Site-specific effects of bisphosphonates on long bones and jawbones

Abstract

Bisphosphonates (BPs) have been extensively used for management of bone diseases with pathologically high resorption. Despite the great clinical benefits, a severe complication known as medication-related osteonecrosis of the jaw (MRONJ) has been reported. It is believed that impaired angiogenesis also contribute to the development of MRONJ while most of the clinical biopsy samples showed avascular necrosis. The present study aims to 1) investigate the effect of BPs treatment on vascularization using a rat model, and 2) to assess the site-specific response of neovessel formation in jawbones and long bones to BPs treatment. In the first experiment, skeletal mature Sprague-Dawley female rats were administered oncologic dose of zoledronic acid (ZA) or normal saline for 4 weeks and then subjected to tooth extraction on the mandible and maxilla, and a bone defect creation on the femur. After surgical procedures, ZA or saline treatment were continued until sacrifice at week 2, week 4 and week 8 post-operatively. The samples were subjected to micro-computed tomography (micro-CT) and histological assessment. Osteonecrosis was only found in jawbones in ZA-treated rats. ZA-treated rats showed significantly higher bone mineral density with greater bone volume in all surgical sites than that in the controls. No significant difference in bone remodeling was found between femur and jawbones in the ZA treated rats. In the second and third studies, the vascular number and density, the expression of angiogenic factors including VEGFA, VEGFR-2 and CD-31 in response to ZA treatment were investigated using the same animal model. Micro-CT examination of vascular perfusion demonstrated a tendency of decreased vessel density and vessel number in ZA-treated groups. Immunohistochemical and real-time polymerase chain reaction analysis had similar findings which showed increased expression of the angiogenic factors in the mandible and decreased expression in the maxilla, while no remarkable change in the femur in the rats treated with ZA. In conclusion, 1) long bones and jawbones undergo similar microstructure changes after surgical intervention in the rat model with ZA treatment; 2) In the ZA treated animals with surgical intervention, osteonecrosis only occurs in jawbones, which suggests that surgical intervention is a risk, but not the sole decisive factor for inducing MRONJ; 3) The neovessel formation is suppressed but not significantly by ZA treatment, indicating that angiogenesis inhibition may contribute to the development of MRONJ but does not play a key role; 4) Expression of the investigated angiogenic factors demonstrates site-specific response to ZA treatment and surgical intervention at protein and gene levels; however, the site-specific difference is not translated to the neovessel formation at functional structure level; 5) The over suppression of bone resorption and angiogenesis inhibition resulted from ZA treatment, which have no site-specific differences between long bones and jawbones, are the underlying risk of osteonecrosis. The unique localization of MRONJ exclusively to the jaws is more related to the invasive surgical intervention and the special condition in oral environment.