Abstract
The microbial aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ) remains undefined. This study investigated the oral microbiota and socket healing after zoledronic acid (ZA) and dexamethasone (DX) administration. Fourteen rats assigned randomly to experimental ( n = 8) and control ( n = 6) groups were injected with ZA+DX or saline, respectively, for 3 weeks prior to and 9 weeks after the extraction of left first upper and lower molars. Whole genomic DNA probes of 38 bacterial species and five Candida species were hybridized to DNA extracted from biofilm samples on exposed bone and adjacent teeth. Only experimental rats exhibited exposed bone at euthanasia. All BRONJ-like lesions were colonized by Staphylococcus pasteuri , Streptococcus parasanguinis , and Streptococcus mitis . A significant correlation was observed between the mean proportions of species colonizing BRONJ-like lesions and the teeth of experimental rats ( r = 0.818, P < 0.001). Significant differences were seen in several species colonizing the teeth of control rats compared to experimental rats ( P < 0.05). Micro-computed tomography analyses revealed higher residual bone in mandibular ( P = 0.001) and maxillary ( P = 0.108) tooth sockets of experimental rats. BRONJ-like lesions were colonized mainly by non-pathogenic bacteria. ZA+DX administered to rats at doses equivalent to those given to cancer patients resulted in changes to the oral biofilm and impaired bone healing following tooth extraction.
The use of high doses of anti-resorptive agents such as bisphosphonates and anti-angiogenic agents like sunitinib to treat cancer has been linked to osteonecrosis. Osteonecrosis of the jaw is a serious clinical problem associated with pain, infection, and bone loss. Bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been defined as the presence of exposed, unhealed bone for more than 8 weeks following surgery in the maxillofacial region of patients treated with bisphosphonates, but with no history of radiation to the head and neck region. Bisphosphonates are a class of drugs originally developed to prevent the bone loss caused by excessive osteoclast activity in post-menopausal women. These drugs are currently prescribed to decrease skeletal complications in the management of metastases from solid tumours such as breast and prostate cancers, as well as in the treatment of metabolic bone disease like osteoporosis.
Bacterial infection has been suggested as a contributing factor in the development of BRONJ, although a direct cause–effect relationship has not been demonstrated. A recent study conducted on BRONJ patients showed that the affected area was heavily colonized by bacteria, with Streptococcus , Eubacterium , and Pseudoramibacter being the most prevalent genera. Preclinical studies using animal models of BRONJ-like disease have not investigated the bacteria colonizing the teeth or the area of exposed bone. Previous work by Mawardi et al. showed that infection of the extraction sockets with Fusobacterium nucleatum in mice treated with high-dose bisphosphonates resulted in delayed wound healing, leaving exposed bone. The BRONJ-like lesions were proposed to arise from reduced proliferation and increased death of gingival fibroblasts, induced by the combination of pamidronate administration and infection with F. nucleatum .
The goal of the present study was to characterize the profile of bacteria colonizing the exposed bone and adjacent teeth in a rat model of BRONJ-like disease and also to assess the impact of the bacteria on exposed bone.
Materials and methods
Rats with BRONJ-like disease
The Animal Use Protocol was approved by the necessary authorities. A total of 14 retired breeder female Sprague-Dawley rats (4–6 months old) were divided randomly into control ( n = 6) and experimental ( n = 8) groups. Control rats received no medication and experimental rats received zoledronic acid (ZA; 125 μg/kg twice a week) and dexamethasone (DX; 5 mg/kg once a week) for a total of 12 weeks. In a previous work by the present investigators, rats exposed to this drug combination consistently exhibited BRONJ-like lesions at the time of euthanasia, at 4 weeks postoperative. The ZA and DX doses were converted from doses of ZA (8 mg/person/3 weeks) and DX (55 mg/person/week) used for humans and were within the limits described in the literature. Doses were converted according to the National Institutes of Health (NIH) guidelines. After 3 weeks, the control and experimental rats were anesthetized and their left maxillary and mandibular first molars extracted (EXO). Drug administration was continued for 9 additional weeks and all control and experimental rats were euthanized using CO 2 inhalation at 12 weeks.
