Abstract
Bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ) is a serious and challenging complication of chronic BP uptake in patients with osteoporosis who require management of skeletal-related events. The efficiency of adjunctive parathyroid hormone (PTH) injection was evaluated after chronic BP administration that was followed by tooth extraction. BRONJ was not observed in any of the subjects in the control groups, while BRONJ was observed in 66% and 22% of the subjects in the tooth extraction group and the tooth extraction with PTH injection group, respectively. In addition the presence and severity of inflammation was lower in the PTH injected group than in the tooth extraction group, but the difference was not statistically significant ( P > 0.01). In conclusion, the administration of 30 μg/kg/day PTH during a period of 8 weeks had positive effects on the resolution of BRONJ, but further studies are required to verify the effectiveness of PTH in the treatment of BRONJ.
Bisphosphonates (BPs) are drugs that are widely used to prevent the skeletal complications of bone metabolism disorders. BPs inhibit the differentiation and activity of osteoclasts (OCs), which reduces bone resorption without affecting mineralization. In recent years, a strong relationship between osteonecrosis of the jaw and BPs has been reported. This condition was initially perceived as very rare, but after a short period of time case series were published. Studies have attempted to discover various methods for the treatment of BP-related osteonecrosis of the jaw (BRONJ), but a fully secured protocol has not been developed for this rare but challenging complication.
Bone tissue performs the most important functions of the skeleton, providing mechanical support, protective and metabolic functions. The continuity of these functions becomes possible through a balance between osteoblast (OB) and OC activities. Parathyroid hormone (PTH) is the major hormone that regulates bone remodelling. Although the continuous infusion of high doses of PTH has catabolic effects, the intermittent administration of PTH at low doses is anabolic for bone formation. A recombinant human parathyroid hormone (PTH 1–34), teriparatide, is the only US Food and Drug Administration (FDA)-approved anabolic agent for the treatment of osteoporosis. In recent years, teriparatide therapy has been widely used either alone or in combination with antiresorptive agents such as BPs.
The intermittent administration of low-dose PTH for the resolution of BRONJ was first described by Harper and Fung in 2007. The authors concluded that a daily injection of teriparatide for 6 months at a dose of 0.3 mg/kg successfully treated the disease. Since then, the potential role of the intermittent administration of teriparatide in BRONJ resolution has been documented in independent case reports.
Although the exact effects of PTH on BRONJ are not clear, case reports have suggested clinical improvement. On the other hand, teriparatide is contraindicated in patients with skeletal malignancies, hypercalcemia, and bone metastases. In addition there is currently no clear evidence on the optimal duration and dosage of PTH in the treatment of BRONJ, especially in patients with previous diseases. Therefore evaluation of the effects of adjunctive teriparatide injection on tissue healing is critical for the clinical and subclinical setting.
The aim of this study was to investigate the effects of adjunctive PTH on OBs, OCs, fibroblasts (FBs), bone healing, and BRONJ in rats after the administration of BP followed by tooth extraction.
Materials and methods
Animal research ethics committee approval was obtained to conduct this study. Thirty-six female Sprague–Dawley rats were divided into four experimental groups. The dosage and duration of zoledronic acid (ZA) injection were as described in an earlier study by Senel et al., and the dosage and duration of PTH injection were calculated by evaluating the life span and body mass index of the animals. In group I, 0.1 mg/kg sterile saline (SS) was injected intraperitoneally (i.p.) three times a week for an 8-week period. In group II, 0.1 mg/kg ZA (Zometa ® , 4 mg flacon, Novartis, Turkey) was injected in the same manner, at the same frequency, and for the same duration. At the end of this period, the animals were sacrificed. In group III, 0.1 mg/kg ZA was injected i.p. three times a week for an 8-week period. Afterwards, the right first molar teeth were extracted and the animals were allowed to recover for 8 weeks before being sacrificed. In group IV, 0.1 mg/kg ZA was injected i.p. three times a week for an 8-week period. Afterwards, the right first molar teeth were extracted and 30 μg/kg/day PTH analogue (Forsteo ® , teriparatide 250 μg/ml, 3 ml injectable, Lilly, Turkey) was injected subcutaneously for 8 weeks. At the end of this 16-week period, the animals were sacrificed. The hemimandibles were revealed for histomorphometrical analysis. All animals were evaluated at the time of sacrifice for mucosal healing and exposed bone in the oral cavity. The criterion for BRONJ was the persistence of exposed bone at the extraction site at 8 weeks after extraction, confirmed by histological analysis.
