Thirty years ago, Gotcher and Jee(30)
used the rice rat model of periodontitis to determine whether dichloromethylene diphosphonate (Cl2
MDP), a non-nitrogen containing BP, could retard or prevent the bone loss associated with periodontal disease. They observed that Cl2
MDP indeed prevented alveolar bone loss. However, and perhaps relevant to the pathophysiology of ONJ, they found an abnormal amount, location, and morphologic pattern of the alveolar bone in mandibles of rats that were treated for 18 wks. The authors(30)
referred to these lesions as “areas of alveolar bone protruded and exposed in the oral cavity with portions that were devoid of bone cells and devitalized”.
This description attracted our attention since it has similarities to the ONJ lesions described in N-BP-treated patients. Nevertheless, the study(30)
did not provide conclusive evidence to support the existence of ONJ, leaving open the possibility to infer that a different bone disorder occurred. Inspired by these observations, we reexamined the periodontitis model in rice rats, previously described,(22-24,27,29)
with methodologies now commonly used in periodontal research, including morphometry, histomorphometry, immunocytochemistry, histochemistry, and microCT techniques.(25,47)
We next performed the present study to determine the effects of two N-BPs now approved for the treatment of osteoporosis and, in the latter case, for metastatic cancer, ALN and ZOL, on: the progression of periodontitis, the integrity of alveolar bone, bone resorption and formation, vascularity, and osteocyte viability.
To our knowledge, very few studies(30,48)
have considered periodontal disease as a comorbid factor in the development of an animal model for ONJ. Taking this risk factor into account improves the potential relevance of an animal model of ONJ, since clinically and radiographically evident periodontitis has been identified as one of the most important risk factors for the development of ONJ in humans.(1,4,49)
Our study shows that about 40% of the rice rats (males and females) treated with oncologic doses of ZOL for 18 wks developed gross mandibular lesions that appear to resemble those observed in ONJ patients. In contrast, only 20% of these animals developed ONJ-like lesions in maxillae. This observation represents an interesting epizootiologic aspect of the model, since it is well established that the incidence of ONJ in humans is two-fold greater in the mandible than in the maxilla.(1,5,17)
Similar incidence of ONJ-like lesions, including osteonecrosis (47%) and exposed bone (21%), were found and elegantly illustrated by Aghaloo et al(48,49)
in maxillae of ZOL-treated rats with ligature-induced periodontitis. Our study also shows that none of the rats treated with ALN developed ONJ-like lesions. Our findings are in agreement with epidemiological data in humans showing a high incidence (6-10%) of ONJ lesions in N-BP-treated cancer patients(3,18,19)
and a very low incidence (~1/10,000) in postmenopausal women undergoing treatment for osteoporosis.(4,5,19,20)
This study did not investigate the effects of N-BPs on rice rats that were fed a standard diet instead of the H-SC diet. Data obtained from such an experiment, in which rice rats would have little or no periodontitis, could contribute to determining whether predisposing factors, in this particular case, moderate/severe periodontitis, are sine qua non
conditions for the development of ONJ. This study also did not investigate the effect of introducing N-BPs into rice rats with periodontitis that had been established by 18wks administration of the H-SC diet. Data obtained from such an experiment, would more closely mimic the situation in adult humans with established periodontitis who begin to take N-BPs as oncologic or osteoporosis therapy.
Remarkably, we observed that rice rats that had mandibular histologic lesions with an inflammatory score of 5 (ONJ-like lesions), were those that displayed evident serious gross lesions including erosion or ulceration of the gingiva, exposed alveolar bone, molar furcation, and loss of alveolar bone supporting the teeth. This correspondence suggests that in our model, gross examination of mandibles can be used similar to a clinical examination, to study the progression of ONJ or to test different treatment modalities for ONJ in vivo before euthanizing the animals for ex vivo tissue analysis.
We further observed that ONJ-like lesions were not only present in rats that received HD-ZOL for 18 wks but also in those treated for 24 wks. This is important, because it suggests that ONJ-like lesions in this model were not transient, since they likely persisted for at least 6 wks after their identification at 18 wks of treatment. Although this time period does not fully comply with the most recent definitions of N-BP-associated ONJ,(15,16)
it could represent a comparable persistence for a rodent animal model.
The definition of ONJ only specifies “an area of exposed bone in the maxillofacial region.” However, numerous clinical reports in humans have described additional bone lesions present in ONJ patients, including osteolysis, osteosclerosis, presence of woven bone, honeycomb-like appearance of affected areas of jaw bones, and pathologic fractures.(4,50-57)
Importantly, we observed some of these lesions in our model. Rice rats treated with oncologic doses of ZOL for 18-24 wks displayed, concomitantly with or without exposed bone, extensive areas of osteolysis, and alveolar bone areas with a honey comb-like appearance as evidenced by microCT. The histologic and histomorphometric analyses of these honey comb-like areas showed a correspondence to alveolar bone areas that were replaced by woven bone, periodontal ligament and/or fibrous tissue, and were sometimes mineralized. Taken together, these data suggest that our model can reproduce not only the presence of exposed necrotic bone in the oral cavity, but also other pathological bone lesions frequently observed in patients with ONJ.
