The Effect Of GSK-3β/β-catenin Signaling Pathway On Orthodontic Tooth Movement And Its Mechanism

PartⅠ GSK-3β/β-catenin pathway regulate the bone formation on tension site during orthodontic tooth movementObjective: To investigate the important role of GSK-3β/β-catenin signaling in tension-induced bone formation during orthodontic tooth movement.METHODS: Thirty-five male Sprague-Dawley rats aged 8 weeks were selected from experimental animal research center of Soochow University.The rats were divided into seven groups: control group(sham,n = 5),7 days tooth movement(7d,n=5),14 days tooth movement(14d,n=5);tooth movement using PBS(OTM,n = 5),tooth movement using LICL intervention(OTM +Li Cl group,n = 5),tooth movement using ICG-001 intervention(OTM+ ICG001 group,n = 5))And Li Cl + ICG-001(OTM +Li Cl + ICG001 group,n = 5).The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 3 mg / ml,0.25 mm diameter ligature was passed through between the first and second molar on the left maxillary of rat,It produces 0.49 N force.Rats in Li Cl group and Li Cl + ICG001 group were gavaged at the dose of 200 mg/kg/d LICL.The ICG group was injected daily at a dose of 10μg into the submucous of the mesial palatal root of the upper left and first left molar until sacrifice.In addition,the LICL gavage group also received the same dose of PBS or local injection of ICG001(10μg)daily until the onset of orthodontic force until they were sacrificed.Then micro-CT,histology and immunohistochemistry were detected.Results: The results of micro-CT and H & E staining showed that in the mechanical stress-induced rats,the distance between the first molar and the second molar was significantly larger than that of the control group(P <0.05)at 14 days after operation.,The periodontal space became larger and the fiber stretched and refined,but not observed in the control group.Some bone parameters such as BMD,BV / TV and Tb.Th increased significantly,while with orthodontic force,Tb.Sp Significantly decreased in GSK-3β inhibitor stimulated rats,Both BV / TV and Tb.Th indicators were significantly increased,but Tb.Sp was significantly reduced;immunohistochemistry results showed that more alkaline Phosphatase and Osterix positive cells as cause of new bone formation were observed in the mechanical tension-induced rat’s tension position but not in the control group.Compared with the control group,the phospho-Ser9-GSK-3β had a stronger staining signal,however,mechanical load had little effect on GSK-3β staining in rats.On the 7 day of staining,the expression of phospho-Ser9-GSK-3β in Day 14 group began to decline,but still higher than the untreated rat body The expression of phospho-Ser9-GSK-3β was significantly increased by mechanical loading,while the decreased GSK-3β significantly increased the BMD of alveolar bone tension(132.3 versus 160.8mg / cm3;P <0.01).ALP and Osterix positive cells significantly increased in alveolar bone in the ROI area of OTM+LICL group.The results of LICL intervention showed that compared with untreated rats,In lithium chloride treatment,the expression of phospho-ser9-gsk-3βwere significantly increased in rats exposed to loading force.There were many β-catenin positive cells in the GSK-3β inhibitor treatment group.After ICG-001 topical treatment,The effects of lithium chloride on bone mass,ALP and Osterix expression were attenuated;The effect of lithium chloride on bone mass,ALP and Osterix expression was greatly impaired after topical treatment with ICG-001.Conclusion: The activity of GSK-3β/β-catenin signaling pathway is related to the orthodontic force induced bone formation during orthodontic tooth movement.Pharmacological inhibition of GSK-3βcan accelerate tension-induced bone formation without affecting tooth movement speed.Can be seen,GSK-3β/β-catenin signaling pathway is activated contribute to orthodontic bone remodeling,can be used as a potential target for clinical treatment.Part Ⅱ GSK-3β/β-catenin pathway regulate the bone absorption on compression site during orthodontic tooth movementObjective: To explore the important role of GSK-3 β/β-catenin signaling pathway in the bone absorption of compression side during orthodontic tooth movement.Methods: Thirty-five male Sprague-Dawley rats aged 8 weeks were selected from