Abstract
BackgroundBisphosphonate (BP)-associated osteonecrosis of the jaw (ONJ) has been reported in patients receiving intravenous BP, particularly zoledronic acid (ZA). The purpose of this study was to develop an in vitro model representative of the effects BP has on soft tissue secondary to its release from bone. Human gingival fibroblasts and oral epithelial cell lines were exposed to various concentrations (0-10 μM) of ZA using dentine discs (DDs) as a direct carrier of BP, which were exposed for 24 hours to ZA in normal medium (NM), washed in phosphate-buffered saline (PBS) and placed in a new co-culture with the cells. The cells were allowed to proliferate until they grew over the bone discs and then the discs either were left unchelated, or were chelated using 0.001% EDTA or EGTA to release BP from the discs and to observe the cellular effects. Direct effects were determined using direct and fluorescent imaging. Apoptotic effects were determined by vital stain, terminal dUTP nick-end labeling, and annexin V studies. The effect on cell proliferation was determined by mitochondrial tetrazolium salt assay. The level of BP release was determined based on the effect of BP directly on cells, using the DDs or the supernatant fluids resulting from chelation.ResultsA dose-response effect was seen on imaging, and effects on apoptosis and cell proliferation were observed with increasing ZA concentrations liberated from the DDs, particularly after calcium cleavage and release of ZA from the DDs with a variety of chelating agents. Apoptotic effects were observed microscopically after chelation at 24 hours. Release of ZA was confirmed by extracting medium from non-chelated and chelated cell culture models with DDs and applying this medium to untreated fresh cell cultures, providing appropriate controls.ConclusionsThe results from this study demonstrate that low concentrations of ZA released from bone can rapidly and directly affect the oral mucosal tissues, initially through the induction of apoptosis and long term through the inhibition of cell proliferation. These findings provide an in vitro model for a soft-tissue mechanistic component in the initiation and/or progression of ONJ.
Highlights
Bisphosphonate (BP)-associated osteonecrosis of the jaw (ONJ) has been reported in patients receiving intravenous BP, zoledronic acid (ZA)
ZA-induced apoptosis and cell-growth inhibition Initial analysis of apoptosis, fluorescent confirmation with rhodamine Physical signs of apoptosis were assayed in individual human gingival fibroblast (HGF) (Figure 1a) and HaCaT (Figure 1b) cells exposed to dentine discs (DDs) treated with normal medium (NM) alone or ZA (0.5, 1, 3 or 5 μM), which either were left unchelated (Figure 1a,b; top rows) or exposed to EDTA 0.001% (Figure 1a,b; bottom rows), Results were determined using fluorescent microscopy on rhodamine-stained cells
The chelated model, representing ZA released from bone, showed HGF and HaCaT cells entering apoptosis at a low concentration of ZA (0.5 μM), and there was a marked ring of dead cells visible at 3 and 5 μM
Summary
Bisphosphonate (BP)-associated osteonecrosis of the jaw (ONJ) has been reported in patients receiving intravenous BP, zoledronic acid (ZA). The cells were allowed to proliferate until they grew over the bone discs and the discs either were left unchelated, or were chelated using 0.001% EDTA or EGTA to release BP from the discs and to observe the cellular effects. The level of BP release was determined based on the effect of BP directly on cells, using the DDs or the supernatant fluids resulting from chelation. One of the more recently reported serious adverse effects of ZA treatment is osteonecrosis of the jaw (ONJ), a condition first recognized in 2003 as a complication associated with BP therapy [9] and originally thought to involve the anti-angiogenic effects of ZA [10,11,12].
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