Ⅰ. INTRODUCTION

Osteonecrosis of the jaws is a notable complication in patients taking bisphosphonates (1-3). Bisphosphonates are widely used to prevent bone loss in patients with osteoporosis (mainly women but in some cases also men) or cancers such as multiple myeloma. Various bisphosphonates have been approved for clinical use, such as alendronate (Fosamax®), risedronate (Actonel®), ibandronate (Boniva®), and zoledronate (Zometa®) (4). Bisphosphonates have two phosphonate [PO (OH) 2] groups that are metabolic resistant to metabolism. Thus, high concentrations of bisphosphonates are maintained for a long time in the bone. These anti-resorptive agents reduce bone turnover by inhibiting osteoclast function (5-6). There are several hypotheses as to how bisphosphonaterelated osteonecrosis of the jaw(BRONJ) develops; (i) inhibition of osteoclastic bone resorption and remodeling, (ii) inflammation/ infection, (iii) inhibition of angiogenesis, (iv) soft tissue toxicity, and (v) innate or acquired immune dysfunction (4). The exact pathogenesis of BRONJ is unclear, however infection has long been considered an important risk factor.

The oral cavity contains more than 700 species of microorganisms that contribute to oral health (7). When the balance is disrupted, harmful bacteria can proliferate and cause infectious diseases such as tooth decay and periodontitis. Bacteria adhere to each other and attach to surfaces such as teeth, bone, or implanted devices (9). In oral cavity, bacteria are presented in the form of biofilms, which consists of microbial populations embedded in a matrix of extracellular polymeric substances. Biofilm theory has been presented to explain infection etiology; dental plaque, for example, is a biofilm closely associated with dental caries and periodontitis (reference).

As mentioned, infection is one of the possible causes of BRONJ. However, there are few reports on the bacterial species involved in BRONJ, so further research is needed to clarify the role of bacteria in this disease. This study identifies the oral bacterial species in the necrotic bones from patients with BRONJ. Sequestration is the most common clinical feature in established BRONJ. These data suggest a bacterial role in the pathophysiology of BRONJ.

Ⅱ. MATERIALS & METHODS

1. Patients & sample collection

The study protocol was reviewed and approved by the Pusan National University Dental Hospital Institutional Review Board (IRB_PNUDH-2018-018). All patients provided their informed consent before they were allowed to participate in this research.

Fifteen patients (2 males and 13 females) were evaluated. The age range of patients was 53 to 86 years (mean: 74.87 years). Their demographic and clinicopathological data are summarized in Table 1. Patients who met all of the following conditions were diagnosed with BRONJ: (i) current or previous treatment with a bisphosphonate; (ii) exposed bone or bone that could be probed through an intra-oral or extra-oral fistula in the maxillofacial region and persisting for more than 8 weeks; and (iii) no history of radiation therapy of the jaws or of obvious metastatic disease in the jaws (4).

Clinical features, radiographic findings, and bisphosphonate intake history were investigated. Patients were excluded if they had systemic signs of infection or, at the time of the study, had cancer or were receiving chemotherapy, radiation, antiretroviral, or steroid therapy (all are presumed risk factors for bone necrosis). All patients were scheduled for surgical treatment (curettage or sequestrectomy) under general or local anesthesia. During surgeries, infected bone samples of similar size were collected carefully, under sterile conditions and without rinsing from the center of the lesion. Samples containing pus, exudate, or granulation tissue were excluded. Collected samples were stored in specimen containers at − 20℃ for genomic DNA extraction.

For histological evaluation, sequestra were rinsed with phosphate-buffered saline (PBS). The H&E stain method was used. Photographs were taken using a photomicroscope at the magnifications indicated.

2. DNA extraction and PCR amplification

Ten bacterial species were selected for DNA extraction and PCR amplification: Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Prevotella nigrescens, Eikenella corrodens, Streptococcus intermedius, Capnocytophaga gingivalis, Porphyromonas endodontalis, and Actinomyces israelii. DNA was extracted using the Qiagen DNeasy protocol (Qiagen, Valencia, CA, USA). DNA concentrations in clinical samples and reference DNA were determined from absorbance at 260 nm using a spectrophotometer (Nanodrop, Thermo, Wilmington, DE, USA).

The standard polymerase chain reaction (PCR) protocol was used to assess the occurrence of all target taxa. Each PCR reaction mixture (20 μL) contained 1 pM of species-specific primers and a double concentration of master mix. Amplification was performed in a PCR Thermal Cycler (Eppendorf, Hamburg, Germany). The program included 35 amplification cycles (denaturation for 30 s at 95℃, annealing for 30 s at 58℃, and extension for 30 s at 72℃) followed by 10 min at 72℃ and finally 10 min at 4℃. The predicted sizes of the PCR products and species-specific primers are listed in Table 2. The gene for 16S ribosomal RNA (rRNA) was used as a reference. PCR products were subjected to electrophoresis on 2% agarose gels and stained with 1 mg/ml ethidium bromide. The separated DNA was viewed under UV light. Digital images of the gels were analyzed using Total Lab TL120 software (Nonlinear Dynamics, Newcastle upon Tyne, UK). The PCR bands were sorted based on their density. The value of each band was expressed relative to that of the 16S rRNA gene, which was assigned a value of 1. Bands were classified as strong (≥ 0.4) or weak (0.2–0.4).