Histomorphometrical analysis
The specimens were fixed in 10% buffered formalin solution. After fixation, the bone samples were decalcified in 25% formic acid. The specimens were processed for paraffin embedding, and cut sections of 4-μm thickness were stained with haematoxylin and eosin and Masson’s trichrome stain. All histopathological examinations were performed by the same blinded pathologist. The sections were examined for new bone formation in the fields of tooth extraction. In these areas, OB, OC, and FB activity and surrounding soft tissue inflammation and necrosis were also evaluated. Cellular bone formation with osteoid formation was defined as osteoblastic activity, and bone resorption lacunae with hypernucleated OC defined as osteoclastic activity, as described by Sonis et al.
Stromal inflammatory cell infiltration was evaluated on a 0–2+ scale, as follows: 0 = no inflammatory cell infiltration; 1 = inflammatory cell infiltration in less than 30% of the specimens; 2 = inflammatory cell infiltration in more than 30% of the specimens.
The presence or absence of OB and OC activity and BRONJ were evaluated by histopathological analysis. In the histomorphometrical analysis the numbers of OBs, OCs, and FBs were counted by examining 4-μm thickness cut sections that included the tooth extraction region, an area of 1 mm 2 . Cell counting was performed using the stereologic analysis method. In this study, the images obtained from stained sections were transferred to the computer screen by video camera mounted on a light microscope and analyzed using a stereological workstation (Stereo Investigator 6.0; Microbrightfield Inc., Williston, VT, USA). This workstation includes a personal computer and computer-controlled motorized specimen stage (BioPrecision MAC 5000 controller system) and a light microscope (Leica DM4000 B). OBs, OCs, and FBs were separately counted using a 20× Leica Plan Apo objective, which allowed accurate recognition. All cells were counted according to the unbiased counting rules of optical fractionators. All cells were counted in each sampled disector probe on the sections during stereological analysis. BRONJ was defined as the combined findings of nonviable bone trabeculae with empty lacunae, chronic or acute inflammatory cells, abscesses, and bacteria. The presence of Actinomyces was also evaluated using periodic acid–Schiff (PAS) staining.
Statistical analysis
The Fisher’s exact test was used for analysis of the categorical variables. The Kruskal–Wallis test and Mann–Whitney U -test with Bonferroni correction were used for the intergroup analysis. The categorical variables are presented as percentages (%), and the other variables are presented as the mean ± standard deviation. Differences were considered significant at P < 0.01. SPSS 17.0 was used for the analysis (SSPS Inc., Chicago, IL, USA).
Results
Activity and number of osteoblasts
A statistically significant difference was observed between the groups for OB number ( P < 0.01). An increased number of OBs was noted in groups III and IV compared with groups I and II ( P < 0.01). OB activity was higher in groups I and II than in groups III and IV, but the difference was not statistically significant ( P > 0.01). Intergroup analysis indicated no significant differences between groups I and II ( P > 0.01), and in addition, no significant differences were noted between groups III and IV with regard to OB number and activity ( Table 1 ).
| Group | OB Min | OB Max | OB Mean | SD | Presence of OB activity | Absence of OB activity | |
|---|---|---|---|---|---|---|---|
| I | SS | 5.15 | 64.99 | 30.22 a | ±20.32 | 100% | 0% |
| II | ZA | 14.35 | 160.28 | 84.14 a | ±66.37 | 100% | 0% |
| III | ZA + Ext | 87.78 | 398.37 | 215.47 a | ±92.86 | 55.5% | 44.4% |
| IV | ZA + Ext + PTH | 149.57 | 377.19 | 279.23 a | ±74.29 | 77.7% | 22.2% |
a An increased number of OBs was noted in groups III and IV compared with groups I and II ( P < 0.01).