Immediately after CO 2 inhalation, samples of biofilm were collected from sites of exposed bone and from the supragingival region of the adjacent teeth by rubbing with a microbrush (Microbrush International, Grafton, WI, USA) for 30 s until saturation (6 μl), as described previously. The use of a microbrush has been shown to be a suitable method for oral microbial biofilm collection. Given the possible inaccuracy in measuring the weight of the samples, standard-sized microbrush tips that are capable of absorbing a volume of about 6 μl were used. Individual specimens were placed in microtubes containing 150 μl of TE (10 mM Tris–HCl, 1 mM ethylenediaminetetraacetic acid (EDTA) pH 7.6) to suspend the bacteria, and 150 μl of 0.5 M sodium hydroxide was added to lyse the cells and expose DNA. Samples were stored at 4 °C until processing by DNA checkerboard hybridization.
DNA checkerboard hybridization
Hybridization to bacterial and fungal DNA probes was performed using the checkerboard method, as described previously. Hybridization probes were prepared from whole genomic DNA of 38 bacterial species and five Candida species ( Table 1 ). Probes were selected based on the relevance of the species to human oral health and disease, and their specificity and sensitivity previously optimized for the detection of 10 4 cells. Whole genomic DNA extracted from the biofilm samples as described above was denatured, precipitated, and blotted onto a hybridization membrane (Hybond N+; Amersham Biosciences, Buckinghamshire, UK) using a MiniSlot 30 apparatus (Immunetics, Cambridge, MA, USA). Defined amounts of genomic DNA corresponding to 10 5 and 10 6 cells of each target species were mixed in a single tube, denatured, precipitated, and applied to the membranes as reference samples. All samples were vacuum-fixed onto the hybridization membrane, which was then baked for 2 h at 80 °C. Membranes were pre-hybridized using an oven shaker (Amersham Biosciences) to control for temperature and humidity. Labelled probes were then introduced using a Miniblotter 45 device (Immunetics) and the membranes hybridized overnight at controlled temperature and humidity under gentle agitation. After washing, hybridization signals were detected by chemiluminescence using CDP-Star reagent (GE Healthcare, Amersham, Buckinghamshire, UK), and exposed to ECL Hyperfilm-MP for 60 min (GE Healthcare). The hyperfilm images were digitized and quantified using TotalLab Quant analysis software (TotalLab Life Science Analysis Essentials, Newcastle upon Tyne, UK).
| Species | Reference | |
|---|---|---|
| Aggregatibacter actinomycetemcomitans a | ATCC | 29523 |
| Aggregatibacter actinomycetemcomitans b | ATCC | 29522 |
| Bacteroides fragilis | ATCC | 25285 |
| Campylobacter rectus | ATCC | 33238 |
| Candida albicans | ATCC | 10231 |
| Candida dubliniensis | ATCC | MYA 646 |
| Candida glabrata | ATCC | 90030 |
| Candida krusei | ATCC | 6258 |
| Candida tropicalis | ATCC | 750 |
| Capnocytophaga gingivalis | ATCC | 33624 |
| Eikenella corrodens | ATCC | 23834 |
| Enterococcus faecalis | ATCC | 51299 |
| Escherichia coli | ATCC | 10798 |
| Fusobacterium nucleatum | ATCC | 25586 |
| Fusobacterium periodonticum | ATCC | 33693 |
| Klebsiella pneumoniae | ATCC | 700721 |
| Lactobacillus casei | ATCC | 393 |
| Mycoplasma salivarium | ATCC | 23064 |
| Neisseria mucosa | ATCC | 25996 |
| Parvimonas micra | ATCC | 33270 |
| Peptostreptococcus anaerobius | ATCC | 49031 |
| Porphyromonas endodontalis | ATCC | 35406 |
| Porphyromonas gingivalis | ATCC | 33277 |
