The effects of chronic zoledronate usage on the jaw and long bones evaluated using RANKL and osteoprotegerin levels in an animal model

Abstract

The discovery of the receptor activator of nuclear factor kappaB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) system (RANK/RANKL/OPG system) has been one of the most important advances in bone biology in the last decade. We investigated how the chronic application of bisphosphonate affects the RANKL and OPG levels in an animal model and whether this effect may be related to bisphosphonate-related osteonecrosis of the jaws (BRONJ). Thirty female Sprague–Dawley rats were used in this study. The rats were randomly divided into three groups (10 in each): Z, the zolendronate group, injected with zolendronate for 10 weeks; S, a control group, injected with saline solution for 10 weeks; and C, a control group, in which no injection was given. RANKL values in the tibia were increased in the Z group when compared with the two controls; however, the RANKL values in the mandible were decreased when compared with the controls. Although the differences did not reach statistical significance, the mandibular OPG values were increased in the Z group when compared with the C and S groups. The mechanism of RANKL negation and absence in osteoclastic activation could be a predisposing factor for the development of BRONJ.

Introduction

Bone tissue is continuously remodelled through bone resorption by osteoclasts and through bone formation by osteoblasts, thereby maintaining a dynamic balance in bone quantity. The discovery of the receptor activator of nuclear factor kappaB (RANK), the RANK ligand (RANKL), and osteoprotegerin (OPG) system (RANK/RANKL/OPG system) has been one of the most important advances in bone biology in the last decade.

RANKL is a 317-amino acid polypeptide that belongs to the tumour necrosis factor (TNF) superfamily and whose mRNA is largely expressed in bone, bone marrow, and lymphoid tissues. RANKL is essential for osteoclast formation, activity, and survival in normal and pathological states of bone remodelling.

Anderson et al. identified RANK as a type I transmembrane protein that can be found on osteoclasts, dendritic cells, fibroblasts, and B and T cell lines. The specific binding of RANKL to the RANK receptor is necessary for osteoclastogenesis. Genetic ablation of RANK or RANKL results in severe osteopetrosis in association with a nearly complete lack of osteoclasts.

OPG acts as a decoy receptor by sequestering RANKL and inhibiting RANK signalling, and it inhibits the development of osteoclasts. Several studies in a variety of animal models have confirmed not only the effect of RANKL blockade with OPG in inhibiting osteoclastogenesis and bone resorption, but also the role of the deregulation of the RANK/RANKL/OPG system in the pathophysiology of multiple bone remodelling disorders, such as osteoporosis, glucocorticoid-induced bone loss, erosive arthritis including rheumatoid arthritis, hypercalcaemia of malignancy, Paget’s disease, and metastatic bone diseases.

Given their ability to suppress osteoclastic function, bisphosphonates are being developed as drugs to treat diseases involving elevated bone turnover. Bisphosphonates are approved for clinical use, the most potent of which is zolendronate.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is characterized by non-healing exposed bone in the mandible or maxilla that persists for more than 8 weeks in a patient who has taken or is currently taking a bisphosphonate and who has no history of radiation therapy on the jaw. The use of the intravenous (i.v.) formulation of zoledronate appears to be associated with an increased risk of BRONJ. Although different theories have been published, the pathogenesis of this phenomenon remains unclear.

We investigated how the chronic application of bisphosphonate affects the RANKL and OPG levels in an animal model, and whether this effect may be related to BRONJ.

Methods

Ethical approval was obtained for the experimental procedures. After their menstrual phase was controlled, 30 female Sprague–Dawley rats were used in this study. The animals were 12 weeks old and weighed 210 ± 15 g. All of the animals were given laboratory food and water ad libitum. They were kept in a quiet and temperature/humidity-controlled room (22 ± 2 °C and 40–60%, respectively) in which a 12:12-h light and dark cycle was maintained. Experiments were conducted between 9:00 and 17:00 h to minimize the diurnal variation.

The rats were randomly divided into three groups ( n = 10): Z, the zolendronate group, injected with zolendronate for 10 weeks; S, a control group, injected with saline solution for 10 weeks; and C, a control group, in which no injection was given. The Z group received injected zolendronate at a dose of 0.1 mg/kg, 3 times a week. All injections were performed intraperitoneally by the same surgeon ( Fig. 1 ). After 10 weeks, without any dental intervention, the rats were sacrificed. The posterior region of the mandible, including the first and second molars, and the upper third of the tibia, were dissected subperiosteally and frozen immediately on dry ice.

Fig. 1
Intraperitoneal application of zoledronate and saline injection at a dose of 0.1 mg/kg.

