound healing in a dose-dependent manner, although this therapy did not affect soft tissue wound healing. Moreover, we demonstrated that CY/ZA combination therapy prevalently induced BRONJ-like lesions in a CY dose-dependent manner by inhibiting both osseous and soft tissue wound healing following tooth extraction.

First, we investigated the effect of CY dose on hard and soft tissue wound healing following tooth extraction. In

Conclusions

In the present study, we demonstrated that CY therapy alone did not induce open wounds in mice, irrespective of CY dose, although CY monotherapy delayed osseous healing in a CY dose-dependent manner. ZA monotherapy did not induce BRONJ-like lesions. Moreover, we showed that BRONJ-like lesions were prevalently induced in a CY dose-dependent manner when tooth extraction was performed in mice treated with CY/ZA combination therapy.

Acknowledgements

This work was supported by JSPS KAKENHI Grant Numbers 25870523, 15K11258 and 18H02994.

Conflict of interest

The authors declare that they have no conflicts of interest.

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To assess the outcomes of several rodent animal models for studying tooth extraction-related medication-related osteonecrosis of the jaw (MRONJ).
After a search of the databases, 2004 articles were located, and 118 corroborated the inclusion factors (in vivo studies in rodents evaluating tooth extraction as a risk factor for the development of MRONJ).
Numerous studies attempting to establish an optimal protocol to induce MRONJ were found. Zoledronic acid (ZA) was the most used drug, followed by alendronate (ALN). Even when ZA did not lead to the development of MRONJ, its effect compromised the homeostasis of the bone and soft tissue. The association of other risk factors (dexamethasone, diabetes, and tooth-related inflammatory dental disease) besides tooth extraction also played a role in the development of MRONJ. In addition, studies demonstrated a relationship between cumulative dose and MRONJ.
Both ZA and ALN can lead to MRONJ in rodents when equivalent human doses (in osteoporosis or cancer treatment) are used. Local oral risk factors and tooth-related inflammatory dental disease increase the incidence of MRONJ in a tooth extraction-related rodent model.
Depletion of macrophages deteriorates bisphosphonate-related osteonecrosis of the jaw-like lesions in mice
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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.
Clinical, CT profile of wistar rats receiving therapeutic doses of alendronate alone and in combination with cyclophosphamide and dexamethasone
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The exact pathogenesis of drug-induced osteonecrosis of the jaw is still unknown. The aim of this study is to develop a reliable rat model of alendronate-related osteonecrosis of the jaw and to determine the CT profile of bisphosphonate-induced osteonecrosis of the jaw.
98 Wistar rats were divided into 4 groups. Rats in the test groups received, in addition to 0.05 mg/kg alendronic acid orally (twice a week for 8 consecutive weeks), 1 mg/kg/day subcutaneously of dexamethasone for 2 weeks and 150 mg/kg cyclophosphamide orally twice and once a week before and after dental avulsion.
From week 4 to week 8 after drug administration, the frequency of stage 3 osteonecrotic lesions increased in the test groups from (G1 = 5 (18.5%); G2 = 11 (39.2%); G3 = 6 (21.4%)) to (G1 = 8 (32%); G2 = 12 (42.8%); G3 = 13 (46.4%)) respectively. However, healing was not impaired in the control group (p = 0.003).
Scannographic analysis showed that the difference in bone mineral density was greater in rats exposed (G2 = 486.22 ± 94.76; G3 = 584.9 ± 42.47) to the drug combinations than in rats exposed (G1 = 421.61 ± 125.81) to alendronate alone.
Based on this study, the rat treated with alendronate in combination or not with dexamethasone and cyclophosphamide and dental extraction can be considered as a new, reliable and reproducible model to better understand the pathophysiology of osteonecrosis of the jaws in the context of osteoporosis and prostate cancer. Furthermore, bone mineral density has been shown to be an important diagnostic criterion.
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Furthermore, the combination of ZOL, dexamethasone and docetaxel (a potent chemotherapeutic agent) worsened MRONJ like-lesions and increased the prevalence of exposed bone compared to mice treated with ZOL and dexamethasone [171]. Another study showed that when mice were subjected to healthy molar extraction and treated with combination therapy of ZOL and cyclophosphamide (cytotoxic chemotherapy), the oral lesions tended to be more severe and manifest histopathologic necrotic exposed bone and/or bony sequestrum [174]. Notably, when pARs, such as ZOL [26,150,152], RANK-Fc, or OPG-FC [23,43,152] were administered to mice with natural periodontitis or experimentally induced inflammatory dental diseases (periapical or periradicular disease), exposed necrotic bone was a distinctive histopathologic feature of the oral lesions, except in one study where mice developed non-exposed necrotic bone [150] (Table 2).
Medication-related osteonecrosis of the jaw (MRONJ) is a potentially severe adverse event affecting patients with cancer and patients with osteoporosis who have been treated with powerful antiresorptives (pARs) or angiogenesis inhibitors (AgIs). pARs, including nitrogen-containing bisphosphonates (N-BPs; e.g., zoledronic acid, alendronate) and anti-RANKL antibodies (e.g., denosumab), are used to manage bone metastases in patients with cancer or to prevent fragility fractures in patients with osteoporosis.
Though significant advances have been made in understanding MRONJ, its pathophysiology is still not fully elucidated. Multiple species have been used in preclinical MRONJ research, including the rat, mouse, rice rat, rabbit, dog, sheep, and pig. Animal research has contributed immensely to advancing the MRONJ field, particularly, but not limited to, in developing models and investigating risk factors that were first observed in humans. MRONJ models have been developed using clinically relevant doses of systemic risk factors, like N-BPs, anti-RANKL antibodies, or AgIs. Specific local oral risk factors first noted in humans, including tooth extraction and inflammatory dental disease (e.g., periodontitis, periapical infection, etc.), were then added. Research in rodents, particularly the rat, and, to some extent, the mouse, across multiple laboratories, has contributed to establishing multiple relevant and complementary preclinical models. Models in larger species produced accurate clinical and histopathologic outcomes suggesting a potential role for confirming specific crucial findings from rodent research. We view the current state of animal models for MRONJ as good. The rodent models are now reliable enough to produce large numbers of MRONJ cases that could be applied in experiments testing treatment modalities. The course of MRONJ, including stage 0 MRONJ, is characterized well enough that basic studies of the molecular or enzyme-level findings in different MRONJ stages are possible.
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¹: S Kuroshima and M Sasaki equally contributed to this work.

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Full Length ArticlePrevalence of bisphosphonate-related osteonecrosis of the jaw-like lesions is increased in a chemotherapeutic dose-dependent manner in mice