Topographic cues of a novel bilayered scaffold modulate dental pulp stem cells differentiation by regulating YAP signalling through cytoskeleton adjustments

Objectives: Topographic cues can modulate morphology and differentiation of mesenchymal stem cells. This study aimed to determine how topographic cues of a novel bilayered poly (lactic-co-glycolic acid) (PLGA) scaffold affect osteogenic/odontogenic differentiation of dental pulp stem cells (DPSCs). Methods: The surface morphology of the scaffolds was visualized by scanning electron microscope, and the surface roughness was measured by profilometry. DPSCs were cultured on each side of the scaffolds. Cell morphology, expression of Yes-associated protein (YAP) and osteogenic/odontogenic differentiation were analysed by immunohistochemistry, real-time polymerase chain reaction, and Alizarin Red S staining. In addition, cytochalasin D (CytoD), an F-actin disruptor, was used to examine the effects of F-actin on intracellular YAP localisation. Verteporfin, a YAP transcriptional inhibitor, was used to explore the effects of YAP signalling on osteogenic/odontogenic differentiation of DPSCs. Results: The closed side of our scaffold showed smaller pores and less roughness than the open side. On the closed side, DPSCs exhibited enhanced F-actin stress fibre alignment, larger spreading area, more elongated appearance, predominant nuclear YAP localization and spontaneous osteogenic differentiation. Inhibition of F-actin alignments was correlated with nuclear YAP exclusion of DPSCs. Verteporfin restricted YAP localisation to the cytoplasm, down-regulated expression of early osteogenic/odontogenic markers and inhibited mineralization of DPSCs cultures. Conclusions: The surface topographic cues changed F-actin alignment and morphology of DPSCs, which in turn regulated YAP signalling to control osteogenic/odontogenic differentiation.