The highest incidence of BRONJ is reported in oncology patients receiving immunosuppressive therapy. A systematic review of reported cases of BRONJ from 2003 to 2006 revealed that 94% of these patients were treated with intravenous BPs (primarily pamidronate and Zol), and 85% were diagnosed with multiple myeloma or metastatic breast cancer.(15
) It is recognized that 60% of cases occurred following a tooth extraction or other dentoalveolar surgery, and the remaining cases occurred spontaneously. Several important predisposing factors for BRONJ have been identified, including the type and total dose of BP, history of trauma, dental surgery, impaired immunity, and development of dental infection. The estimated incidence of BRONJ in patients receiving intravenous BPs for malignant diseases ranges from 0.8% to 12%,(18
) an incidence that is 900 times higher than that of general osteoporosis patients.(19
) Despite these identified clinical correlates with the incidence of BRONJ, no evidence-based study has established a true direct causal effect. Therefore, the establishment of a BRONJ-like animal model would be a critical milestone for defining the pathogenesis of the disease, its etiology, and potential risk factors. Recent studies in rats showed that administration of Zol acid and Dex preceding a dental extraction resulted in bone and soft tissue changes that resembled those noted in human BRONJ.(32
) The doses for oncologic indications of BP are 10 to 12 times higher than those used in osteoporosis(3
) and usually in combination with an immunosuppressive agent such as corticosteroids.(15
) In this study, we adapt a similar treatment regimen for cancer patients to induce BRONJ in mice. Recognizing that the cumulative high dose of BP has an effect on the progressive course of BRONJ and the relatively shorter lifespan of the mouse compared with human, we titrated Zol dose to achieve a concentration that produced a repeatable clinical effect in the generation of our murine BRONJ-like model without renal toxicity (data not shown). The use of high-dose Dex in conjunction with BPs has been adapted to mice using the equivalent treatment regimen for advanced multiple myeloma.(15
) The addition of Dex in conjunction with BP treatment significantly increased the incidence of BRONJ in wild-type mice compared with Zol alone, suggesting that immunosuppressive therapy may render mice more susceptible to BRONJ lesions. This observation is in accordance with human epidemiologic studies showing a much higher incidence of BRONJ in cancer patients receiving immunosuppresive therapy.(15
We showed that BRONJ-like lesions exhibit similar characteristics to human disease involving mucosal ulceration or open sockets, osseous sclerosis, exposed necrotic bone and sequestra, and radiopaque alveolar bone in the jaw, as demonstrated by µCT and histologic studies. The alveolar bone of BRONJ-like lesion failed to remodel up to 7 weeks after extraction and in some cases up to 12 weeks (Supplemental Fig. 3
), resulting in accumulation of necrotic bone. The clinical manifestations of BRONJ, specifically open sockets with exposed necrotic bone and no mucosal lining, persisted beyond the 2- to 3-week normal course of healing seen in nontreated control mice and therefore met the current diagnosis criteria of human BRONJ.(5
) The hallmark of persistent presence of BRONJ-like clinical, radiographic, and histologic features at the extraction site supports our model of BRONJ-like lesion in mice and allows accurate delineation of the pathophysiologic mechanisms of the disease, leading to the future development of new clinical and laboratory endpoints for accurate diagnosis and prediction of BRONJ in humans.
Since most cancer patients are on multiple immunosuppressant drugs, including Dex, and chemotherapeutic agents and therefore experience some degree of impaired immunity, it is postulated that immunosuppression may contribute to an increased susceptibility to BRONJ. Recently, increasing evidence, both in vitro and in vivo, supports the idea that BPs are able to regulate the immune system by modulating both innate and adaptive immune responses(20
) and impairing monocyte/macrophage and dendritic cell maturation and function.(30
) Immunomodulation by BP treatment can result in either immunosuppression or generalized enhanced immune responses(19
) that subsequently may promote the development of BRONJ. More important, in our preliminary studies, attempts to develop BRONJ-like animal models using different strains of mice revealed that nearly 100% of immunocompromised mice versus 33% of wild-type C57BL/6J mice treated with oncologic doses of Zol and Dex developed BRONJ-like pathologic lesions at 7 weeks after tooth extraction. Furthermore, studies using adaptive immune transfer with pan-T-lymphocytes prior to surgical tooth extraction in Zol- and Dex-treated immunocompromised mice resulted in a reduced incidence of BRONJ from 100% to 50% at 2 weeks and to 14.3% at 8 weeks after extraction. This effect was not observed when Tregs were depleted from the pan-T-lymphocyte pool. This compelling evidence confirms our hypothesis that altered immune homeostasis, specifically a lack of functional T cells in immunocompromised mice, renders these mice more susceptible to BRONJ-like lesions.
