Abstract
Clinically, bisphosphonate-related osteonecrosis (BRON) only occurs in the jaw (BRONJ). We aimed to determine differences between the jaw and other bones, as well as the relationship between periodontal pathogens and BRON. Twelve male Wistar rats were divided into two groups: group Z ( n = 6) were subcutaneously injected with zoledronic acid weekly for 4 weeks; group C ( n = 6) were injected with saline. One week after the final injection, rats in both groups were divided into three subgroups: Aa subgroup was injected with freeze-dried Aggregatibacter actinomycetemcomitans into bone marrow of the mandibles and femurs, while CFA and saline subgroups were injected with complete Freund’s adjuvant (CFA) or saline using the same protocol. Four weeks after those injections, the rats were euthanized. Areas of osteonecrosis were measured histologically. Statistically, rats in group Z showed significantly wider osteonecrosis areas in both mandibles and femurs with each type of local injection than rats in group C. In group Z, mandibles and femurs stimulated with A. actinomycetemcomitans or CFA had significantly wider osteonecrosis areas than those stimulated with saline. We have developed a rat model with BRONJ-like lesions. Our results showed BRON to develop not only in the jaw but also in other types of bone following an inflammatory stimulus.
Bisphosphonates (BP) are used in patients with multiple myeloma, bone metastasis, and hypercalcemia, and are also effective for the treatment of Paget’s disease of the bone and osteoporosis. Zoledronic acid (ZOL) is a third-generation BP and the most potent BP in general clinical use, as it shows strong anti-osteoclastic activity.
Since 2003, growing numbers of case reports and series regarding bisphosphonate-related osteonecrosis of the jaw (BRONJ) have been published, linking the administration of BP, particularly when given intravenously, with this previously rare condition of osteonecrosis of the jaw.
BRONJ is defined as a condition characterized by non-healing exposed bone in the mandible or maxilla persisting for more than 8 weeks in a patient who has taken or is currently taking a BP and has no history of irradiation therapy to the jaws.
The frequency of BRONJ is estimated to be 0.07/10,000 patients receiving oral medicine and 1–12% of patients receiving intravenous administration. Despite numerous studies, little is known about the relationship between BP treatment and osteonecrosis of the jaw, while prophylaxis and management of BRONJ have not been established. Once BRONJ develops, it is very refractory, and wide areas of osteonecrosis of the jaw are seen in some cases, including those with exposed bone and infection, which affects the quality of life of the patient.
The low frequency of BRONJ has prevented adequate development of prophylaxis and treatment for affected individuals, as it is clinically difficult to study a disease with such a low incidence to reveal the pathogenesis. Therefore, it is important to develop adequate animal models in order to develop effective prophylaxis and management strategies for this condition from the models.
Originally there was confusion regarding BRONJ, with the most important question being why, in spite of medicine administered for the whole body, only the jaw shows the disorder. As a result of this, some hypotheses have been presented including: (1) high remodelling speed in the jaw leading to greater accumulation of BP, and (2) bacterial infection that easily occurs in the jaw, thus leading to a rapid advance of osteonecrosis. However, scientific support is still lacking.
We speculated that if bone tissue other than the jaw was affected by BP and then acquired a bacterial infection, it may develop osteonecrosis. Furthermore, to examine whether the jaw has a specific property related with BP, it was compared to the femur. The specific aim of this study was to examine the relationship between osteonecrosis and periodontal pathogen infection, thus jaw bones and femurs of rats were stimulated with a freeze-dried periodontal pathogen.
In the present study, we aimed to develop an effective BP-induced osteonecrosis rat model and to determine the relationship between bacterial infection and BRONJ.
Materials and methods
The experiment was performed with the approval of the animal care committee.
