In vitro synergistic effects of zoledronic acid and calcium on viability of human epithelial cells

Objective: Bisphosphonate-related osteonecrosis of the jaw is a common complication with defective wound healing of oral mucosa and frequently occurs in patients receiving zoledronic acid (ZA). The aim of this in vitro study was to investigate whether ZA has a cytotoxic effect at clinically relevant concentrations on epithelial cells when calcium conditions are altered.

Methods: HaCaT human keratinocyte cells were treated with ZA in the presence of various concentrations of calcium. The concentrations of ZA included submicromolar ones, which are comparable with those found in the plasma of patients. Cell viability and apoptosis were assessed using MTT assay and annexin V flow cytometry.

Results: Under standard culture conditions, cell growth was inhibited at 1 μM of ZA or above, but was unaffected by lower concentrations. However, when calcium concentrations were moderately increased, cell viability was decreased and apoptosis was induced at 0.2-0.3 μM of ZA. Moreover, a 50% reduction in serum in the hypercalcemic medium resulted in a significant decrease in cell viability at a much lower concentration (0.05 μM).

Conclusion: These results suggest that clinically relevant concentrations of ZA, which alone have little effects, can be toxic to the epithelial cells depending on the conditions of extracellular calcium.