The Role Of Alpl Gene In Alveolar Bone Remodeling Under Orthodontic Forces

With the improvement of the people’s understanding of aesthetics,the tooth orthodontic treatment has gained more and more widely attention.Orthodontic treatment,which indicates the teeth to move in the alveolar bone under orthodontic force and eventually reach the normal position.Therefore,a growing number of studies have worded on how force mediated the periodontal ligament and alveolar bone reconstruction during tooth movement.Alpl gene is the tissue nonspecific alkaline phosphatase（TNALP）encoding gene.TNALP is a type of alkaline phosphatase（ALP）,which mainly expressed in bone and liver.In addition,TNALP plays a key role in the process of osteoblast differentiation and new bone formation.In vitro studies show that the higher amount of Alpl gene expression can be used as important indicator of mesenchymal stem cells osteogenesis differentiation.However,there is still lack of in vivo evidence of how Alpl gene participate in orthodontic tooth movement.In this study,Alpl knockout mice were applied to build the orthodontic tooth movement model,finding that the lack of Alpl gene affects the normal reconstruction of alveolar bone.Through cultivation of bone marrow mesenchymal stem cells（BMMSCs）derived from Alpl knockout and wild type mice,this study found that osteogenesis ability of BMMSCs decline after knockout Alpl gene.It has been reported that adenosine triphosphate（ATP）is a classic substrate of ALP.Therefore,the author further discusses the purine signaling pathway after Alpl knock out and found increase of P2Y2 receptors during orthodontic tooth movement.This study for the first time observed the crucial role of Alpl gene in the alveolar bone reconstruction and further discussed the downstream mechanism,providing theoretical basis for orthodontic tooth movement mechanism research.Part one:Alpl knockout inhibits orthodontic tooth movementObjective:To study the role of Alpl gene in orthodontic tooth movement.Methods:Alpl knockout mice B6.129 S7-Alpl^tm1Sor/J mice were generated.Because of the perinatal death of homozygous mice,the Alpl knockout mice used in this study were heterozygote.Agarose gel electrophoresis was applied to test mice genotype,heterozygous mice were chosen for further experiment,and wild type mice were used as control.Homemade torque device was applied to build orthodontic tooth movement model.The mice body weight was recorded.To measure maxillary first molar mesial movement distance,micro-CT analysis was utilized for three-dimensional reconstruction of maxilla.Histological staining was applied to observe the morphological changes of the two groups after alveolar bone remodeling.Immunofluorescence staining detected Alpl gene encoding protein ALP,early osteogenesis related transcription factor RUNX2,and the expression of Ki-67 protein involved in cell proliferation.Results:Successful breeding Alpl knockout mice and its weight and activity have no obvious difference compared with wild type.Successful build orthodontic tooth movement in mice model.The weight of two groups decreased obviously after 4 days of orthodontic force loading.Micro-CT shows maxillary first molar movement distance decreases after Alpl gene reduction.Immunofluorescence shows wild type mice ALP⁺cells increased on tension side,consisting with previous reports.While after reducing of Alpl gene,ALP⁺cells significant reduced in periodontal tissue.Osteogenesis early transcription factor RUNX2 expression decreased significantly after knockout of Alpl.In addition,the Ki-67⁺proliferate cells decreased obviously on tension side.Conclusion:Alpl knockout inhibits orthodontic tooth movement,indicating the important role of Alpl gene in periodontal remodeling.Part two:Alpl knockout after bone marrow mesenchymal stem cells between the osteogenesis ability declineObjective:To test and compare the BMMSCs biological performance of Alpl knockout mice and the wild-type mice.Methods:BMMSCs derived from of wild type and Alpl knockout mice were cultivated.Flow cytometry was used to detect mesenchymal stem cell surface markers CD146 and CD73,and CD34 hematopoietic stem cell surface markers.Real time quantitative PCR was applied to test Alpl gene expression differences between two groups of cells.Clone formation unit experiment was applied to test colony forming ability of two groups of cells.Osteogenesis induction of BMMSCs were used to compare alkaline phosphatase activity and mineralized nodule formation.Results:BMMSCs Alpl mice were polygon,less long spindle cell type.Flow detection between two groups of cells visible expressed CD146 and cd73 on the surface of the mesenchymal stem cells,and CD34 hematopoietic stem cell surface marker was barely expressed.Real time quantitative PCR results shows BMMSCs from Alpl mice express low level of Alpl gene.Two groups of cells have clone forming ability,but Alpl group cell colony formation rate was lower.Osteogenesis induction study showed that Alpl reduction leading to decrease of ALP activity and mineralized nodules formation.Conclusion:Alpl knockout inhibits osteogenesis ability of BMMSCs.Part three:Alpl gene affects orthodontic tooth movement through purine signaling pathwayObjective:Screening purine signaling pathway.Methods:Real time quantitative PCR screened P2X and P2Y receptor in purine signaling pathways.Adding extracellular ATP to select consistent change of P2X and P2Y receptor family.Immunofluorescence staining to observe its distribution in the process of orthodontic tooth movement.Results:Real time quantitative PCR shows the increase of P2Y2 and P2Y6 gene in Alpl and wide type BMMSCs treatment with ATP.Western Blot result shows P2Y2 and P2Y6 receptors after adding ATP were increased,but P2Y2 protein expression quantity increases more obviously.Immunofluorescence shows that P2Y2⁺cells increased significantly in Alpl mice.Conclusion:P2Y2 receptor participates in the alveolar bone reconstruction,which may be mediated via Alpl.However,the related mechanism still needs further research.