Ⅲ. RESULTS

1. Evaluation of patients

All patients showed radiographic evidence of an illdefined lytic lesion in the bone (Fig. 1) and clinical evidence of exposed and necrotic jaw bones. They showed inflammatory reactions in soft tissues around the lesion (Fig. 2). Their underling systemic disease was osteoporosis, which they were treating with bisphosphonate. Three patients had a history of cancer.

The patients were taking different bisphosphonates via peroral (PO) or intravenous (IV) routes. More than half were taking PO alendronate (n = 8, 54%). Three patients were taking risedronate (20%), two were taking ibandronate (13%), and two zoledronate (13%). All patients used bisphosphonates more than 2 years (2–10 years, mean: 4 years). Patient 9 used alendronate for more than 10 years.

All patients had previously had alveolar bone surgery. Most had tooth extraction (13/15, 81%). Two patients received an oral incision and drainage (I & D). One patient had an implantation surgery. BRONJ appeared almost three times more often in the mandible (11/15, 73%) than in the maxilla (4/15, 27%).

2. Histopathologic Finding

Histopathological examination of the obituaries was performed. The infiltration of many acute and chronic inflammatory cells around the necrotic bone tissue can be observed(Fig. 3).

3. Detection of oral bacterial species

Two to nine bacterial species were detected by PCR in each patient (Fig. 4). Two species were gram-positive and eight were gram-negative; all identified bacteria were anaerobes (Table 3). To estimate the relative proportions of each bacterial species, the optical density of PCR bands in each patient was normalized to the optical density of the 16S rRNA gene (Table 4).

The results of a semiquantitative analysis of 10 bacterial species present in bone specimens are shown schematically in (Fig. 5). P. gingivalis was the most frequently detected species (12/15, 80.0%), with strong DNA bands in all patients. T. forsythia was observed observed next most frequently (10/15, 66.7%), followed by T. denticola, S. intermedius, and P. endodontalis (9/15, 60.0%). P. intermedia, E. corrodens, S. intermedius, C. gingivalis, P. endodontalis, and A. israelii were also identified.

As mentioned, patients used different types of bisphosphonates. The detected oral microbial species differed according to the type of bisphosphonate used. In the alendronate group (n = 8), P. gingivalis and T. forsythia were most frequently detected (85.7%). T. denticola, S. intermedius, and P. endodontalis were the most frequently detected in the second band (57.1%). P. intermedia, P. nigrescens, E. corrodens, C. gingivalis, and A. israelii were also identified, although only in small amounts. In the risedronate group (n = 3), P. gingivalis and S. intermedius were most frequently detected (100%). T. forsythia, T. denticola, and P. endodontalis were the most frequently detected in the second band (57.1%). In the ibandronate group (n = 2), P. gingivalis, T. denticola, P. nigrescens, S. intermedius, and P. endodontalis were detected in half of the samples. In the zoledronate group (n = 2), P. gingivalis, T. forsythia, T. denticola, and P. endodontalis were detected in all the samples, whereas P. intermedia, P. nigrescens, and S. intermedius were detected in half of the samples.

Microbial species also differed according to the duration of bisphosphonate intake. In the short-duration group (2–3 years), P. gingivalis was detected in all patients (n = 6, 100%), and T. forsythia, S. intermedius, and P. endodontalis were also frequently detected (83.3%). In the intermediate- duration group (4–5 years, n = 8), P. gingivalis, T. forsythia, and T. denticola were frequently detected (62.5%). In the patient who took alendronate for over 10 years, only P. gingivalis (strong) and T. forsythia (weak) were identified.

Microbial species also differed according to systemic disease. In the osteoporosis-only group (n = 10), P. gingivalis and T. forsythia were most frequently detected (70%), followed by T. denticola, S. intermedius, and P. endodontalis (60%). In the cancer and osteoporosis group (n = 3), P. gingivalis, T. forsythia, T. denticola, and P. endodontalis were detected in all patients (100%), and S. intermedius was also detected (66.7%). P. gingivalis (strong) and T. forsythia (weak) were identified in the patient with diabetes and osteoporosis, and P. gingivalis, P. nigrescens, and S. intermedius were identified in the patient with rheumatoid arthritis and osteoporosis.

Microbial species also differed according to the predisposing dental procedure. In the tooth-extraction group (n = 12), P. gingivalis was most frequently detected (83.3%) and T. forsythia was also frequent (75.0%). P. endodontalis was detected in all the samples from the patients (n = 2) who underwent intra-oral I & D. P. gingivalis, T. denticola, S. intermedius, and P. endodontalis were identified in the patient who had implant surgery.

Ⅳ. DISCUSSION

The pathophysiology of BRONJ remains unclear, however, infection is considered as one of the major risk factors. In this study, several bacterial species were detected in the BRONJ sequestra. Most of them were gram-negative anaerobes associated with periodontitis. Panoramic radiographs provided a view of generalized alveolar bone loss in most patients. Angular bone defects or root furcation defects were also present at the BRONJ site. These radiographic findings provide evidence of chronic periodontitis as a pre-existing inflammatory disease.

