Evaluation of an in-vitro Wounding Model for Human Gingival Fibroblasts

Background: Bisphosphonate prevents excessive bone loss and is used to treat patients with osteoporosis and metastatic bone cancer. However its use has become a significant issue in recent years, following numerous case reports of bisphosphonate-related-osteonecrosis-of-the-jaw(BRONJ). While conventional studies consider altered osteoclastic function as the primary factor in its pathogenesis, recent studies suggest that bisphosphonate may also have an anti-angiogenic effect in soft tissue. Objectives: The aim of this study is to develop an in-vitro wounding model to investigate the effects of zoledronic acid(ZA)(Zometa®,Novartis) on soft tissue healing, in this case the migration of human gingival fibroblasts(HGF). Methods: Primary cultures of HGF were developed from gingival tissues excised during crown-lengthening surgery. HGF were seeded in 12-well plates and wounded with pipette tips once they became 100% confluent. Tissue culture media containing ZA(1µM, 10 µM, 30 µM and 100 µM) was added immediately after wounding and HGF were incubated for 17 hours before being fixed and stained. Photographs of the 12-well plates were then taken and the software ImageJ(NIH) was used to measure the remaining wounding site at fixed locations in all wells. Results: While developing the model, it was discovered that the concentration of fetal bovine serum used in the treatment groups had to be reduced to 1% to reveal the inhibitory effect of ZA on migration. For ZA, the concentration dependant pattern was not observed but all concentrations exhibited a 125% change from the controls. Conclusions:All concentrations of ZA appeared to have an inhibitory effect on the migration of HGF despite the lack of concentration dependant response. Further research is required to see if this result is reproducible with HGF cells from different patients and whether longer exposure to ZA will increase that amplitude of the inhibitory effect. This research was supported by the New Zealand Dental Association Research Foundation.