Expression Of Plexin-A1 During Mouse Molar Development And Its Effect On Odontoblastic Differentiation Of Dental Pulp Stem Cells

ObjectivesGeneration and development of teeth depends on the interaction between epithelium and mesenchyme.It is a complex progress that is regulated by a series of complex gene cascades.The formation of dental arch depends on the co-regulation of multiple signaling molecules and position signals,which are involved in the initiation,morphogenesis and dentition of dental development.However,the molecular and signal molecular regulatory networks that regulate the morphogenesis of the initiation,morphogenesis and pattern formation of tooth development are very complex.And the exact mechanism of tooth development is not fully understood at present,and many signal molecules may be involved.Plexins,a transmembrane receptor,are the major cell-surface receptors for semaphorins and play an important role in axon-guided,angiogenesis and immune response regulation.Nine different plexins have been found:Plexin-A1-A4,Plexin-B1－B3,Plexin-C1 and Plexin-D1.Although Plexin-A1 has been shown to play a role in lung and heart morphogenesis,its expression and function in non-neuronal tissues is unclear.The purpose of this study was to detect the expression of Plexin-A1 during tooth development and the function of Plexin-Al in odontoblastic differentiation of dental pulp stem cells.Materials and methods1.The expression of Plexin-A1 at different stages of mouse tooth development was detected by immunohistochemistry.2.Human pulp stem cells were obtained by tissue block-enzyme digestion method.Human pulp stem cells were obtained by monoclonal culture.The flow cytometry was used to detect surface markers of hDPSCs.Alizarin red staining and oil red O staining was used to detect the osteogenic,adipogenic differentiation potential of hDPSCs respectively.3.Lentivirus transfected human dental pulp stem cells and inhibited endogenous expression of Plexin-A1.And then cells were cultured in osteogenic induction solution for 3,7 and 14 days.The mRNA expression of mineralization related factors including ALP,OCN,Runx2 and DSPP were detected by qRT-PCR.The effect of silenced Plexin-A1 on alkaline phosphatase activity of hDPSCs was detected by ALP staining.Results1.Immunohistochemical results showed that Plexin-A1 was spatio-temporal specific in the development of mouse teeth.Plexin-A1 was expressed in the enamel organ at embryonic day 13.5(E13.5)of mouse embryo development.At E14.5 and E16.5,Plexin-A1 was found in the outer enamel epithelium,inner enamel epithelium,stellate reticulum and in the dental papilla.Plexin-Al was expressed in the ameloblast and odontoblast layer at E18.5.On the second day after birth(P2.5),Plexin-Al was mainly expressed in the odontoblast,ameloblast and matrix.On the 5th and 7th day after birth,Plexin-A1 was expressed in ameloblast layer,odontoblast layer and immature enamel.On the 13th day after birth,Plexin-A1 was mainly expressed in the reduced dental epithelium and the odontoblast layer.Plexin-Al was mainly expressed in the odontoblast layer and periodontal ligament at P21 and P282.The primary human pulp stem cells were cultured by the method of tissue mass and enzyme digestion successfully.The cells grew adherent to the bottle wall.Human dental pulp stem cells were obtained by monoclonal culture.CD44,CD90 and CD 105 were highly expressed and CD34 and CD45 were lowly expressed,which were detected by flow cytometry method.After 21 days of mineralization induction,alizarin red staining showed the formation of mineralized nodules.After 21 days of adipogenesis,oil red O staining showed droplet formation.3.After human dental pulp stem cells were infected with lentivirus,endogenous Plexin-Al expression was inhibited,and mRNA expression of mineralization related factors ALP,Runx2,OCN and DSPP was decreased after mineralization induction in transfected human dental pulp stem cells.ConclusionPlexin-A1 was spatio-temporal specific during mouse molar development.Inhibition of endogenous Plexin-Al expression in human dental pulp stem cells decreased the mRNA expression of mineralization related factors,including alkaline phosphatase(ALP),osteocalcin(OCN),Runt-related transcription factor 2(Runx2)and dentin sialophosphoprotein(DSPP),suggesting that Plexin-Al may play a necessary role in cell differentiation,morphogenesis and tissue mineralization during tooth development.