The clinical staging system updated on the 2009 AAOMS expanded abbreviation guidelines (16)
has served to more accurately categorize patients with ONJ. We attempted to use this system for ONJ-like lesions found in rice rats. We observed that HD-ZOL administered for 18 wks induced the development of ONJ-like lesions that could be comparable to a stage 1/2, since the rats did not lose weight (as an indicator of well-being and lack of pain or distress), but had periodontal lesions frequently accompanied by infection. On the other hand, ONJ-like lesions present in rice rats treated with HD-ZOL for 24 wks could be comparable to those in stage 2/3, because exposed necrotic bone was frequently accompanied by infection, osteolytic lesions, and the loss of a significant amount of body weight (data not shown). The development of a staging system for the rice rat model would be highly desirable in order to advance the development of early diagnostic protocols and treatments, particularly in the early stages of the disease (phase 0), when no clinical evidence of necrotic bone but nonspecific clinical findings and symptoms appear to be present in humans.
In contrast to the consistent and potent antiresorptive effect of ALN and ZOL observed at the distal femoral metaphyses, different responses were seen in jaw bones. Responses were associated with the degree of histological lesions found at these locations. When periodontal lesions were absent or present to a low degree, as found in maxillae at 18 wks and in mandibles at 12 wks, HD-ZOL exerted a potent antiresorptive effect. In contrast, the antiresorptive effect of HD-ZOL was lost when a high degree of damage, destruction and commensal bacterial (Actinomyces
-like) growth were present. It is well established that oral bacterial infection, particularly with Actinomyces sp
., is a common finding in patients with ONJ.(52,58-62)
When this takes place, microorganisms can stimulate bone resorption by inducing cytokines and/or RANKL.(63-65)
Our findings are in agreement with what Reid and Cornish(52)
defined as the “ONJ paradox”, where aggressive local osteolysis could be present in patients treated with antiresorptives as a result of infection-induced bone resorption.
We also observed that rice rats treated with oncologic doses of ZOL for 18 wks have areas of exposed alveolar bone that contain empty osteocyte lacunae and/or osteolytic lesions. In line with these findings, we also observed decreased reactivity to LDH in osteocyte lacunae in males and an increased percentage of osteocyte apoptosis in female rice rats at this time point. Taken together, these data suggest that indeed, HD-ZOL is associated with increased levels of apoptosis/necrosis in alveolar bone osteocytes of rats with induced periodontitis. There are some differences in the assessments of these tests. The assessment of LDH negative osteocyte lacunae considers not only the presence of unreactive osteocytes (necrotic), but also empty osteocyte lacunae. Therefore, the presence of cellular remnants and absence of osteocytes in lacunae are also expressions of necrotic cells. In contrast, the percentage of TUNEL positive osteocytes, accounted only for those cells that have fragmented DNA, a characteristic of both apoptotic as well as necrotic cells, from the total of DAPI positive osteocytes in the ROI. Therefore, empty osteocyte lacunae were excluded from this calculation. The presence of empty osteocyte lacunae is a characteristic of the alveolar bone described in ONJ patients.(19,59,62)
Taken together, these findings suggest that the LDH assay would be a better indicator of the index of osteocyte necrosis in the alveolar bone compared to TUNEL staining, which underestimates the real number of necrotic/apoptotic osteocytes. Whether apoptosis/necrosis of osteocytes results from side effects of the inflammatory response, bacterial endotoxins and/or end-products, a direct toxic effect of HD-ZOL, or other mechanisms, remains to be defined.
Finally, we observed a tendency toward reduced blood vessel number in the periodontium of rice rats treated with HD-ZOL, but only after 24 wks of treatment. We have previously described an antiangiogenic effect of another N-BP, ALN, in a tooth extraction rat model.(32)
However, in the latter study, the effect appeared early and was transient.
Zoledronic acid has been reported to inhibit oral keratinocytes proliferation,(66)
induce oral epithelial cell apoptosis,(67)
and exert a potent antiangiogenic effect.(68-70)
It is well established that the integrity of the gingival epithelium is a physical barrier for the oral microbiota to invade, colonize and induce inflammation and disease. Taken together, our data suggest that high doses of ZOL could enhance or promote the persistence of periodontitis and tissue destruction by inhibiting re-epithelization of damaged gingival epithelium, allowing commensal bacterial growth and periodontal infection. Bacterial infection can then stimulate production of pro inflammatory cytokines and increase the expression of RANKL by different cell types in the periodontal tissue, resulting in more tissue destruction, decreased angiogenesis, and the paradoxical local elimination of the antiresorptive effect of this potent N-BP.
Most ONJ cases reported in the literature of the past five years have been associated with the use of oncologic doses of N-BP's(1-5)
. The more recent observation of ONJ in N-BP-naïve patients receiving RANKL antibodies(6-9)
suggests that it may be the physiologic effect of strongly inhibiting bone resorption by any means, rather than through the specific use of an N-BP, that drives much of ONJ's pathophysiology. In this report, we show that rice rats with moderate/severe periodontitis develop ONJ-like lesions when co-administered oncologic doses of ZOL. A similar experiment in which RANKL antibodies or osteoprotogerin were administered to rice rats with moderate/severe periodontitis would test this hypothesis.
In summary, our data show that rice rats treated with oncologic doses of ZOL appear to be a promising animal model for studies of the pathogenesis and treatment of ONJ. Our study also showed that osteoporosis doses of ALN and ZOL produced no such lesions in a similar timeframe. Our study suggests that HD-ZOL exacerbates the inflammatory response and periodontal tissue damage in rice rats, inducing lesions that resemble ONJ. Our data show that HD-ZOL inhibits bone resorption in the jaw of rice rats when the integrity of the periodontium is preserved. However, the antiresorptive effect is lost simultaneously with extensive ulceration of gingival epithelium, exposed necrotic bone and/or osteolysis, severe inflammatory cell infiltration of the lamina propria, and presence of bacterial biofilm adhered to alveolar bone or deep in the lamina propria.