The effects of a systemic single dose of zoledronic acid on post-implantation bone remodelling and inflammation in an ovariectomised rat model

Abstract

Bisphosphonates reverse the negative effects of ovariectomy on bone, but they have also been associated with adverse processes in human jawbone. The molecular events determining bone regeneration and implant integration in osteoporotic conditions, with and without bisphosphonate treatment, are unclear. In this study, ovariectomised rats, to which a single dose of saline (NaCl) or zoledronic acid (Zol) was administered, received titanium alloy implants in their tibiae and mandibles. An enzyme-linked immunosorbent assay, gene expression analysis and histomorphometry were performed. The results show that ovariectomy, per se, upregulated the expression of genes denoting bone formation in the tibia, bone remodelling in the mandible and apoptosis in the tibia and mandible. Zoledronic acid administration resulted in lower levels of a remodelling marker in serum and downregulated gene expression for inflammation, bone formation, angiogenesis and apoptosis, mainly in the mandible, after 28 d of healing. Histomorphometry revealed improved bone-to-implant contact in the tibia, while the opposite was observed in the mandible. The present data show that a systemic single dose of zoledronic acid, in ovariectomised animals, results in site-specific differences in the regulation of genes involved in bone healing and regeneration in association with implant installation. These events occur in parallel with site-specific differences in the rate of osseointegration, indicating diverse tissue responses in the tibia and mandible after zoledronic acid treatment. The zoledronic acid effect on gene expression, during the late phase of healing in the mandible, suggests negative effects by the anti-resorptive agent on osseointegration at that particular site.

Introduction

Metabolic disorders, such as postmenopausal osteoporosis, have a major impact on bone structure [1] and may affect the processes involved in fracture healing as well as osseointegration [2], [3]. Appropriate management strategies require further knowledge and understanding of the effects of these conditions, and their medications, on biological mechanisms involved in bone healing and regeneration. The ovariectomised rat is a well-established model for osteoporosis, with the advantages that is easy to manipulate, highly reproducible and representative of peri- and post-menopausal osteoporosis in women [4]. Negative effects on bone healing around titanium implants have been revealed in ovariectomised rats [5], [6], [7], [8]. However, few studies have been reported on osseointegration in treated osteoporosis and information about the biological events at the interface after the therapeutic administration of anti-osteoporotic agents are still lacking.

Bisphosphonates (BPs) are potent anti-resorptives, routinely used as a first treatment choice for osteoporosis and also to reduce the risk of resorptive skeletal-related events in patients with bone metastases [1], [9], [10]. A highly potent nitrogen-containing bisphosphonate, zoledronic acid (Zol), used to treat postmenopausal bone loss and administered as a single dose once a year, has been shown to improve bone density, reduce bone remodelling and reduce the risk of fractures in osteoporosis [11], [12], [13]. In relation to biomaterial integration, it has been observed that systemically administered Zol [14] and another nitrogen-containing bisphosphonate (alendronate) [15], [16] improve bone formation and osseointegration around titanium implants in ovariectomised animal models. Apart from these findings, the exact cellular and molecular mechanisms governing the improved bone content, structure and strength, induced by the systemically administered nitrogen-containing bisphosphonates, have not been described.

The sequence of events that take place at fracture healing has been closely studied in rats, where the inflammatory response initiated by damage to the tissue is present during the first week and is then followed by the repair and remodelling phases [17]. Several biological mediators and biomolecules temporally and spatially affect the different stages of bone healing, including inflammation, angiogenesis, osteogenesis and remodelling. In osteoporosis, many of these mediators were shown to be influenced systematically [18] or locally [19] at skeletal bone sites. Interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are major proinflammatory cytokines that have been shown to be active in the development of osteoporosis and their expression was inhibited by oestrogen [18], [20], [21], [22], [23]. Bone formation markers, such as alkaline phosphatase (ALP) and osteocalcin (OC), were shown to be increased in the serum of osteoporotic patients [24]. Furthermore, the expression of collagen type 1 (Col1a1) has been observed to increase in the tibia of OVX rats [25]. The osteoclast differentiation and bone remodelling activity is controlled by many factors, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CatK), receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG) [26], [27], [28]. Increased expression levels of OPG and RANKL were observed in ovariectomised rat bone compared with normal bone [25], [29]. Vascular endothelial growth factor (VEGF) is an angiogenic factor that induces not only angiogenesis but also bone formation and remodelling activities [30]. The expression of VEGF was significantly reduced in the tibial metaphysis of ovariectomised mice [19]. The levels of apoptosis markers, such as Caspase 3, have been reported to increase after ovariectomy in rodents [31], [32], [33]. Given the evident roles of these factors, it can be hypothesised that the mechanisms of action of bisphosphonates, in reversing the osteoporotic conditions, may involve their regulation at the sites of bone healing, which may subsequently affect the osseointegration events and bone formation at biomaterials.

Recently, an adverse effect, osteonecrosis of the jaw (ONJ), has been described as a result of the systemic administration of bisphosphonate [34], [35], [36], characterised by pain, exposed bone, sequesters and sometimes severe infection and fractures [37]. The condition is usually preceded by surgical trauma, such as dental extraction or dental implant installations [37]. The implantation of titanium implants is associated with unavoidable localised trauma to the recipient bone site. Self-drilling mini-screws have produced microfractures of the bone at the bone-implant interface at the ultrastructural level, compared with self-tapping mini-screws [38]. Numerous theories relating to the pathogenesis of ONJ have been suggested; they include suppressed angiogenesis, suppressed bone remodelling and/or a cytotoxic effect on fibroblasts, keratinocytes and osteocytes, [39], [40], [41]. As the exact mechanisms causing ONJ are not known, a proper understanding of the molecular signatures elicited by bisphosphonates could provide clues to the pathogenic changes and complications that might be associated with nitrogen-containing bisphosphonate treatments. In this study, we hypothesised firstly that a single dose of a highly potent nitrogen-containing bisphosphonate could alter the molecular events of bone healing after surgically induced trauma and implant installation and, secondly, that the anatomical site may be important for osseointegration in the bisphosphonate-treated model. The objective of this study was to evaluate bone response to a single dose of zoledronic acid, followed by a surgical trauma with implant insertion at different locations in osteopenic rats. Molecular techniques, such as serum analysis by enzyme-linked immunosorbent assay and gene expression analysis of bone samples from implantation sites, were used to study the sequence of molecular events and the results were corroborated by histomorphometric measurements of osseointegration.