Methods: DPSCs isolated from human adult third molar were stably transduced with canonical Wnt-1 or the active form of b-catenin using retrovirus-mediated infection. Following selection, confluent cell layers were induced to differentiate toward an odontoblast-like lineage with ascorbic acid, dexamethasone and b-glycerophosphate. Post-differentiation analysis included staining for calcium mineral and alkaline phosphatase (ALP) and analysis of osteogenic lineage markers osteonectin, osteopontin, bone sialoprotein, and collagen I.
Results: Northern blot analysis found that Wnt-1 strongly induced the expression of matricellular protein osteopontin (OPN) and modestly enhanced the expression of type I collagen in DPSCs. Unexpectedly, Wnt-1 inhibited alkaline phosphatase (ALP) activity and the formation of mineralized nodules by more than 80% when compared to control DPSCs. Moreover, over-expression of b-catenin was also sufficient to suppress differentiation and mineralization.
Conclusion: Our results are the first demonstration that canonical Wnt/b-catenin signaling negatively regulates the odontoblast-like differentiation of DPSCs and may help to maintain their stem cell properties. Studies of crystal nucleation suggest that OPN is a potent inhibitor of mineral crystal growth at high concentrations. Thus, it is possible that the induction of OPN by Wnt-1 may play a role in the suppression of mineral formation in DPSCs.
Research supported by R01DE-016513 and T32DE-007057.