In this study, MRONJ incidence was investigated when ZA was administered and tooth extraction was performed after inducing periodontitis in OVX rats. Although ZA improved the osteoporosis induced in the long bones, it also induced MRONJ onset.
Periodontal disease is one of the most common causes of tooth extraction. MRONJ occurs in patients after tooth extraction, and the teeth cannot be restored owing to periapical infection with advanced dental caries or periodontal disease [16, 26, 27]. Inducing periodontal lesions was attempted by placing tooth ligation on the cervical portion of the teeth for 8 weeks before extraction. Duarte et al. reported that bacterial species commonly observed in humans were found in the biofilm around the ligature 42 days after ligation in the rats [28]. Liu et al. reported that a 28-day ligature could cause significant loss in the trabecular pattern of the alveolar bone in rats [29]. Therefore, the tooth ligation was kept for 8 weeks in our study, and this duration was adequate to induce periodontal lesions. Alveolar bone resorption and furcation involvement around the M1 and M2 areas were observed in all the rats. However, there was no visible gingival inflammation or tooth mobility observed 8 weeks after ligation, which was an unexpected outcome. Additionally, the amount of alveolar bone resorption was less than expected. This could be explained by the rapid metabolism in rats and their remarkable ability to regenerate. Many previous studies have attempted to overcome the limitations of tooth ligation used to induce periodontal disease in animal models in various ways [30, 31]. It is necessary to include experimental data using different methods of inducing periodontal disease [32–34].
In the micro-CT analysis, as seen in previous studies, osteoporosis was induced in the long bone by performing OVX, and improved bone quality was observed with ZA administration; however, the mandible showed different findings. When MRONJ occurs, the BV/TV decreases because bone loss increases due to bone resorption and sequestrum formation. Due to the inhibitory action of ZA on osteoclasts, bone resorption is suppressed, BS/BV is lowered, and BS/TV is increased. Thus, there was substantial inflammation in and around the tooth extraction sockets in the ZA group in our study. In the ZA group, the Tb.Th and Tb.N values were high, but the Tb.Sp was low. These values are used to evaluate the characteristics of trabecular bone in osteoporosis, which shows decreased BV/TV, Tb.Th, and Tb.N and increased Tb.Sp; therefore, they can be used to evaluate the effectiveness of osteoporosis drugs. However, it is considered that the bone in a state of increased inflammation due to MRONJ should not be interpreted as an evaluation criterion for non-inflammatory trabecular bone. Summarizing the results of the tibia and jaw, we found that the BV/TV, Tb.Th, and Tb.N were high and Tb.Sp was low in the non-inflammatory state (tibia), but the BV/TV showed contrasting findings in the inflamed state (jaw) (Figs. 3 and 4). This is because osteosclerosis of the trabecular bone occurs when osteomyelitis develops.
MRONJ was diagnosed according to the histological criteria (exposed bone, necrotic bone, inflammatory cell infiltration, or sequestrum) at the extraction site. This was because there were MRONJ lesions in some rat specimens that could not be clinically detected and were only observed in the microscopic analysis. Although the definition of MRONJ is based on the macroscopic appearance in the clinic, MRONJ was diagnosed based on the following histological criteria under experimental conditions: (1) presence of ulcerative lesions with exposed and necrotic bone and/or osteolysis [35]; (2) presence of pseudo-epitheliomatous-like hyperplasia of the epithelium accompanied by inflammatory infiltration [36]; and (3) presence of sequestrum and bacterial colonies [8, 37]. The incidence of MRONJ in the ZA group was significantly higher than that in the CONT group. Notably, the incidence of 7.7% in the CONT group was unexpected. These results could be attributed to the general condition and root morphology of the rats. The rats used in this experiment could be considered immunosuppressed because they represented postmenopausal women with osteoporosis. Therefore, the extraction sites of the CONT group may have also shown inflammatory findings. The other reason is the morphological characteristics of the rat molars. Rat molars have more divergent roots than those of humans, resulting in more chances of remnant root pieces, which may affect the healing of the extraction sites.
Many studies have been conducted to elucidate the pathogenesis of MRONJ, and there are several reports on those related to periodontal and pulp diseases in rats [38–48]. Particularly, when a surgical procedure such as tooth extraction is performed in a state of chronic inflammation such as periodontitis, the incidence of MRONJ is high [38–45]. Although various hypotheses have been proposed regarding the pathogenesis of MRONJ, the mechanism related to macrophage activation is considered the most influential [38]. It has been reported that MRONJ develops when ZA is administered in a periodontitis-induced rodent model [49]. Moreover, if tooth extraction is performed in the presence of periodontitis, the incidence of MRONJ increases, and if tooth extraction is performed after periodontitis treatment, its incidence decreases [50]. Interleukin-17 cytokine was found to increase the M1/M2 macrophage ratio in the mucosa surrounding the MRONJ affected area, suggesting that the activation of M1 macrophages acts as a major risk factor for MRONJ.
A limitation of this study is that a comparison of the incidence of MRONJ between ligated and non-ligated teeth was not performed. To investigate whether periodontal disease-induced extraction is a risk factor for MRONJ, ligated and non-ligated teeth should be extracted simultaneously from one specimen and analyzed to compare the differences of the extraction sites. The ligation period and ZA dose should be further adjusted in future studies. Moreover, an experimental design reflecting the withdrawal period before and after the extraction to simulate the patient's situation is warranted.