| In recent years,the oral health economy and value economy have been upgraded and the penetration rate of the orthodontic market has gradually increased,but there are still problems in the process of orthodontic treatment,such as long treatment,"black triangle" after orthodontic treatment,orthodontic root resorption,etc.These are all the challenges faced by orthodontists.Orthodontic tooth movement(OTM)is the process of mechanical force induced periodontal tissue remodeling.After stimulated by force,the tooth was transmitting the force signal to the periodontal tissue,and then the periodontal tissue cells converted the force signal into biochemical signals to regulate bone resorption.Bone cells are mechanosensitive cells that can receive mechanical signals through mechanical stimulus receptors on the cell membrane surface.In the process of OTM,mechanical force stimulated the compressive side of the alveolar bone,promoting osteoclast differentiation and inhibiting osteogenesis,making bone resorption activity more active than bone deposition,while on the tensile side,tensile force promoted osteogenic differentiation more actively,making bone deposition dominant.Many cytokines were involved in bone remodeling,among which erythropoietin hepatocyte kinase receptor B4 and its ligand B2(Eph B4/ephrin B2)were ligands and receptors expressed on the membrane surface of osteoblasts and osteoclasts,which has a regulatory effect on bone remodeling.Therefore,in order to investigate the molecular mechanism of bone remodeling during orthodontic tooth movement,the present study was based on the compress-tensile theory that "bone resorption was active on the compressive side and bone deposition was active on the tensile side",and combined with the important role of ephrin B2-Eph B4 signaling pathway in bone remodeling,we proposed the hypothesis that The ephrin B2-Eph B4 positive signaling correlates with active bone formation on the distraction side of orthodontics.Part I: The effect of mechanical tensile force on osteogenic differentiation OBJECTIVE: To investigate the effects of mechanical tensile force on osteoblast proliferation,differentiation and related signaling pathways.METHODS: Mouse osteogenic precursor cells MC3T3-E1 were cultured,and MC3T3-E1 were loaded with mechanical tensile force of 1000,2000 and 4000 με(physiological low,medium and high intensity)using a four-point bending cell force system with stimulation times of 0,2,6 and 12 h to simulate in vitro orthodontic force.The morphological changes of the cells after loading mechanical tensile force were observed by a microscopy,and the changes of cell proliferation were detected by the Cell CCK-8.After osteogenesis induction,the osteogenic differentiation of MC3T3-E1 was measured by ALP activity,ALP staining,q PCR and western blot to detect the expression of ALP,RUNX2,OPN and OCN in MC3T3-E1.The changes of ephrin B2-Eph B4 signaling,MAPKs and PI3K/Akt in MC3T3-E1 were examined after tensile force stimulation,too.RESULTS: Microscopic observation showed that mechanical tensile force elongated the morphology of MC3T3-E1 and significantly lengthened cell synapses.CCK-8results showed that,compared with the control group,mechanical tensile force of1000 and 2000 με had no effect on MC3T3-E1 proliferation,and force stimulation of4000 με significantly inhibited cell proliferation.ALP activity results showed that,compared with the control group,mechanical tensile force of 1000,2000 με promoted the expression of ALP in MC3T3-E1 compared with the control.the ALP and ARS staining results showed that 1000 and 2000 με of mechanical tensile force significantly promoted the osteogenic differentiation of MC3T3-E1.Therefore,in the subsequent experiments,we chose 2000 με as the optimal intensity to apply mechanical tensile force to MC3T3-E1.q PCR and western blot results showed that mechanical tensile force promoted the gene and protein level expression of osteogenic differentiation-related factors RUNX2,OPN and OCN in MC3T3-E1 compared with the control group.The expression levels were significantly increased at 6 h and 12 h of tensile force stimulation.In addition,q PCR and western blot results showed that mechanical tensile force promoted gene and protein expression of ephrin B2 and Eph B4 in MC3T3-E1,while the phosphorylation level of Eph B4 was also significantly increased.Meanwhile,the protein levels of p-ERK1/2,p-p38,p-JNK,p-PI3 K and p-Akt were also significantly changed by mechanical tension stimulation,all of which were upregulated.CONCLUSION: Mechanical tensile force of 1000 and 2000 με had no effect on osteoblast proliferation in vitro and significantly promoted osteogenic differentiation.Mechanical tensile force promoted the activation of ephrin B2-Eph B4 signaling,MAPKs and PI3K/Akt pathway in osteoblasts.Part II: Mechanical tensile force promoted osteoblast differentiation through ephrin B2-Eph B4 forward signalingOBJECTIVE: To verify the mediating effect of ephrin B2-Eph B4 signaling on the osteogenic differentiation by mechanical tensile force.METHODS: After osteogenesis induction,ephrin B2-Eph B4 forward signaling in MC3T3-E1 was blocked using the Eph B4 receptor-specific blocker NVP-BHG712.A mechanical tensile force of 2000 με was applied to MC3T3-E1 using a four-point bending cell force system,and the force was applied for 6 h.Subsequently,the expression levels of osteogenic differentiation-related factors ALP,RUNX2,OPN and OCN in MC3T3-E1 were measured by ALP activity,ALP staining,q PCR and western blot.RESULTS: The results of blocking Eph B4 receptor showed that NVP-BHG712 effectively inhibited the expression level of p-Eph B4 in MC3T3-E1,but had no effect on Eph B4.ALP activity,ALP staining and ARS staining results showed that after blocking ephrin B2-Eph