Study On Expression And Roles Of Osterix During Orthodontic Periodontium Remodeling

Background and ObjectiveOrthodontic tooth movement is resulted from periodontal tissue remodeling induced by mechanical strength stimulation,moving in a certain direction.It is the forefront in the orthodontics study to realize the effect of orthodontic force on the periodontal tissue and to investigate the relationship between the mechanical force and bone remodeling.Periodontal ligament(PDL)is the connective tissue located between the tooth root and alveolar bone.The transmission of the force applied to the teeth to alveolar bone is mediated by the response of PDL to the force.PDL functions in bone remodeling during orthodontic tooth movement and has the ability of bone resorption and formation,inducing adaptation of periodontal tissues to the mechanical stress.The cells in PDL have been suggested to be multipotent cells and be composed of heterogenous cell populations that have the capacity to differentiate into either osteoblasts or cementoblasts depending on the microenvironment.These cells respond directly to orthodontic force and mechanical stimulation alone can induce the diferentiation of PDL cells to osteoblast-like cells,suggesting that the increased osteogenic differentiation and activity is responsible for the mechanical stress-induced bone remodeling.This attracts the interests of many researchers to investigate their behavioral change and roles in orthodontics.However,the molecular mechanism by which mechanical stress enhances osteoblast proliferation and diferentiation is complex and involves multiple signaling molecules and pathways,including,but not limited to,bone morphogenetic protein 2(BMP-2),insulin-like growth factorâ… (IGF-I),transforming growth factorβ1 (TGF-β1),vasoactive peptide,nitric oxide,prostaglandin E2 and 12(PGE2 and PG12),pertussis toxin-sensitive heterotrimeric G proteins,stretch-activated ion channels,integrins.Recent data indicate that intranuclear transcription factors are associated with the intracellular regulatory pathways that convert extracellular physical or mechanical stimuli into a coordinated cellular response.Osterix(Osx),also named Sp7 is a novel osteoblast-specific internuclear transcription factor belonging to the Sp/XKLF Family,which contains three C2H2-type zinc fingers at its C terminus.There are growing evidences indicating that Osx acts downstream of Runx2/Cbfa1 and plays an essential role in osteogenic differentiation and bone formation.Osx regulates the expression of a number of important osteoblastic marker genes such as osteocalcin(OC),osteonectin, osteopontin(OPN),bone sialoprotein(BSP),and collagen typeâ… (Col I).In Osx null embryos,despite the presence of Runx2/Cbfa1 expression at levels comparable to those in wild-type embryos,both early and late markers of osteogenic differentiation were absent or severely reduced and,thus,osteogenic differentiation and maturation were completely arrested.Osx null mice also showed the complete lack of both endochondral and intramembranous bone formation due to the absence of osteogenic differentiation.So,Osx has been considered essential for osteogenic differentiation and bone formation.Furthermore,the overexpression of Osx has been shown to be sufficient to guide differentiation of murine embryonic stem cells,bone marrow stromal cells,and adipose-derived stem cells towards the osteoblastic lineage in vitro.Previous studies have detected a positive expression of Osx mRNA and protein in tooth germs,which suggested that Osx may be involved in the tooth term development and periodontal tissue formation.But whether Osx participate in the differentiation of PDL cells and the periodontal tissue remodeling induced by mechanical strength stimulation has not been reported.Thus,the aim of the study is to examine the expression of Osx subjected to mechanical force in periodontal tissues and human PDL cells by the application of orthodontic force in vivo and in vitro,and to investigate the roles of Osx during orthodontic periodontium remodeling.This study will contribute to a better understanding of the molecular biology mechanism of periodontal tissue bone remodeling during tooth movement which may set the basis for clinical work.Methods1.Observe the changes of Osx expression in periodontal tissues during orthodontic tooth movement in ratsA titanium-nickel closed-coil spring was applied to the rat maxillary first molar (M1)and the upper incisors with a ligature wire to realize the M1 movement mesially with 40g force.The coil spring was kept constant and recorded for 0h,1h,2h,4h,8h, 12h,1d,3d,5d,7d or 14d.Upon completion of experiments,the maxillae were removed.The specimens were fixed in 4%paraformaldehyde in 0.1 M phosphate buffer for 12h and decalcified in 10%ethylene diamine tetraacetic acid(EDTA)at 4℃for 8 wks.After being dehydrated,cleared in xylene,and paraffin-embedded, 4μm serial sections were cut parasagittally.Immunohistochemical staining was carried out with anti-Osx goat polyclonal antibody to examine the expression of Osx.2.Observe the expression of Osx mRNA and protein after application of mechanical force on human PDL cells.Human PDL cells were isolated and cultured in vitro with explant method. Experiments were carried out with cells between passages 2 and 3.Approximately 2.5×10⁵ cells were seeded onto six-well cell culture plates and cultivated until they reached 80%confluence.The medium was then changed to DMEM supplemented with 2%FBS,to remain quiescent for 24h.Then the six-well cell culture plates were inserted into a centrifuge with a horizontal microplate rotor and centrifuged for 1,2,4, 6,8 or 12 h at 631 rpm,which corresponds to 