Phagocytosis of titanium particles and necrosis in TNF-alpha-resistant mouse sarcoma L929 cells

In the oral cavity, titanium is an excellent biocompatible material. However, it is reported that high ratios of intracellular titanium particles can cause cell apoptosis or necrosis by as-yet unknown mechanisms. The purpose of this study was to investigate the response of tumor necrosis factor alpha (TNF-alpha)-resistant L929 fibroblasts to titanium particles. Cells were cultured in Eagle's medium supplemented with fetal bovine serum and L-glutamine. Titanium particle sizes were less than 9 micro. Cytotoxicity was assayed by a cell counting kit, trypan blue dye exclusion test and lactate dehydrogenase (LDH) leakage. The production of reactive oxygen species (ROS) was detected by a confocal laser scanning microscope (CLSM) using dichlorofluorescein diacetate as a fluorescent probe. Morphology was viewed by a CLSM and with an X-ray microanalyser (XMA). When titanium particles were added to cells, the viability decreased to around 50% at a particle concentration of 2.0%. The number of dead cells and LDH activity in the culture media increased significantly between 1 and 2 days. However, formation of active oxygen species did not occur, since no dichlorofluorescein fluorescence was observed. A scanning electron photomicrograph (SEM) revealed a large number of particles covering or adhering to cellular components in lysed cells compared with flattened control cells attached to the substrate. The XMA showed that the titanium accumulation was coincident with the deformed cell shape. The CLSM also confirmed that particles were within the cells. From these results it was concluded that titanium particles ingested in large quantities into the cell induced necrosis by a pathway other than by producing ROS.