Methods: DPSC isolated from human dental pulp were cultured and the cells between p3 and p5 were used. Cells grown to 60-70% confluence were treated with TNF-α (5 or 10 ng/ml) in 0.3% serum-containing medium for varying time points. Subsequently, the cells were examined for apoptosis (propidium iodide staining), NFκB activation, proliferation (using MTT and CFSE staining), gene expression analysis for osteogenic lineage commitment (using quantitative real time PCR [qPCR]) and differentiation analysis (using Alizarin Red staining).
Results:Acute TNF-α treatment (4h) induced apoptosis dose dependently. Interestingly, we observed an upregulation of NF-κB expression in addition to an increase in the phosphorylation of p65 subunit at 4h of treatment. However, prolonged treatment of DPSC with TNF-α (9 days) showed an increase in proliferation. Strikingly, the differentiation potential of DPSC was markedly abrogated at day 9. In particular, the expression levels of bone morphogenic proteins (BMP)-1 & 2, BMR receptor isoforms-1&2 and the tissue growth factor (TGF) family of proteins were observed to be significantly down-regulated upon treatment with TNF-α. Additionally, the levels of osteoactivin and osteocalcin were also observed to be decreased. In parallel, Alizarin Red-S staining was also found to be abrogated markedly when compared to control.
Conclusions: Our studies provide a comprehensive picture of inflammation-mediated emergence of apoptotic-resistant DPSC, with a lack of differentiation potential. Thus, we conclude that treatment approaches to prevent prolonged inflammation will protect DPSC from undergoing phenotypic alterations