Signaling Pathways in DPSC Osteogenic Differentiation on Fluorapatite Modified Scaffolds

Objectives:
Previously, we showed that the fluorapatite (FA) coated polycaprolactone (PCL) nanofiber scaffold stimulated the differentiation and mineralization of dental pulp stem cells (DPSCs). Objective: The present study aims to explore the potential signaling pathways involved in mediating this differentiation and mineralization process.
Methods:
Methods: The Human Signal Transduction PathwayFinder™ RT²Profiler™ PCR Array was used to analyze the involvement of potential signal transduction pathways during the DPSC osteogenic differentiation. Based on the PCR array results and our previous data, a perturbation study of 4 signaling pathways, i.e. Hedgehog (cyclopamine), Insulin (PPP), Wnt (FH535) and FGF (SU5402), was performed. Moreover, the autophagy process, indicated by the expression of the microtubule-associated proteins 1 light chain 3A/B-II (LC3-II), and the cell osteogenic phenotypic changes were also studied.
Results:
Results: Comparing the cells grown on PCL+FA scaffolds to those on PCL only scaffolds, the transcript expression of BMP2, BMP4, PTCH1 and WNT2 (Hedgehog) and CEBPB (Insulin) decreased by more than half at day 7, whereas the expression of BMP2, BMP4, FOXA2, PTCH1, WNT1 and WNT2 (Hedgehog), CEBPB, FASN, HK2 (Insulin), CCND1 and MYC (Wnt) more than doubled at day 14 when obvious cell osteogenic differentiation occurred. Phenotypically, the ALP activity, OPN expression and the expression of autophagy marker, LC3-II were coincidently decreased in all the perturbation groups at day 14. Consistently, no positive Alizarin red staining was observed in the specimens from these four perturbation groups at day 28.
Conclusions: Conclusion: When obvious cell differentiation occurred at day 14, the inhibition of Wnt, Insulin, FGF and Hedgehog pathways significantly decreased the DPSCs osteogenic differentiation and mineralization. The osteogenic differentiation of DPSCs grown on FA modified PCL scaffolds appears to be positively modulated by the Wnt, Insulin, FGF and Hedgehog signaling pathways, which were further coordinated with and/or mediated through the cell autophagy process.