B4 signaling,the ability of mechanical tensile force promoting the osteogenic differentiation of MC3T3-E1 was significantly decreased.q PCR and western blot results showed that after blocking ephrin B2-Eph B4 signaling,the ability of mechanical tensile force to promote MC3T3-E1 expression of RUNX2,OPN and OCN was also diminished.CONCLUSION: NVP-BHG712 effectively inhibits the phosphorylation of Eph B4 receptors in MC3T3-E1.ephrin B2-Eph B4 forward signaling mediated mechanical tensile force-induced osteogenic differentiation.Part III: Mechanical tensile force promotes bone deposition on the distraction side of OTM in rats via ephrin B2-Eph B4 positive signalingOBJECTIVE: To clarify the effect of ephrin B2-Eph B4 signaling on bone deposition on the tensile side of OTM in rats.METHODS: An OTM rat model was established: the ends of orthodontic nickel-titanium tension springs were fixed to the maxillary first molar and incisor of SD male rats with a tensile force of 50 g.Animal models were grouped(n=5): OTM group(modeling,periodontal injection of saline),ephrin B2-Fc group(modeling,periodontal injection of Eph B4 receptor-specific agonist ephrin B2-Fc,2 mg/kg/day),NVP-BHG712 group(modeled,periodontal injection of NVP-BHG712,5 mg/kg/day),and ephrin B2-Fc + NVP-BHG712 group(modeled,periodontal injection of ephrin B2-Fc and NVP-BHG712,2 mg/kg/day + 5 mg/kg/day).Samples were taken 7days after treatment,and the rats’ maxillary alveolar bone was scanned by micro-CT to measure the distance of maxillary first molar movement.The expression levels of ephrin B2,Eph B4 and p-Eph B4 in the periodontal tissues on the tensile side of OTM in rats were measured by immunohistochemistry(IHC).Hematoxylin-eosin(H&E)staining was used to observe the morphological changes of the alveolar bone on the tensile side of OTM in rats.The changes of alveolar bone structure on the tendile side of OTM in rats were observed by micro-CT scan reconstruction,and the parameters of alveolar bone trabeculae were analyzed by the analysis software.RESULTS: Micro-CT measurements showed no significant difference in the OTM distance of the maxillary first molar in the experimental groups compared with the OTM group.IHC results showed that the number of ephrin B2,Eph B4,and p-Eph B4 positive cells was significantly increased in the periodontal tissue of the maxillary OTM-tensile side of the rats compared with the control side.H&E staining and measurements showed that the ephrin B2-Fc group had more bone volume and higher density of trabeculae in the alveolar bone on the tensile side of the OTM compared with the OTM group;the NVP-BHG712 group had less bone volume and sparse trabeculae;the ephrin B2-Fc + NVP-BHG712 group had slightly higher alveolar bone than the NVP-BHG712 group but still lower than the ephrin B2-Fc group.Micro-CT results showed that compared with the OTM group,the alveolar bone density on the tensile side of OTM in the ephrin B2-Fc group was significantly increased,and the bone volume fraction(BV/TV),bone trabecular thickness(Tb.Th.),and bone trabecular number(Tb.N.)were significantly increased,and the bone trabecular spacing(Tb.Sp.)was significantly decreased;the alveolar bone density in the NVP-BHG712 group was significantly reduced,BV/TV and Tb.N.were decreased,while Tb.Sp.was increased;compared with the NVP-BHG712 group,the BV/TV and Tb.N.in the ephrin B2-Fc + NVP-BHG712 group were increased,but were still lower than those in the ephrin B2-Fc group.CONCLUSION: Orthodontic tensile force promoted the expression of ephrin B2 and Eph B4 in periodontal tissues,and at the same time,tensile force promoted the activation of ephrin B2-Eph B4 forward signaling in periodontal tissues.Ephrin B2-Eph B4 forward signal had a promotional effect on bone deposition on the tensile side of OTM in rats.Part IV: Ephrin B2-Eph B4 forward signaling mediate mechanical tensile force-induced osteogenic differentiation via ERK1/2and PI3K/ Akt pathways OBJECTIVE: To verify that ephrin B2-Eph B4 forward signaling mediate mechanical tensile force-induced osteogenic differentiation through ERK1/2 and PI3K/Akt pathways.METHODS: The Eph B4 receptor-specific blocker NVP-BHG712 was used to block ephrin B2-Eph B4 forward signaling.A mechanical tensile force of 2000 με was applied to MC3T3-E1 using a four-point bending cellular force system.The protein levels of p-ERK1/2,p-p38,p-JNK,p-PI3 K and p-Akt in MC3T3-E1 were detected by western blot.After osteogenesis induction,MC3T3-E1 was treated with ERK1/2pathway blocker trametinib and PI3 K pathway blocker LY294002,respectively.Then loading mechanical tensile force to the cells,and the expression levels of osteogenesis-related factors RUNX2,OPN and OCN in the cells were detected by q PCR and western blot.RESULTS: The western blot results showed that after blocking ephrin B2-Eph B4 signaling,the phosphorylation of ERK1/2,PI3 K and Akt in MC3T3-E1 was inhibited which induced by mechanical tensile force,while the phosphorylation levels of p38 and JNK were not significantly changed compared with those in the tensile force group.After blocking ERK1/2 and PI3 K pathways,q PCR and western blot results showed that mechanical tensile force-induced the osteogenic differentiation in MC3T3-E1 was also inhibited compared with the tensile force group,and this inhibition could not be reversed by ephrin B2-Eph B4 signaling-specific agonist ephrin B2-Fc.CONCLUSION: Ephrin B2-Eph B4 forward signaling mediates the activation of ERK1/2 and PI3K/Akt pathways in MC3T3-E1 which induced by tensile force.ERK1/2 and PI3K/Akt pathways were the downstream pathways of ephrin B2-Eph B4 forward signaling to mediate mechanical tensile force on inducing the osteogenic differentiation in MC3T3-E1. |
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