Methods: Human DPSC harvested from extracted third molars, between passages 3 and 5 were used in this study. Human DPSC were cultured in odontogenic differentiation medium containing 100 μg/mL ascorbic acid, 10 mM β-glycerophosphate, and 10 mM dexamethasone for up to 21 days. Actin cytoskeleton was stained using TRITC phalloidin. Gene expression analysis was performed to quantify differentiation markers, using real time quantitative PCR (qRT-PCR) analysis. Alizarin Red staining was carried out to examine mineralization potential of DPSC. Additionally, alkaline phosphatase activity was measured to determine the differentiation potential, using spectrophotometry analysis.
Results: Our findings demonstrate that human DPSC underwent differentiation after 21 days of exposure to odontogenic medium, as observed in alkaline phosphatase activity assay. qRT-PCR analysis revealed an up-regulation of differentiation markers in DPSC exposed to odontogenic medium. Interestingly, our investigations in DPSC with odontogenic lineage commitment displayed fewer and thicker actin filaments mostly located at the peripheral regions. Whereas, the undifferentiated cells showed large number of thin, and parallel actin filaments. On the contrary, inhibition of actin polymerization blocked odontogenic differentiation.
Conclusions: Actin cytoskeleton reorganization is an essential process in the differentiation of hDPSC along the odontogenic lineage. Thus, studies delineating the mechanisms driving the cytoskeletal changes may bring novel insight to our current understanding of regenerative endodontic therapies.