Role of Zoledronic Acid in the Apoptotic Cell Death of Oral Fibroblasts and Epithelial Cells: A Mechanism of Bisphosphonate-Associated Osteonecrosis

Purpose: Osteonecrosis of the jaw is reported in patients receiving IV zoledronic acid (ZA). The purpose of this study was to investigate the effects of ZA on oral mucosal cells using an in vitro model as an alternative mode for the induction of bone necrosis.

Experimental Design: Human gingival fibroblast and keratinocyte cell lines were exposed to different concentrations (0.25–3μM) of ZA and siRNA versus caspase 3 or 9, using 1μM as the expected baseline concentration. Apoptotic effects were determined by TUNEL and Annexin V studies. Effects on cell proliferation were determined by Coulter Counter and MTS assay. Validation of the apoptotic effects of ZA were obtained via RT2 Profiler PCR Arrays of human apoptosis and Western blot.

Results: A dose response effect on apoptosis and cell proliferation was observed with increasing ZA concentrations; both reversed using siRNA against caspase 3 or 9. Initial apoptotic effects were observed microscopically at 8 hours, with increasing apoptotic events measured out to 24 hours. Gene expression analysis demonstrated the differential expression of multiple genes involved in apoptosis including: TNF, Bcl-2, caspase, IAP, TRAF, and death domain families. Western blot analysis confirmed the presence of activated forms of caspase 3 and 9 and underexpression of survivin protein expression.

Conclusions: This study demonstrates that low concentrations of ZA rapidly and directly affect the oral mucosal tissues though the induction of a gene-regulated apoptotic process. These findings provide the establishment of an oral cell based model supporting a soft tissue mechanism in the pathogenesis of osteonecrosis.