To further delineate the specific immune components contributing to BRONJ susceptibility in our model, we explore the role of Tregs, a subpopulation of T cells capable of suppressing various immune responses and thereby regulating immune homeostasis and tolerance to antigens.(33
) Tregs have been shown in vitro and in vivo to suppress IL-17-producing T-helper cells, Th17s, and their reciprocal relationship is modulated by the acute inflammatory protein IL-6.(35
) Th17 cells are important players in the etiopathogenesis of several inflammatory and autoimmune diseases, including rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), psoriasis,(36
) and chronic periodontal lesions.(30
) There exist two major subtypes of CD4+
Tregs, the naturally occurring CD4+
Tregs (nTregs) that originate in the thymus and the induced CD4+
Tregs (iTregs) that are generated in the periphery. In our study, we observed that systemic infusion of Tregs completely prevented the development of BRONJ-like lesions at 7 weeks after extraction. To further confirm our hypothesis, we treated BRONJ-like mice with anti-CD25Ab and observed exacerbation of BRONJ-like disease, shown as an increased incidence of open socket with exposed bone from 50% to 66% at 2 weeks. Mechanistically, we demonstrated that treatment with Zol/Dex suppressed both Tregs and the Treg/Th17 ratio in peripheral blood.
Recently, the immunomodulatory and anti-inflammatory effects of MSCs have been reported in a variety of animal models, and it is speculated that they play a potential role in certain human diseases. Interestingly, our data showed that treatment with systemic infusion of MSCs resulted in a marked suppression of the Zol- and Dex-induced increase in Th17 cells in peripheral blood and restoration of Treg levels in the treated BRONJ-like mice. At 2 weeks after extraction, BRONJ-like wild-type C57BL/6J mice receiving MSC infusions showed complete mucosal healing and bone regeneration at the extracted alveolar socket. To our knowledge, these promising treatment outcomes provide the first evidence that immunotherapy with MSCs and Tregs can reverse Treg deficiency in BRONJ-like mice and is capable of preventing and curing BRONJ-like lesions in mice.
In our model, we observed BRONJ-like lesions only at the site induced by dental extraction, making BRONJ a condition that affected only the oral cavity. However, it is not surprising that the underlying Zol- and Dex-induced necrotic bone presumably could occur at other skeletal sites if they were traumatized. Further studies are in progress to determine the potential systemic effect of Zol. We recognized that several other mechanisms, including osteoblast function, angiogenesis, dental infection, and conditions linked to altered immune homeostasis, potentially contribute to the pathogenesis of BRONJ. Even though bacterial colonization has been reported in BRONJ, as shown at the bottom of the extraction socket (Supplemental Fig. 4
), it is still unknown whether the infection arises initially in the bone or soft tissue or whether it primarily plays a causative role in the pathogenesis of BRONJ.(43
) In this study, we observed that necrotic bone was often found adjacent to area of intense local inflammatory infiltrates, suggesting an association between inflammation, microbial flora, and tissue degeneration/necrosis in BRONJ-like disease. The underlying host immunocompromise in conjunction with other risk factors unique for the oral cavity, specifically the open wound and the oral microbial flora, may contribute to the high incidence of BRONJ in patients undergoing dental surgeries. Further studies are needed to verify whether infection constitutes a risk factor in BRONJ development.
In summary, despite the potential risk of developing BRONJ in a subpopulation of patients, BP has greatly benefited a large number of patients suffering from skeletal complications related to bone metabolism and neoplasms. Our animal models will allow the direct evaluation of clinically identified risk factors in the development of BRONJ and the garnering of evidence-based guidance leading to the development of preventive, diagnostic, and therapeutic tools in the management of human BRONJ. More important, cell-based immunotherapy using systemic MSCs and Tregs potentially can offer a safe and effective novel therapeutic modality in preventing the development of BRONJ disease in vulnerable cancer patients who have to undergo BP treatment for their cancer, as well as the larger group of patients who are on oral BPs for bone-related metabolism conditions.