experimental animal research center of Soochow University.The rats were divided into seven groups: control group(sham,n = 5),7 days tooth movement(7d,n=5),14 days tooth movement(14d,n=5);tooth movement using PBS(OTM,n = 5),tooth movement using LICL intervention(OTM +Li Cl group,n = 5),tooth movement using ICG-001 intervention(OTM +ICG001 group,n = 5),And Li Cl + ICG-001(OTM +Li Cl + ICG group,n = 5).From the morphological point of view,using micro-CT detection of osteoclast staining results,and by immunohistochemical staining periodontal membrane markers of MMP-9,RANKL and canonical Wnt signaling pathway molecules GSK,P-GSK,β-catenin and Sclerostin in order to determine whether MMP-9 and RANKL are related to osteoclasts,whether GSK,P-GSK,β-catenin are involved in Wnt signaling pathway,Whether the classic Wnt signaling pathway is activated.Results: The results of HE and microscopical CT showed that when the orthodontic force was loaded,the first molar deviated from the near middle movement,and the distance between the second molar was increasing.In the fourteenth day after the operation,the distance between the first molar and the second molar was significantly greater than that of the control group.Under the action of mechanical load pressure,the periodontal ligament fibers in the rat were arranged irregular and incomplete.After 7 days,the periodontal gap narrowed,alveolar bone destruction and bone edge resorption of bone resorption.While the control group arranged regularly,periodontal fiber width uniform,uniform distribution of fibroblasts in the periodontal fibers,both cytoplasm and nucleus with clear structure;TRAP staining showed that all groups of mobile teeth rat periodontal tissue were observed in osteoclasts,osteoclasts are less visible at day 0,with the increase of loading the rats moved tooth pressure side of alveolar bone edge,multinucleated red cytoplasm,osteoclast blue nucleus,mostly in bone resorption and osteoclast number increased with increasing days after 0 days: after only less amount of osteoclasts,when reached the maximum the value of force for 7 days,while the visible alveolar bone obvious absorption phenomenon,beginning 14 days when the afterburner began to reduce the number of osteoclasts.Immunohistochemical staining showed that the number of MMP-9 and RANKL positive cells were brown yellow were increase with the increase of days: and when the afterburner afterburner 0 days,MMP-9 and RANKL positive cells were less in quantity,when after 7 days,the number of MMP-9 and RANKL positive cells were significantly increased when added to force 14 days later,the number of MMP-9 and RANKL positive cells were significantly reduced again,a statistically significant difference between the groups(P<0.05);the number of GSK,P-GSK,-catenin,scerostin β four positive cells with different changes: when the afterburner days after 0 days,the pressure side of alveolar bone,the number of-catenin positive cells in P-GSK β is not much;7 days of orthodontic tooth loading.P-GSK,the number of β-catenin positive cells increased,the expression of the most,the number of GSK positive cells decreased,the expression of at least 14 days;afterburner,compared with afterburner 7 days,P-GSK,β-catenin positive cells The number of cells decreased,but more than 0 days,the number of GSK-3βpositive cells increased or had no significant difference;for the GSK-3β inhibitor stimulation in the rat,two indicators of BV/TV and Tb.Th decreased significantly in all experimental group rats were mobile dental periodontal tissue were observed in osteoclasts.But the number of positive cells in each group is different,the most in OTM+Li Cl group;when the pressure of rats induced by intragastric administration of Li Cl for 7 days,the number of β-catenin and P-GSK positive cells increased,the number of GSK-3β positive cells decreased GSK-3β/β-catenin signaling pathway is activated,the number of RANKL cells increased significantly,MMP-9 when the pressure;to rats by gavage for 7 days ICG001,GSK-3 β/β-catenin signaling pathway was inhibited,expressed as the number of β-catenin and P-GSK positive cells decreased,the number of GSK-3β positive cells increased β.It is also found that the GSK-3β inhibitor LICL by gavage every day can significantly reduce