| Prevotella intermedia | ATCC | 25611 |
| Prevotella melaninogenica | ATCC | 25845 |
| Prevotella nigrescens | ATCC | 33563 |
| Pseudomonas aeruginosa | ATCC | 27853 |
| Pseudomonas putida | ATCC | 12633 |
| Solobacterium moorei | CCUG | 39336 |
| Staphylococcus aureus | ATCC | 25923 |
| Staphylococcus pasteuri | ATCC | 51129 |
| Streptococcus constellatus | ATCC | 27823 |
| Streptococcus gordonii | ATCC | 10558 |
| Streptococcus mitis | ATCC | 49456 |
| Streptococcus mutans | ATCC | 25175 |
| Streptococcus oralis | ATCC | 35037 |
| Streptococcus parasanguinis | ATCC | 15911 |
| Streptococcus salivarius | ATCC | 25975 |
| Streptococcus sanguinis | ATCC | 10556 |
| Streptococcus sobrinus | ATCC | 27352 |
| Tannerella forsythia | ATCC | 43037 |
| Treponema denticola | ATCC | 35405 |
| Veillonella parvula | ATCC | 10790 |
Micro-computed tomography (micro-CT) analysis of rat jaw bones
After collection of the biofilm samples, the EXO (left) and non-EXO (right) sides of both the upper and lower jaws were removed, cleaned of muscle attachments and soft tissues, and processed essentially as described previously. Specimens fixed overnight at 4 °C in 4% paraformaldehyde were rinsed thoroughly in three changes of sterile phosphate buffered saline (PBS) and stored at 4 °C. Micro-CT scans were captured on a Bruker Skyscan 1172 instrument (Bruker, Kontich, Belgium) at 8.9-μ resolution with 550 ms of exposure time, 59 kV, and 167 mA, adjusted to allow maximum differentiation between mineralized and non-mineralized tissues, and using a 0.5-mm thickness aluminium filter. The average of seven measurements (in millimetres) separated by 0.5 mm spanning the extraction sites in each jaw was taken to calculate the height of residual exposed bone. The region of interest (ROI) in which to capture quantitative data was defined as described previously. Briefly, for the non-EXO side, the ROI extended from the mesial aspect of the second molar to the mesial aspect of the first molar, including both the cortical and trabecular bone and excluding the first molar itself. For the mandible, the ROI extended towards the lower border of the mandible to cover the bone up to the beginning of the incisor enamel, which provided a clear anatomical reference. For the EXO side, the ROI was extended 3.0 mm from the mesial surface of the second molar to cover the EXO area including the separated bone fragments (sequestra) and excluding the alveolar ridge. Bone volume to tissue volume (BV/TV) fractions of the non-EXO sides and the number, volume, and surface area of the isolated bone fragments at the extraction sites were quantified using Bruker Skyscan CTAn software.
Following micro-CT scanning, non-decalcified samples were embedded in polymethylmethacrylate (PMMA), sectioned serially (5 μ), and dried for 4 days before staining for bone mineral (Von Kossa).
Statistical analyses
To adjust for any potential variation in the volume or weight of the samples, mean proportions were used; these were calculated by averaging the percentages of target species detected within each sample. The Mann–Whitney U -test was employed for between-group comparisons of bacterial proportions, bone height, and micro-CT quantitative data. Non-parametric Spearman correlation was used to assess the within-group relationship of the proportions of species colonizing the BRONJ-like lesion and the teeth. All statistical analyses were performed using IBM SPSS Statistics version 19.0 software (IBM Corp., Armonk, NY, USA), with a significance level of <0.05.