Biofilms can form everywhere. Bacteria adhere to surfaces in all nutrient-sufficient aquatic ecosystems (10), and they can attach to the tooth surface, damaged tissue, and implanted medical devices. (14). These sessile bacterial cells usually exist as polymicrobial species mixtures in the oral cavity. In this study, 10 bacterial species were selected for identification by PCR. The selected species are predominant pathogens responsible for oral infectious diseases, such as periodontitis, dento-alveolar abscess, and endodontic infections. According to the PCR results, sequestra from the BRONJ patients were heterogeneously populated with oral bacteria, with two to nine species detected in each patient.

P. gingivalis, T. forsythia, and T. denticola were detected at high levels. They are important gram-negative periodontopathogenic species (15). P. gingivalis is often found in deep periodontal pockets and is a major causative agent of chronic periodontitis. An in vitro study has shown that P. gingivalis can invade gingival fibroblasts and can survive in them in the presence of substantial concentrations of antibiotics (16). Higher levels of T. forsythia are more frequently associated with gingivitis and chronic and aggressive periodontitis than with health (17). T. denticola is a highly proteolytic bacterium associated with the prevalence and severity of periodontal disease (15). According to Socransky et al. (18), P. gingivalis, T. forsythia, and T. denticola together are called the 'red complex'. This complex is found most frequently in deep periodontal pockets, and its presence is directly related to clinical measures of periodontal disease. The results of this study and the radiographic findings from the BRONJ patients explain the close relationship between periodontal disease and BRONJ.

P. endodontalis is associated with periodontitis, endodontic infections, and tooth pulp necrosis. S. intermedius is also a known periodontal pathogen. As mentioned, these bacteria aggregate and cluster. Streptococci, as early colonizers, have been shown to prepare a favorable environment for late colonizers such as Porphyromonas, Prevotella, and Treponema. These late colonizers are more demanding in terms of growth conditions, binding to the established streptococci. After dento-alveolar surgery, exposed bone provides a surface to which oral bacteria can attach and develop into biofilms, resulting in infection.

In this study, all patients were typically symptomatic. They complained of dull or aching pain and had exposed and necrotic bone or fistulae with evidence of infection. Radiographic findings showed an osteolytic bone region in the mandible or maxilla, however it did not extend to the inferior border or beyond the alveolar bone. These cases were all categorized as Stage 2 BRONJ.

To treat these patients, discontinuation of bisphosphonate intake was first recommended. The patients had drug holidays if their systemic conditions permitted. To relieve their symptoms, pain medication and antibiotics were prescribed. Medications reduce the symptoms of pain and infection. Systemic antibiotics usually help remove bacteria at infection sites; however, if the bacteria exist in the form of biofilms, more active treatment should be considered because biofilms are more resistant to antibiotic therapy than free-living bacteria. More aggressive surgical treatment may relieve symptoms and prevent progression of the disease. For Stage 2 BRONJ, a surgical procedure is necessary. Surgical treatment, such as curettage or sequestrectomy, greatly assists in the removal of biofilms from the necrotic bone by reducing bacterial mass.

In summary, gram-negative anaerobic bacterial species associated with periodontitis were frequently detected in BRONJ sequestra analyzed in this study. Most patients had undergone a predisposing dental procedure, such as tooth extraction. In the panoramic view, alveolar bone loss was observed. On the basis of these results, the possible treatments for BRONJ patients are as follows. (ⅰ) The patient must maintain good oral hygiene to prevent exposure to any pre-infectious dental disease. (ⅱ). Regular dental check- ups and appropriate treatment of dental caries or periodontitis may help prevent BRONJ. (ⅲ) Careful tooth extraction and dental care are necessary for patients taking bisphosphonates. (ⅳ) To treat BRONJ patients, the use of effective antibiotics for anaerobic Gram-negative bacteria is recommended. (ⅴ) For patients who have undergone surgical BRONJ therapy, using a chlorhexidine gargle to reduce oral bacteria is effective.

In conclusion, this study shows the importance of oral bacteria in BRONJ. Further studies, including scanning electron microscopy examination of sequestra in BRONJ patients and detection of additional bacterial species by quantitative PCR, may help further clarify the pathogenesis of BRONJ.

Ⅴ. CONCLUSIONS

This study demonstrates the possibility of a close association between oral bacteria and BRONJ. In this study, several oral bacterial species, mainly gram-negative aneaerobes, were detected in the sequestra from BRONJ patients. High levels of P. gingivalis, T. forsythia, and T. denticola were detected in the samples. Oral bacteria may contribute to the pathogenesis of BRONJ and may worsen osteonecrosis of the jaw at the same time. Sequestra infected with oral bacterial species are suspected to be an important risk factor for BRONJ. Thus, the prevention and management of BRONJ may depend on the effective control of oral bacteria. Particularly, at stages 2 or 3, surgical debridement and systemic antibiotics may be necessary and good oral hygiene is likely very important for the control of BRONJ.