Activity and number of osteoclasts
A statistically significant difference was observed between the groups for OC number ( P < 0.01). An increased number of OCs was noted in groups III and IV compared with groups I and II ( P < 0.01). OC activity was higher in groups I and II than in groups III and IV, but the difference was not statistically significant ( P > 0.01). Intergroup analysis indicated no significant differences between groups I and II ( P > 0.01), and in addition, no significant differences were noted between groups III and IV with regard to OC number and activity ( Table 2 ).
| Group | OC Min | OC Max | OC Mean | SD | Presence of OC activity | Absence of OC activity | |
|---|---|---|---|---|---|---|---|
| I | SS | 0.98 | 1.08 | 1.00 a | ±0.02 | 100% | 0% |
| II | ZA | 0.68 | 2.11 | 1.26 a | ±0.47 | 100% | 0% |
| III | ZA + Ext | 0.51 | 12.89 | 4.32 a | ±4.42 | 55.5% | 44.4% |
| IV | ZA + Ext + PTH | 1.07 | 12.09 | 4.34 a | ±3.65 | 66.6% | 33.3% |
a An increased number of OCs was noted in groups III and IV compared with groups I and II ( P < 0.01).
Fibroblast numbers
A statistically significant difference was observed between the groups for FB number ( P < 0.01). An increased number of FBs was noted in groups III and IV compared with groups I and II ( P < 0.01). Intergroup analysis indicated no significant differences between groups I and II ( P > 0.01), and in addition, no significant differences were noted between groups III and IV with regard to FB number ( Table 3 ).
| Group | FB Min | FB Max | FB Mean | SD | |
|---|---|---|---|---|---|
| I | SS | 5.15 | 64.99 | 30.22 a | ±20.32 |
| II | ZA | 14.35 | 160.28 | 84.14 a | ±66.37 |
| III | ZA + Ext | 87.78 | 398.37 | 215.47 a | ±92.86 |
| IV | ZA + Ext + PTH | 149.57 | 377.19 | 279.23 a | ±74.29 |
a An increased number of FBs was noted in groups III and IV compared with groups I and II ( P < 0.01).
BRONJ
Although no mucosal ulceration was noted in groups I and II, the mucosa overlying the extraction sites was ulcerated and unhealed exposed bone with gross necrosis was noted in group III. On the other hand complete epithelialization of the extraction sites was noted for 77% of rats in group IV by microscopic and macroscopic evaluation. BRONJ was not observed in any of the subjects in groups I and II, but BRONJ was observed in 66% of the subjects in group III and 22% of the subjects in group IV. A statistically significant degree of BRONJ was observed in group III compared with groups I and II ( P < 0.01). In contrast, no significant difference was observed in group IV compared with groups I and II ( P > 0.01). Intergroup analysis indicated no significant differences between groups I and II ( P > 0.01), and in addition, no significant differences were noted between groups III and IV with regard to BRONJ ( P > 0.01).
Inflammation
A statistically significant difference was observed between the groups for the presence and severity of inflammation (PSI) ( P < 0.01). Intergroup analysis indicated no significant difference between groups I and II ( P > 0.01), but statistically significant differences were observed in groups III and IV compared with group I ( P < 0.01). PSI was significantly higher in group III than in group II, but no significant difference was observed between groups II and IV. Although PSI was lower in group IV than in group III, the difference was not statistically significant ( P > 0.01). In addition, the PSI levels were similar in groups I, II, and IV ( P > 0.01) ( Table 4 ). In group IV, new bone formation was observed with minimal inflammation and recovery areas surrounding the foci of necrosis, whereas in group III, foci of abscesses, excessive inflammation, and BRONJ areas were clearly observed ( Fig. 1 ).
