Neu5Ac Induces Human Dental Pulp Stem Cell Osteo/Odontogenic Differentiation By Enhancing MAPK/ERK Pathway Activation

Sialic acid(SA)is a naturally occurring monosaccharide.SA is a component of cell surface sugar moieties that are associated with N-and O-glycan chains and glycolipids wherein it can be attached via ?2-3,?2-6,and ?2-8 linkages.It is primarily found in two major forms:N-acetylneuraminic acid(Neu5Ac)and Nglycolylneuraminic acid(Neu5Gc).Of these forms,only Neu5Ac is found in healthy humans,wherein it serves as an important regulator of interactions between cells and mediates the function of immune cells.Sialic acid is closely related to many oral diseases such as recurrent aphthous ulcer,oral potentially malignant disorders(OPMD),and oral cancers.Sialic acid plays different roles in different oral diseases.Patients with OPMD and oral cancers exhibit high concentration of sialic acid in serum and saliva,while patients with recurrent aphthous stomatitis and other oral inflammations exhibit a low concentration of sialic acid.Dental pulp stem cells(DPSCs)must undergo odontogenic differentiation in order to facilitate the process of dentin-pulp complex repair.As a kind of mesenchymal stem cells,DPSC has a great prospect in clinical treatment.The specific role plays by sialic acid in the context of DPSCs osteo/odontogenesis,however,has yet to be explored.Herein we sought to explore the ability of Neu5Ac to influence DPSC osteo/odontogenic differentiation and its molecular mechanism.Our study found that DPSCs can be isolated from human third permanent teeth and grown in vitro.Fluorescent microscopy was used to detect the existence of sialic acid on the DPSC membrane.Following the treatment of different concentrations of Neu5Ac and neuraminidase,which specifically removed sialic acid from the cell surface,the osteo/odontogenic differentiation of these cells was evaluated via mineralization,alkaline phosphatase,and in vivo assays.Treatment of DPSCs with Neu5Ac(0.1 mM or 1mM)significantly enhanced their mineralization ability and alkaline phosphatase activity.The expression levels of BSP,DMP1,and DSPP were also increased.Treatment of nude mice with ManNAc(the prerequisite form of Neu5Ac)also enhanced DPSC mineralization activity in vivo.In addition,the expression of genes related to osteo/odontogenic differentiation and MAPK signaling at different stages of this diferentiation process was analyzed in the presence or absence of Neu5Ac.Furthermore,Neu5Ac treatment enhanced p-ERK expression in DPSCs,while ERK pathway inhibition disrupted the ability of Neu5Ac to enhance the osteo/odontogenic differentiation of these cells.In summary,the results of the present study revealed that desialylation of DPSC by neuraminidase inhibits osteo/odontogenic differentiation while Neu5Ac treatment was sufficient to enhance DPSC osteo/odontogenic differentiation.We further determined that ERK signaling was necessary in order for Neu5Ac to mediate such enhanced differentiation in these cells.Future analyses will be essential to fully explore the underlying mechanisms whereby Neu5Ac influences DPSC osteo/odontogenesis.