the pressure side during orthodontic tooth movement and alveolar bone MMP-9 RANKL cells;when the Li Cl intervention to the pressure of rats induced by local injection of ICG001 at the same time,the activation of GSK-3 β / β-catenin signaling pathway is inhibited at the same time,compared with Li Cl group the expression of-catenin and P-GSK,decreased β,GSK-3 expression increased,MMP-9 and RANKL positive cells decreased,bone mass increased,but still more than the control group.Conclusion: With compression force,GSK-3β/β-catenin signaling pathway is involved in orthodontic tooth movement during orthodontic tooth movement and is related to osteoclast formation in orthodontic tooth pressure.The results also suggest that the process of mechanical load-induced bone resorption is mediated by the GSK-3β / β-catenin signaling pathway.However,due to the large research space of stem cells and the complicated signal pathways involved,the mechanism of Wnt signaling pathway regulating osteoclasts remains unclear and remains to be further studied.Part Ⅲ GSK-3β/β-catenin pathway regulate the bone formation of osteoblast with tension force during orthodontic tooth movement in vitro experimentsObjective: To explore the relevant factors and mechanisms of osteogenic differentiation that affect the process of orthodontic tooth movement from the biological point of view,providing a theoretical reference for orthodontic tooth movementMethods: In this study,SAOS2 cells were selected for in vitro experiments.By using the traction force to simulate the stress environment of cells in vitro and molecular biological detection methods,To explore the relevant factors and mechanism of osteogenesis that affect the process of orthodontic tooth movement.Results: The results of micro-CT showed that the cells appeared irregular,mononuclear,circular or oval shape before force(0h),and the whole cell bodies were occupied by these cells.After the cells were stressed,The shape gradually grew from similar circular to fibrous,and arranged with a certain directionality,and with the extension of time,the number of fibrous cells more and more;ELISA test results showed that ALP activity with the force(P <0.05).ALP gene expression increased similarly with the increase of time,ALP gene expression increased to the maximum at 4h,and was significantly higher than that of the control group(P <0.05).The results of RT-PCR showed that ALP increased gradually with the increase of time,When the force time reaches 4h,both ALP and OCN reach the maximum value,RUNX2 decreases with time,and remains basically unchanged at 3h,while OSTERIX changes first and then decreases with the increase of force time,Statistically significant different(P <0.05);Western bolt assay showed that 15% of the mechanical strain RUNX2 and stimulates the expression of the OCN.Compared with the control group,the expression of OCN increased significantly at 1h,3h and 6h in each stress group,the difference was statistically significant(P <0.0.5),while the expression of RUNX2 and OCN were different,The expression of RUNX2 increased first,then decreased and then increased with the increase of time,the expression of RUNX2 was significantly different(P <0.0.5),while the expression of GSK-3B,P-GSK andβ-catenin increased with the increase of the time and increased to the peak at 6h.The results of ALP activity also showed that Li Cl co-cultured with osteoblasts was only affected by Li Cl and was inhibited by Li Cl + Compared with the control group,the activity of ALP in ICG001 intervention group was significantly increased(P <0.05),compared with the control group,the activity of ALP in ICG001 intervention group was significantly lower than that in the control group(P < 0.05).Compared with the control group,the content of GSK-3β,p Ser9-GSK-3β and β-catenin increased significantly(P <0.05),but when ICG001 was added,GSK-3β and p Ser9-GSK-3β and β-catenin levels were significantly decreased;orthodontic tooth movement At the 7th day of intervention,the changes of biochemical indexes such as AST,ALT,ALP,BUN,CRE and UA in the blood samples of the rats in each group showed no significant difference(P> 0.05).Conclusions: Li Cl can activate GSK-3β / β-catenin signaling pathway to regulate osteoblast differentiation.Meanwhile.