Abstract
There is evidence for a link between the use of systemic bisphosphonates and osteonecrosis of the jaw (ONJ). This condition has the appearance of chronic osteomyelitis, and antibiotics prevent the development of ONJ in animal models. Clinically, ONJ can sometimes be treated successfully by mucoperiosteal coverage. If ONJ is indeed primarily caused by bacterial infection, immediate coverage of the extraction alveolus might reduce the risk of ONJ developing in risk patients. Therefore, we studied whether immediate mucoperiosteal coverage after tooth extraction could prevent the development of ONJ in a rat model. Thirty rats were randomly allocated to three groups (10 in each): (1) group I (controls): extraction, no drug treatment; (2) group II (non-coverage): extraction, dexamethasone plus alendronate; (3) group III (coverage): extraction, dexamethasone plus alendronate, plus coverage with a mucoperiosteal flap. Rats were examined for macroscopic ONJ-like wounds after 2 weeks. All animals in the non-coverage group developed large ONJ-like changes. The coverage and control groups showed an intact overlying mucosa in all rats. Findings were confirmed with histology. Bisphosphonates and dexamethasone caused ONJ-like lesions after tooth extraction in a rat model. This was prevented by immediate mucoperiosteal coverage. The risk of ONJ in patients using bisphosphonates might be reduced by mucoperiosteal coverage after tooth extraction.
Bisphosphonates are antiresorptive drugs that act specifically on osteoclasts, thereby maintaining bone density and strength. Bisphosphonates are used in many clinical settings, including the prevention and treatment of primary and secondary osteoporosis, Paget’s disease of bone, hypercalcaemia, multiple myeloma, and osteolysis associated with bone metastases of malignant tumours.
Among patients with cancer, intravenous bisphosphonate therapy has been linked to osteonecrosis of the jaw (ONJ), a serious adverse event that can negatively affect the patient’s quality of life. This condition is defined as non-healing, exposed bone for more than 8 weeks in a patient receiving bisphosphonates and without a history of local radiation therapy. For patients on bisphosphonates, oral surgical procedures exposing bone to the oral cavity increase the risk of ONJ. The calculated frequency of ONJ after tooth extraction in risk patients ranges from 7% to 9%.
As jaw bone containing bisphosphonates can only be resorbed slowly, it is conceivable that bacterially contaminated bone cannot be removed fast enough to prevent the development of chronic osteomyelitis. During surgery or tooth extraction, bone exposure might open the door to bacterial invasion, and microbial contamination seems to play an important role in maintaining the osteomyelitic wounds. Recently, it has been shown that antibiotics can be useful to prevent the development of ONJ in a rat model. If ONJ is indeed primarily caused by bacterial infection, immediate coverage of the extraction alveolus after tooth extraction might reduce the risk of ONJ developing in risk patients. Mucosal coverage of extracted alveoli has been undertaken for treatment purposes in the early stages of ONJ with good results.
Although bisphosphonate therapy may cause ONJ, there is a clear awareness in clinical practice that the administration of bisphosphonates is required for the optimal treatment of bone metastases from solid tumours. The treatment of ONJ has so far been conservative. In view of this, there is a consensus about the difficulty of treating bisphosphonate-associated ONJ and the importance of prevention. Recently published recommendations suggest hygiene control and dental evaluation and treatment before initiating bisphosphonate therapy, similar to recommendations for patients requiring radiotherapy involving the jaws. In severe cases, however, dental surgical procedures may be necessary despite the increased risk. In these cases, the infectious nature of ONJ suggests that surgical coverage of the exposed bone might prevent its development.
The present study was designed to evaluate the role of mucoperiosteal coverage for the prevention of ONJ following dental surgery associated with bisphosphonate therapy in a rat model.
Materials and methods
Animals and study design
Male Sprague-Dawley rats, 10 weeks of age, were used in this study (body weight range 364–420 g). The animals were kept four per cage with free access to water and rat-chow. The study was approved by the regional ethics board for animal research, and institutional guidelines for the care and treatment of laboratory animals were followed. Animals were acclimatized to the study environment 1 week before the start of the study.
Thirty rats were randomly allocated to three groups (10 in each), by use of a lottery ( http://www.graphpad.com/quickcalcs/randomize1.cfm ). All rats underwent extraction of the left maxillary first molar and received the following treatments: (1) group I (controls): extraction, no drug treatment; (2) group II (non-coverage): extraction, alendronate (200 μg/kg) subcutaneously once daily for 14 days, starting the day of surgery, and dexamethasone (0.5 mg/kg) subcutaneously once daily for four consecutive days starting 2 days prior to surgery; (3) group III (coverage): extraction plus mucoperiosteal flap, alendronate plus dexamethasone as above.
