TEGDMA Causes Apoptosis in Primary Human Gingival Fibroblasts
V. Janke1,*,
N. von Neuhoff2,*,
B. Schlegelberger2,
G. Leyhausen1, and
W. Geurtsen3,+
1 Department of Conservative Dentistry & Periodontology
2 Institute of Cell and Molecular Pathology, Medical Univ. Hannover, Germany; and
3 Department of Restorative Dentistry, School of Dentistry, University of Washington, Box 357456, Seattle, WA 98195-7456, USA;
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Figure 1. Analysis of cell growth with the Hoechst 33342 assay after incubation of the human gingival fibroblasts with different concentrations of TEGDMA for 24 hrs. No growth inhibition was found at concentrations of 1 mM and 2.5 mM, whereas 5 mM and 7.5 mM TEGDMA significantly inhibited cell growth. The diagram shows the results of 3 independent experiments. Values are means ± SD. *p < 0.05, **p < 0.01 statistically different in comparison with control (100%).
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Figure 2. "Apoptotic" effects of TEGDMA on HGF. Fibroblasts were stained with APOPercentageTM for 1 hr. (A,D) Untreated control cultures; no apoptotic cells are visible. Cells exposed for 24 hrs to TEGDMA: (B) 2.5 mM and (C,E) 7.5 mM TEGDMA. Apoptotic cells are stained intensely red (A-C bar = 22 µm; D,E bar = 5.5 µm).
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Figure 3. Induction of apoptosis and necrosis by different concentrations of TEGDMA after 24 hrs. FACS analysis after staining with Annexin V-FLUOS/PI. Three distinct cell distribution patterns are visible: normal, viable cells (lower left quadrant); apoptotic fibroblasts (lower right quadrant); and necrotic and/or "apoptotic necrotic" HGF (upper right quadrant). (A) Non-treated cells (negative controls). Most cells are ‘located’ in the lower left quadrant (viable cells). (B) Cells treated with 1 mM TEGDMA (similar to A). (C) Cells treated with 2.5 mM TEGDMA. A slight shift toward the area indicating "apoptotic" cells is noticeable. (D) Cells treated with 5 mM TEGDMA. (E) Cells incubated with 7.5 mM TEGDMA. (F) Cells incubated with 10 mM 5-fluorouracil (positive control); Annexin V-FLUOS/PI staining reveals the shift of the cells toward apoptosis. (D) and (E) clearly reveal a significant increase in the share of apoptotic cells as well as a moderate shift toward the upper right quadrant, which is indicative of necrotic and/or "apoptotic necrotic" cells. (A-F) Diagrams of one representative experiment each.
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Figure 4. Viable, apoptotic, and "apoptotic necrotic" or necrotic cells induced by various concentrations of TEGDMA after 24 hrs. The diagram shows the results of the FACS analysis of 3 independent experiments (means ± SD). normal viable HGF; apoptotic HGF; necrotic and/or "apoptotic necrotic" HGF. ** p < 0.01; statistically significantly different compared with negative controls.
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