The Biologic Effect of Particulate Titanium Contaminants of Dental Implants on Human Osteoblasts and Gingival Fibroblasts

Purpose: To evaluate the effects of different titanium particle concentrations on the viability of human calvarial osteoblasts and human gingival fibroblasts. Materials and Methods: Primary human calvarial osteoblasts (HCO, 3H Biomedical) and human gingival fibroblasts (HGF-1, ATCC) were cultivated and allowed to adhere for 24 hours. Titanium powder concentrations (0.01 to 1.0 mg/mL) were added, and samples were analyzed at three time points (24 hours, 7 days, 21 days). Cell viability was analyzed using living cell count, proliferation (MTT) assay, and live/dead staining. Cytotoxic effects were evaluated using lactated dehydrogenase assay. Qualitative analysis of cell viability was performed. In addition, scanning electron microscopy (SEM) analysis was performed. Release of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) was estimated with human IL-6/human TNF-α ELISA. Results: Titanium concentrations of 0.1 mg/mL and 1.0 mg/mL showed medium- and long-term effects on cell growth and proliferation rates. Cytotoxic effects by release of lactate dehydrogenase were observable during the first 24 hours. Human gingival fibroblast cells showed a release factor between 2.6 and 3.4. Titanium powder seemed to be more cytotoxic to human gingival fibroblast cells than to human calvarial osteoblast cells. For human calvarial osteoblasts, only the highest concentration showed cytotoxic effects with a release factor of 2.7. Human calvarial osteoblasts secreted IL-6 only during the first 24 hours and only in the highest titanium concentration, whereas human gingival fibroblasts secreted IL-6 during the entire period. The lowest titanium concentration showed stronger secretion of IL-6 compared with the control. Incorporation of smaller and single titanium particles by cells was identified under SEM analysis. Conclusion: Cell viability is negatively correlated with titanium concentration. Further, titanium debris might lead to an inflammatory biologic response of dental peri-implant tissue. Also, cells interact with the debris, eg, with incorporation of particles. Keywords: human gingival fibroblasts, human osteoblasts, primary cell culture, titanium debris, titanium particles