The animals were anaesthetized and inspected after 1, 2, and 3 weeks. To reduce animal suffering, those with ONJ-like lesions were euthanized using carbon dioxide after 2 weeks. The others were kept alive until 3 weeks, to ensure that no wounds developed.
Experimental parameters
The main hypothesis was that the mucoperiosteal coverage of newly exposed bone would prevent the development of ONJ-like lesions under circumstances where they would otherwise arise. Therefore, we noted any presence of ONJ-like lesions by clinical observation, and this was confirmed by histology.
Surgery
The rats were anaesthetized with dexmedetomidine (Dexdomitor 0.24 mg/kg) and ketamine (Ketalar 43 mg/kg). Buprenorphine (Temgesic 0.03 mg/kg, Reckitt Benckiser Pharmaceuticals, UK) was administered preoperatively to reduce pain. Buprenorphine was also given every 12 h for 2 days after surgery. No antibiotic or other medication was given. All surgery and evaluation was performed with concealment of treatment allocation. Under general anaesthesia, the left maxillary first molar was removed using a sharpened dental explorer and a luxation technique. A triangular part of the gingiva (2 mm × 2 mm) with its base at the anterior margin of the extraction defect was removed in order to increase the likelihood of ONJ-like changes ( Fig. 1 ).
In the coverage group, a buccal mucoperiosteal sliding flap was created to cover the extraction defect. Two divergent incisions through the mucosa and periosteum were made buccally to the extraction site ( Fig. 2 a ). Thus, a broad-based flap consisting of periosteum and mucosa was created ( Fig. 2 ). The lengthening of the flap was effected by making an incision in the periosteum at the base of the flap ( Fig. 2 b). The flap could thereafter be sutured to the palatal margin of the gingival defect without tension. The surgeon was blinded to the drug treatment allocation.
Clinical assessment of wound healing
Wound healing was assessed macroscopically, and the surgical field was photographed 7, 14, and 21 days after the operation. Mucosal healing was classified into two groups: either complete healing with covering normal mucosa, or mucosal ulceration. The evaluator was blinded to the treatment.
Histological assessment
The maxilla of each animal was excised, divided at the midline, and fixated in 4% formaldehyde solution for 48 h. The left maxilla was then decalcified using formic acid, embedded in paraffin, sectioned, and stained with haematoxylin and eosin. Sectioning was performed in the sagittal plane, each section showing the remaining second molar, the extraction site, and its frontal mucosal area. Several sections from each specimen were then evaluated for epithelial discontinuity, presence of bone sequestra, and accumulation of inflammatory cells, by a blinded examiner.
Statistics
Statistical evaluation was done by χ 2 test for all groups, followed by Fisher’s exact test for pairwise intergroup comparisons.
Results
Initially, the animals tolerated anaesthesia and tooth extraction well. However, at euthanasia, the non-coverage group had lost weight, whereas the other groups had gained weight ( Table 1 ).
| Control (group I) | Non-coverage (group II) * | Coverage (group III) | |
|---|---|---|---|
| Start | 388 (16) | 390 (11) | 387 (18) |
| Day 5 | – | 344 (14) | 350 (14) |
| Diff % | – | −11 (2.6) | −9.5 (3.4) |
| Euthanasia | 432 (22) | 354 (34) | 413 (20) |
| Diff % | 11 (1.8) | −9.0 (9.1) | 6.8 (3.9) |
There were no exclusions and no loss to follow-up. Group II (non-coverage) was significantly different from the other groups for all measured outcome variables ( Table 2 ; P < 0.001 for all).
| Group | Bisphosphonate + dexamethasone | Flap coverage | Macroscopic ONJ-like lesions ( n ) | Epithelial continuity ( n ) * | Inflammatory cells * | Sequestra * | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Yes | No | Yes | No | None | Some | Many | None | Some | Many | |||
| Control (group I) | No | No | 0 | 10 | 9 | 0 | 7 | 2 | 0 | 6 | 3 | 0 |
| Non-coverage (group II) | Yes | No | 10 | 0 | 0 | 10 | 0 | 0 | 10 | 0 | 4 | 6 |
| Coverage (group III) | Yes | Yes | 0 | 10 | 9 | 0 | 7 | 2 | 0 | 7 | 2 | 0 |
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