| ObjectivesBisphosphonates (BPs) such as zoledronic acid (ZA) are widely used to treat complications of bony metastases in cancer patients. Recent studies have revealed a serious adverse event in patients receiving IV BP therapy, bisphosphonate-associated osteonecrosis of the jaw (BPONJ). BPONJ, defined as exposed nonviable maxillomandibular bone, is manifested by poor oral wound healing, oral soft-tissue breakdown, and exposure of the underlying intra-oral bone, culminating in necrosis of the exposed bone. Currently, there is no effective clinical treatment for BPONJ. The purpose of this study was to develop an in vitro model representative of the effects ZA has on soft tissue. We culture human gingival fibroblasts with tissue-explant method. Human gingival fibroblasts were exposed to various concentrations (0-10μM) of ZA to observe the cellular effects.. Then we evaluated the effect of ZA on the proliferation and apoptosis of the cells. Our model provides an in vitro mechanism for studying the initial effect of ZA on cells of the oral mucosa.This model may constitute an initiating mechanism for BRONJ.MethodsCulture human gingival fibroblasts with tissue-explant method. Fibroblasts from gingival samples were obtained during routine surgical procedures,passage3-6cells were used in experiment. These cells were exposed to ZA(0-10μM) in normal medium (NM). Direct effects were determined using direct and fluorescent imaging by Rhodamine-123staining. Apoptotic effects were determined by annexin V and PI double staining studies. The effect on cell proliferation was determined by MTT assay and proliferation test.Results1. Human gingival fibroblasts can be successfully cultured with tissue-explant method; the gingival fibroblasts were tested and exhibited the typical morphologic fibroblast patterns. They grew fast after passage.2. Apoptotic effects were observed microscopically after exposed for24hours to ZA. Physical signs of apoptosis were assayed in individual human gingival fibroblast. Results were determined using fluorescent microscopy on rhodamine-stained cells. At24hours in the control group, only a few HGF cells appeared to aggregate, fragment, and begin showing dead floating cells, indicating apoptosis. By contrast, the ZA groups showed HGF cells entering apoptosis at a low concentration of ZA (1μM). A dose dependent-increase in apoptosis in the ZA-treated HGF cells observed by microscopy was corroborated by the results from annexin V and PI double staining flow cytometry apoptosis assays, The differences between control group and ZA groups were significant (P<0.05) for HGF cells at three concentrations of ZA(1,5,10μM). Comparing with control group, no obvious difference was found at0.5μM concentrations.3. Cell proliferation using MTT assay and proliferation test was performed on HGF cells. The0.5μM group failed to demonstrate a significant difference in cell proliferation from the NM control. The other groups demonstrated a combined decrease in cell proliferation compared with the control group. Proliferation test showed that a dose and time response effect between ZA and HGF in a certain concentration range (0.5-10μM).Conclusion1. Human gingival fibroblasts can be successfully cultured with tissue-explant method; the cells grew fast after passage and can apply enough cells for the following study.2. The results from this study demonstrate that low concentrations of ZA can rapidly and directly affect the human gingival fibroblasts through the induction of apoptosis. Significant differences from controls were seen in ZA groups starting at the1μM level.3. ZA affect the HGF through the inhibition of cell proliferation. After treated with ZA for24hours, a significantly inhibiting effect was seen in ZA groups starting at the1μM level and a dose and time response effect can be found. |
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