Results
Microbiological results
Of the 43 different microbial species investigated, a total of 39 were detected on the exposed bone and 26 on the adjacent teeth of the experimental rats treated with ZA+DX, and 40 were detected on the teeth of control rats that received no treatment. Staphylococcus pasteuri , Streptococcus parasanguinis , Streptococcus mitis , Streptococcus gordonii , and Streptococcus oralis species had the highest mean proportions. S. pasteuri , S. parasanguinis , and S. mitis were detected on all exposed bone in the experimental group. No detectable hybridization signals were seen for Bacteroides fragilis or Campylobacter rectus in any location. Targeted species and their frequencies of occurrence per collection site are shown in Table 2 .
| Control | ZA+DX | |
|---|---|---|
| Teeth ( n = 6) | Teeth ( n = 8) | Exposed bone ( n = 8) |
| P. gingivalis (6) | S. oralis (8) | S. pasteuri (8) |
| M. salivarium (6) | S. mitis (7) | S. parasanguinis (8) |
| S. mutans (6) | S. gordonii (7) | S. mitis (8) |
| S. mitis (6) | S. pasteuri (8) | S. gordonii (7) |
| S. gordonii (6) | S. mutans (8) | S. oralis (7) |
| S. oralis (6) | S. parasanguinis (8) | S. salivarius (7) |
| E. faecalis (3) | S. moorei (8) | S. mutans (7) |
| T. denticola (6) | S. sobrinus (6) | S. sanguinis (7) |
| S. constellatus (6) | S. sanguinis (6) | P. putida (6) |
| L. casei (4) | M. salivarium (5) | C. glabrata (7) |
| S. moorei (6) | T. denticola (4) | S. moorei (7) |
| V. parvula (6) | T. forsythia (6) | S. sobrinus (6) |
| S. aureus (6) | V. parvula (6) | C. tropicalis (7) |
| P. putida (6) | S. salivarius (7) | P. micra (5) |
| C. tropicalis (6) | C. tropicalis (5) | S. aureus (6) |
| S. sanguinis (6) | C. glabrata (6) | F. periodonticum (5) |
| T. forsythia (6) | C. krusei (6) | V. parvula (6) |
| P. nigrescens (5) | S. aureus (3) | P. nigrescens (5) |
| S. pasteuri (6) | P. putida (3) | C. dubliniensis (7) |
| P. intermedia (6) | C. albicans (5) | E. corrodens (6) |
| S. sobrinus (6) | C. dubliniensis (5) | C. krusei (7) |
| P. micra (5) | L. casei (2) | E. faecalis (4) |
| S. parasanguinis (5) | S. constellatus (2) | S. constellatus (5) |
| P. melaninogenica (4) | E. corrodens (1) | K. pneumoniae (6) |
| S. salivarius (6) | P. micra (1) | T. denticola (3) |
| C. albicans (6) | P. nigrescens (1) | M. salivarium (3) |
| C. glabrata (6) | F. periodonticum (0) | C. albicans (6) |
| C. krusei (6) | E. faecalis (0) | P. intermedia (4) |
| C. dubliniensis (6) | K. pneumoniae (0) | T. forsythia (4) |
| C. gingivalis (3) | P. intermedia (0) | P. gingivalis (3) |
| K. pneumoniae (3) | P. gingivalis (0) | L. casei (3) |
| E. corrodens (2) | C. gingivalis (0) | C. gingivalis (3) |
| F. nucleatum (3) | N. mucosa (0) | N. mucosa (4) |
| P. endodontalis (4) | E. coli (0) | E. coli (4) |
| Aa_b (2) | P. aeruginosa (0) | P. aeruginosa (4) |
| P. aeruginosa (3) | Aa_a (0) | Aa_a (2) |
| Aa_a (1) | P. melaninogenica (0) | P. melaninogenica (2) |
| F. periodonticum (3) | P. endodontalis (0) | P. endodontalis (2) |
| P. anaerobius (3) | F. nucleatum (0) | F. nucleatum (1) |
| N. mucosa (1) | P. anaerobius (0) | P. anaerobius (0) |
| E. coli (0) | Aa_b (0) | Aa_b (0) |
| B. fragilis (0) | B. fragilis (0) | B. fragilis (0) |
| C. rectus (0) | C. rectus (0) | C. rectus (0) |
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