Effects of Necdin on odontogenic differentiation and mineralization of rat dental germ ectodermal mesenchymal stem cells

To investigate the regulative effect of neurally differentiated EC-cell-derived protein, Necdin, on the odontogenic differentiation and mineralization of rat dental germ ectomesenchymal stem cells（EMSCs）.

Immunofluorescence staining was used to detect the expression and distribution of Necdin in E13.5d and E19.5d rat dental germ. EMSCs derived from E19.5d rat dental germ were isolated and cultured, and identified with flow cytometry for cell surface antigens. The EMSCs were stably transfected with lentiviral vector of Necdin silencing. Then, the apoptosis of EMSCs was detected by flow cytometry in the negative control group（sh-NC）and silenced group（sh-Necdin）. The proliferation capacity of EMSCs was detected by CCK-8 assay, and the migration ability of EMSCs was detected by cell scratching assay. After mineralization induction, the regulative effect of Necdin on the odontogenic differentiation and mineralization of EMSCs was observed by alkaline phosphatase staining, alizarin red staining, RT-PCR and Western blotting.

The immunofluorescence results showed that Necdin was strongly expressed and concentrated in the inner enamel epithelium, outer enamel epithelium and in the contact area between epithelium and mesenchyme. After Necdin silencing, the EMSCs showed higher apoptotic rate, enhanced proliferation ability but weaker migration ability when compared with the cells from the sh-NC group（all P<0.01）. What's more, Necdin silencing also resulted in lighter alkaline phosphatase stain and less mineralized nodule formation, and up-regulations of the molecules related to tooth differentiation and mineralization at mRNA and protein levels（P<0.05）in the cells from the sh-Necdin group than those from the sh-NC group.

Necdin is specifically expressed in the future odontogenic differentiation and mineralization regions in rat dental germ. Its silencing promotes the apoptosis and proliferation, but inhibits the migration and the abilities of odontogenesis differentiation and mineralization in EMSCs. Necdin might play an important role in tooth development and mineralization.