The Mechanism Of PIEZO1 Mediates Mechanotransduction In Odontoblasts And VEGFA Mediates Human Dental Pulp Stem Cells Migration

Part Ⅰ:The Mechanism of PIEZO1 Ion Channels Mediates Mechanotransduction in OdontoblastsAim: Dentin hypersensitivity is a high-morbidity disease with an unknown mechanism.Current research suggests that odontoblasts are potential sensory cells that sense external stimuli and transmit pain signals.PIEZO1,as a specific mechanically activated ion channel,may play an important role in mechanical transduction in odontoblasts.Therefore,this study focuses on the function and mechanism of PIEZO1 ion channel in odontoblasts mechanotransduction.Methodology: DPSCs were cultured and induced into odontoblast-like cells.The expression of PIEZO1 in human dental section and odontoblast-like cells were observed by immunohistochemical(IHC)and immunofluorescence(IF)staining.The spatiotemporal expression of Piezo1 in odontoblasts during mouse tooth germ development was observed by IHC staining.Calcium ion fluorescence were real-time detected in odontoblast-like cells after stimulation with Yoda1 and Gs MTx4,fluid shear model with or without Calcium ion extracellular solution.The patch current clamp technique was used to record the current changes of odontoblast-like cells under mechanical stimulation,Yoda1 or Gs MTx4.ATP release were detected in odontoblastlike cells after stimulation with Yoda1 and/or Gs MTx4.The patch voltage clamp technique was used to record the action potential changes after ATP stimulation on the trigeminal neurons(TG),the current changes of TG under ATP stimulation was also recorded by patch current clamp technique.Results: IHC and IF staining showed that PIEZO1 ion channels was positive expressed in odontoblasts soma and odontoblastic process,but weakly positive expressed in dental pulp cells,DSPP and PIEZO1 was positive expressed in odontoblast-like cells in vitro.Piezo1 is gradually expressed in odontoblasts in the tooth germ of mouse after birth.After the shearing stress mechanical stimulation of the fluid shear model or Yoda1 on the odontoblast-like cells,the calcium ion fluorescence was increased rapidly,but with no obvious change under Gs MTx4 or non-Calcium ion extracellular solution.When single-cell mechanical stimuli were applied to odontoblast-like cells,evoked inward currents were recorded by patch-clamp electrophysiology.The peak of inward current was increased and the current inactivation became slow after mechanical stimulation with Yoda1,but the mechanical-stimulation inward current was almost disappeared after adding Gs MTx4.After adding Yoda1,the ATP released by odontoblasts was increased significantly,while Gs MTx4 reversed the release of ATP.Patch clamp detection showed that ATP evoked slow inward currents and increased the frequency of action potentials of trigeminal neurons.Conclusions: Odontoblasts were evoked fast inward current via PIEZO1 ion channel and released ATP neurotransmitter after external mechanical stimuli.ATP neurotransmitter induced slow inward currents and increased the frequency of action potentials of trigeminal neurons.These findings suggest PIEZO1 as a mechanical transducer in odontoblasts to mediate signal transduction under diverse pathophysiological conditions of the dentin.Part Ⅱ:The Mechanism of VEGFA Mediates Human Dental Pulp Stem Cells MigrationObjective: Dental pulp stem cells(DPSCs)play an important role in the repair and reconstruction of damaged dental pulp and dentin.DPSCs are located in the stem cell niches and need migrate to the damaged area to repair the impaired tissue.Stem cell migration is regulated by various cytokines secreted by the cells.VEGFA mediates the invasion of tumor cells and migration of endothelial cells,suggesting that VEGFA may play a key role in the homing and migration of DPSCs.In the present study,we investigated the effects and underlying molecular mechanisms of VEGFA on the migration of DPSCs,and aimed to unravel novel molecular drivers of DPSCs migration and offer new treatment targets for the repair of dentin damage.Methodology: The expression of VEGF in inflammatory pulp tissue and LPS-stimulated dental pulp cells was examined by q RT-PCR and IF staining.The migration of DPSCs was evaluated using transwell migration and wound healing assays.The activation of PI3 K,Akt,p38 and FAK signalling was detected by Western blot.Silence RNA(si RNA)technology was utilized to knockdown the expression of VEGFR1(Flt-1)and VEGFR2(Flk-1/KDR).PF573228(inhibitor of FAK),LY294002(inhibitor of PI3K),SB203580(inhibitor of p38)and SU5416(inhibitor of VEGFR2)were employed to investigate the effect of VEGFA on the migratory mechanism of DPSCs.Results: The expression of VEGF in inflammatory pulp tissue in vivo and LPSstimulated dental pulp cells in vitro were greater than control groups(P<0.05).VEGFA promoted the migration of DPSCs in a concentration-dependent manner.Several signalling pathways,including FAK,PI3 K,Akt and p38,were activated by VEGFA in a dose-and time-dependent manner in DPSCs.The VEGFA-induced migration of DPSCs was significantly inhibited with drug inhibitors such as PF573228,LY294002,SB203580 or SU5416(P<0.05).These signalling pathways activated by VEGFA were significantly suppressed by SU5416.The VEGFA-induced migration of DPSCs was significantly inhibited by SU5416 or transfection with si RNA of VRGFR2(P<0.05)but not VEGFR1 si RNA.Conclusion: VEGFA/VEGFR2 axis promoted the migration of DPSCs via the FAK/PI3K/Akt and p38 MAPK signalling pathways.These findings reveal a novel molecular mechanism for cell migration of DPSCs,which may contribute to the remodelling of pulp tissue and dentine.