Sonic Hedgehog Signaling Regulates Odontoblast Proliferation and Differentiation In Vitro

Objectives: Recent studies indicate that Sonic hedgehog (Shh) plays important roles in developing tooth germs. In this study, we investigate the effects of Shh on odontoblast proliferation and differentiation in vitro using primary human dental pulp stem cells (hDPSC)(Gronthos et al, 2000). Methods: To determine whether Shh stimulates odontoblast proliferation, hDPSC were cultured for 1 and 5 days in medium containing different doses of recombinant Shh(rShh, 50 and 100ng/ml) and/or 5 mM cyclopamine which is Shh inhibitor, and were analyzed for cell proliferation by MTS assay. Next, to test whether Shh stimulates odontoblast differentiation, hDPSC were cultured for 15 or 18 days in different doses of rShh(50 and 100ng/ml) and/or 5 mM cyclopamine, and were analyzed for alkaline phosphatase (ALP) activity, alizarin red staining and RT-PCR analysis of dentinsialophosph protein (dspp). The ALP activity and cell proliferation assay data were analyzed by one-way factorial ANOVA followed by Scheffe's multiple comparison tests. Results: Shh induced apparent change in hDPSC proliferation at 1 and 5 days after treatment. As expected, cyclopamine inhibited the Shh-induced hDPSC proliferation. ALP activity and dspp expression were increased in cultures receiving rShh in hDPSC significantly (p<0.05). Treatment with rShh accelerated calcification of hDPSC dose-dependently, but did not accelerate calcification of the cells with cyclopamine. Conclusion: These results indicate that Shh promotes cell proliferation and differentiation in odontoblastic cell cultures. This work was partially supported by Grant-in-Aid for Scientific Research 17209062 and 18390154 from the Ministry for Education, Science, Sports and Culture of Japan.