Twelve male Wistar rats (8 weeks old) were utilized as experimental animals and divided into two groups: six were injected subcutaneously with ZOL (Novartis Pharma, Basel, Switzerland) at 0.1 mg/kg of body weight weekly for 4 weeks (group Z), and six injected with an equal volume of saline in the same manner (group C). The dose of ZOL given was determined on the basis of the dose used for the clinical treatment of cancer patients. One week after the final injection, both groups were divided into three experimental subgroups: (1) the Aa subgroup, which received freeze-dried Aggregatibacter actinomycetemcomitans into the bone marrow of the bilateral mandibles and femurs; (2) the CFA subgroup, which received complete Freund’s adjuvant (CFA) using the same protocol; and (3) the saline subgroup, which received saline using the same protocol.
For these experimental procedures, the animals were anesthetized with chloral hydrate (3.5 mg/kg) and placed in a supine position. After shaving and antisepsis, the skin over the mandibles and femurs was cut bilaterally, and a blunt dissection over the bone was performed. The periosteum of the mandibular border and femur was cut, and then reflected in a longitudinal direction along the bone. Next, a small hole was drilled through the cortical bone in the posterior portion of the mandibular border and in the distal two-thirds of the ventral surface of the femur using a 1.8-mm diameter round bur with sufficient irrigation. After making bilateral drill holes, microfibrillar collagen hemostat (ZERIA Pharmaceutical, Tokyo, Japan) soaked with freeze-dried A. actinomycetemcomitans (40 μg/site, dissolved in 4 μl saline), CFA (4 μl/site), or saline (4 μl/site) was packed into each hole in the rats in the Aa, CFA, and saline subgroups, respectively. Finally, the holes were covered with yellow bone wax (Alfresa Pharma Corporation, Osaka, Japan). After thorough irrigation of the wounds, the incisions were closed in layers.
Four weeks after performing these procedures, the rats were euthanized with an overdose of chloral hydrate and fixed with a perfusion of 2% paraformaldehyde. The bilateral mandibles and femurs of each rat were extracted and fixed with 10% buffered formalin solution. After fixation, the bone samples were decalcified in 10% ethylenediaminetetraacetic acid (pH 7.2) at room temperature for 4 weeks and processed for paraffin embedding. Jaw specimens were sliced in a sagittal direction into 6-μm thick sections, while femur specimens were sliced in the direction of the long axis at the same thickness. All specimens were stained with haematoxylin and eosin (H&E). All sections were blinded for identity and evaluated in a random order.
An area of osteonecrosis was defined as a region of bone that had empty lacunae > 500 μm 2 , in accordance with the definition of A llen and B urr . We determined areas of osteonecrosis as bone areas with empty lacunae > 500 μm 2 with continuity to the drilled site. Measurements were performed using a DP2-BSW (Olympus, Tokyo, Japan). Differences among the groups were evaluated using the Mann–Whitney U -test, with a P -value of 0.05 considered to be statistically significant.
Results
No exposed bone or abnormal behaviour was observed during the observation period, and no significant differences in weight were noted among the groups.
Histopathological findings
Mandible
Extensive osteonecrosis was observed in groups Z-Aa and Z-CFA, while this was seen in only a limited area around the drill hole in group Z-saline. In contrast, scant osteonecrosis was observed in group C with all three local stimulations ( Figs. 1 and 2 ).
Femur
Extensive osteonecrosis was observed in groups Z-Aa and Z-CFA, while small amounts of necrosis were observed in group Z-saline and some specimens in group C-Aa. Scant osteonecrosis was observed in groups C-CFA and C-saline ( Figs. 3 and 4 ).
Statistical analysis
Mandible
The area of osteonecrosis was 3.19 ± 1.76 mm 2 in group Z-Aa, 3.60 ± 1.26 mm 2 in group Z-CFA, 0.85 ± 0.51 mm 2 in group Z-saline, 0.18 ± 0.16 mm 2 in group C-Aa, 0.22 ± 0.14 mm 2 in group C-CFA, and 0.00 ± 0.01 mm 2 in group C-saline. Groups Z-Aa and Z-CFA had significantly wider areas of osteonecrosis as compared to the other groups, while group Z-saline had a wider area of osteonecrosis than group C-saline ( Fig. 5 ).
