TNF-alpha Promotes an Odontoblastic Phenotype in Dental Pulp Cells

Objectives: To examine the effects of the pro-inflammatory mediator, tumor necrosis factor-alpha (TNF-alpha) on pulp cells by evaluating mineralization and expression of mineralization-associated proteins. Material & Methods: Primary human dental pulp and periodontal ligament cells were stimulated with recombinant human TNF-alpha for 6, 12, 24, 48 and 96 hours. 10-30 µg of total protein were used for Western blotting analysis of matrix metalloproteinases (MMP-1, MMP-2, MMP-13), dentin sialoprotein (DSP), dentin phosphoprotein (DPP), dentin matrix protein-1 and osteocalcin. Immunoblot bands were analyzed by densitometry using the Image J 1.34s Software. Mineralized nodule formation was performed by culturing confluent dental pulp cells in media containing 10 mM beta-glycerophosphate and 50 µg/mL ascorbic acid for 28 days. Nodules were visualized by staining with a von Kossa 5% silver nitrate solution and exposure to bright light. Quantification of insoluble calcium in cell/ matrix layers was performed using a calcium assay kit. To compare the protein levels expressed after TNF-alpha stimulation over time, quantitative densitometric data were derived as fold change relative to baseline control levels. One-way ANOVA and post hoc comparisons were performed (alpha= 0.05). Results: TNF-alpha-challenged pulp cells exhibited increased mineralization and early and increased expression of dentin phosphoprotein, dentin sialoprotein, dentin matrix protein-1 and osteocalcin during a phase of reduced matrix metalloproteinase expression. MMP-1 and MMP-13 were detected at later time points in pulp cells in response to TNF-alpha stimulation compared to dentinogenesis related proteins. We investigated whether these events were related and found that p38, a mitogen-activated protein kinase, differentially regulated MMP-1 and DSP/ DPP expression upon TNF-alpha treatment. Conclusion: These findings indicate that TNF-alpha stimulates differentiation of dental pulp cells towards an odontoblastic phenotype via p38, while negatively regulating MMP-1 expression (Study supported by Grant NIH-R01-13725 to YLK and CAPES Foundation Fellowship to FWGPS)