All tissues were cut into fine pieces in ice-cold phosphate-buffered saline (PBS), pH 7.4, containing protease inhibitors (aprotinin 2 μg/ml, leupeptin 5 μg/ml, pepstatin 0.7 μg/ml, and phenylmethylsulfonyl fluoride (PMSF) 100 μg/ml; Sigma, St. Louis, MO, USA), followed by homogenization using a rotor homogenizer. The homogenates were centrifuged for 5 min at 14,000 × g and 4 °C. The supernatants were transferred to fresh pre-cooled Eppendorf tubes. Triton X-100 was added at a final concentration of 0.01%. The samples were centrifuged again for an additional 5 min at 14,000 × g at 4 °C. The supernatants were aliquoted and stored at −80 °C. The RANKL and OPG concentrations were determined using rat ELISA kits (Uscn Life Inc., Wuhan, China) in accordance with the manufacturer’s instructions.

The statistical analysis consisted of the Kruskal–Wallis test followed by the Mann–Whitney U -test. All data are presented as the mean ± standard error of the mean, and differences were considered significant at P < 0.05.

Methods

Ethical approval was obtained for the experimental procedures. After their menstrual phase was controlled, 30 female Sprague–Dawley rats were used in this study. The animals were 12 weeks old and weighed 210 ± 15 g. All of the animals were given laboratory food and water ad libitum. They were kept in a quiet and temperature/humidity-controlled room (22 ± 2 °C and 40–60%, respectively) in which a 12:12-h light and dark cycle was maintained. Experiments were conducted between 9:00 and 17:00 h to minimize the diurnal variation.

The rats were randomly divided into three groups ( n = 10): Z, the zolendronate group, injected with zolendronate for 10 weeks; S, a control group, injected with saline solution for 10 weeks; and C, a control group, in which no injection was given. The Z group received injected zolendronate at a dose of 0.1 mg/kg, 3 times a week. All injections were performed intraperitoneally by the same surgeon ( Fig. 1 ). After 10 weeks, without any dental intervention, the rats were sacrificed. The posterior region of the mandible, including the first and second molars, and the upper third of the tibia, were dissected subperiosteally and frozen immediately on dry ice.

Fig. 1
Intraperitoneal application of zoledronate and saline injection at a dose of 0.1 mg/kg.

All tissues were cut into fine pieces in ice-cold phosphate-buffered saline (PBS), pH 7.4, containing protease inhibitors (aprotinin 2 μg/ml, leupeptin 5 μg/ml, pepstatin 0.7 μg/ml, and phenylmethylsulfonyl fluoride (PMSF) 100 μg/ml; Sigma, St. Louis, MO, USA), followed by homogenization using a rotor homogenizer. The homogenates were centrifuged for 5 min at 14,000 × g and 4 °C. The supernatants were transferred to fresh pre-cooled Eppendorf tubes. Triton X-100 was added at a final concentration of 0.01%. The samples were centrifuged again for an additional 5 min at 14,000 × g at 4 °C. The supernatants were aliquoted and stored at −80 °C. The RANKL and OPG concentrations were determined using rat ELISA kits (Uscn Life Inc., Wuhan, China) in accordance with the manufacturer’s instructions.

The statistical analysis consisted of the Kruskal–Wallis test followed by the Mann–Whitney U -test. All data are presented as the mean ± standard error of the mean, and differences were considered significant at P < 0.05.

Results

The descriptive statistical values for RANKL in the mandible and tibia are shown in Table 1 . The RANKL values in the tibia were increased in the Z group when compared with the two controls; however, the RANKL values in the mandible were decreased when compared with the controls. The increase in tibia values and the decrease in mandible values were not statistically significant (tibia P = 0.055; mandible P = 0.44; Figs. 2 and 3 ).

Table 1
Mean and standard deviation of RANKL values.
Group a Mandible Tibia
Mean SD Mean SD
Z 584.15 144.17 341.58 55.75
C 723.21 324.53 236.86 85.48
S 719.47 224.41 296.25 38.84
RANKL: receptor activator of nuclear factor kappaB ligand.

a Z: zolendronate injection; C: no injection; S: saline injection.

Fig. 2
RANKL tibia values (group 1: zolendronate injection (Z); group 2: no injection (C); group 3: saline injection (S)).

Fig. 3
RANKL mandible values (group 1: zolendronate injection (Z); group 2: no injection (C); group 3: saline injection (S)).

The descriptive statistical values for OPG in the mandible and tibia are shown in Table 2 . Although the differences did not reach statistical significance, the mandibular OPG values were increased in the Z group when compared with the C and S groups ( P = 0.74). The OPG values in the tibia were increased significantly in the Z group ( P = 0.045). In double comparisons, there was a significant difference between the Z and C groups ( P = 0.037) ( Figs. 4 and 5 ).

Jan 24, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on The effects of chronic zoledronate usage on the jaw and long bones evaluated using RANKL and osteoprotegerin levels in an animal model

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