Inhibition of oral mucosal cell wound healing by bisphosphonates

Abstract

Purpose: Bisphosphonates (BPs) are a widely used class of drugs that are effective in the treatment and prevention of osteoporosis, hypercalcemia of malignancy, and bone metastases associated with multiple myeloma, breast cancer, and other solid tumors. In the past several years there have been numerous reports describing the occurrence of osteonecrosis of the jaws (ONJ) associated with these drugs. Whether the ONJ lesion initiates in the oral mucosa or derives from the underlying bone is not well understood. In this report we describe the effect of pamidronate, a second-generation BP, on oral mucosal cells.

Materials and methods: Murine oral keratinocytes were isolated and exposed to pamidronate at a range of clinically relevant doses. Cellular proliferation was measured using a MTS/PMS reagent-based kit and wound healing was examined with a scratch assay. To determine whether oral keratinocytes undergo apoptosis following exposure to pamidronate, TUNEL, caspase-3, and DAPI apoptosis assays were performed.

Results: We show that BP pretreatment of oral mucosal cells inhibits proliferation and wound healing at clinically relevant doses, and that this inhibition is not due to cellular apoptosis.

Conclusions: To our knowledge this is the first report investigating the effect of nitrogen-containing BPs on oral mucosal cells. This study suggests that BPs inhibit oral keratinocyte wound healing which may play a significant role in the initiation of ONJ.

Figure 1. Cellular proliferation of oral keratinocytes with pamidronate

Low concentrations of pamidronate (0.003 - 0.03 mM) did not effect cell proliferation over the course of 7 days. A higher dose (0.1mM) significantly inhibited proliferation. * - p<0.05 when compared to controls.

Figure 2. Cellular proliferation of oral keratinocytes with pamidronate

Cells exposed to 0.1mM pamidronate did not proliferate from 24 hours to 7 days. * - p<0.05 when compared to controls.

Figure 3. Caspase-3 activity in oral keratinocytes incubated with pamidronate

Pamidronate did not significantly increase caspase-3 activity in cells over a 48 hour period when compared with controls. The positive control (+) was obtained by incubating cells with staurosporine and cycloheximide. * - Significant increase compared with controls, p<0.05.

Figure 4. Apoptosis in oral keratinocytes incubated with pamidronate

TUNEL assay performed on cells incubated with 0.03, 0.1, and 0.3 mM pamidronate did not increase the percentage of TUNEL positive cells when compared to nontreated cells. Staurosporine and cycloheximide treated positive control cells were all apoptotic by 24 hours.

Figure 5. Wound healing in oral keratinocytes with pamidronate

Cells were observed for 96 hours after wounding in cell culture plates. Control wells exhibited significant migration into the wounded area from time of injury (A) to 96 hours (B). Cells pre-incubated for 72 hrs with 0.1mM pamidronate prior to wounding had greatly reduced migration from 0 (C) to 96 hours (D). Data shown is representative of 3 independent experiments.