80g.Total RNA and nuclear extracts were isolated.The expression of Osx mRNA and protein was measured by Real-time RT-PCR and Western Blot respectively.And Immunofluorescence assay was used to detect the expression and subcellular localization of Osx protein by green fluorescence.3.Observe the role of Osx in the mechanical stress-induced human PDL cells differentiationHuman PDL cells between passages 2 and 3 were seeded at 2.5×10⁵ cell/well into six-well plates.After the cultures reached approximately 80%confluence,human PDL cells were transfected with either an Osx expression vector pcDNA3.1 flag-Osx or the mock control vector pcDNA3.1 flag using Lipofectamine^TM2000,following the manufacturer's protocol.Four hours later,the medium was changed,and the cells were cultured in normal DMEM containing 10%FBS for 24h.Then,cells were centrifuged for 6h at 631 rpm.Before and after centrification,the expression of Osx mRNA and protein in untransfected cells,mock-transfected cells and Osx-transfected cells was measured by Real-time RT-PCR and Western Blot respectively. Furthermore,the changes of alkaline phosphatase(ALP)activity and mRNA expressions of Cbfal,ALP,OPN,OC,BSP and Col I genes were measured to assess the diferentiation of human PDL cells.4.Observe the expression of Osx mRNA and protein induced by recombinant human BMP-2(rh BMP-2)in human PDL cellsHuman PDL cells were seeded at 1.0×10⁵ cell/well into six-well plates and cultured in DMEM with 10%FBS.After 24 h,cells were cultured in 10%-FBS medium with or without 200ng/ml rh BMP-2 for 2,3,5,7,10,14 and 21 days.After incubation of each cell type,the cells were collected for total mRNA and protein extraction.For BMP-2 dose-response experiments,human PDL cells were cultured at 1.0×10⁵ cell/well and exposed to varying concentration of rhBMP-2 from 50,100, 150,200,250,300,400,to 600ng/ml.After 7 days of culture,the cells were collected for detection of mRNA and protein.Results1.The expression of Osx in periodontal tissues after orthodontic tooth movement in ratsIn the untreated control teeth,Osx was expressed at a low level in the rat periodontal ligament,especially located near the cementum.Application of orthodontic loading simultaneously induced a significant increase of Osx expression which reached the maximum at day 5.Strong direct Osx expression was observed in the periodontal ligament near the root surface at the compression site as well as near the root and alveolar bone surface in strain area.Osx immunoreactivity was stronger in periodontal ligament at the tension side compared to that at the compression side. Besides,the new bone at the tension side also showed an obvious Osx expression.2.The expression of Osx mRNA and protein after application of mechanical force on human PDL cells.In primary cultured human PDL cells,Osx mRNA proved to have a very weak expression.After 2h of mechanical stimulation,the mRNA expression of Osx increased slightly,but no statistical significance(P＞0.05)was noted.Subsequently, the expression drastically increased from 4h to 8h(P＜0.01),followed by a slight increase up to 12h.Based on Western blot analysis,Osx protein was not detected at the initial time point.After 4h of mechanical stimulation,a weak immunoreactive band of Osx protein about 46 kDa could be observed(P＜0.05).Subsequently,the protein expression increased gradually and peaked at 12h(P＜0.01).On the basis of immunofluorescence assay,no green fluorescence of Osx protein was detected in primary human PDL cells.After 4h,faint green fluorescence began to show in the cytoplasm of a small percentage of cells(P＜0.01).After 8h,green fluorescence was distributed in 40 to 50%of the cells,and many were clearly present in the nucleus;By 12h,intensive green fluorescence had translocated into the nucleus of most cells(about 87%).3.The role of Osx in the mechanical stress-induced human PDL cells differentiationAt 24h after transfection,Osx mRNA level in Osx-transfected cells increased significantly by 28.1- fold when compared with that in untransfected cells(P＜0.01), with the Osx protein also showing an intensive expression.In contrast,there was no significant difference in Osx mRNA and protein levels between mock-transfected cells and untransfected cells(P＞0.05).After 6h of mechanical stimulus,a significant increase in Osx expression was shown in all three groups.However,Compared to mock-transfected and untransfected cells,Osx-transfected cells further showed the highest Osx expression level in its mRNA and protein.The ALP activity in Osx-transfected cells exhibited a 2.5- fold increase when compared with that in untransfected or mock-transfected cells.After mechanical stimulus,ALP activity increased in mock-transfected cells as well as in untransfected cells.The overexpression of Osx in osx-transfected cells showed a more significant up-regulation of ALP activity,which was about 2.3- fold higher than the untransfected cells in response to mechanical force(P＜0.01).There was no difference in mRNA expressions of Cbfal and all the five osteogenic marker genes between mock-transfected and untransfected cells. Compared to untransfected cells,Cbfal mRNA level showed no change in Osx-transfected cells(P＞0.05),while the upregulated mRNA expressions of all the five osteogenic genes were observed(^*P＜0.05,^**P＜0.01).The 6h mechanical stimulus did not affect mRNA expression of Cbfal,but significantly enhanced mRNA expressions of all the five osteogenic marker genes in the three groups.Furthermore, the mRNA expressions of all five osteogenic markers in Osx-transfected cells still exhibited the highest levels(^*P＜0.05,^**P＜0.01).4.The expression of Osx mRNA and protein induced by rhBMP-2 in human PDL cellsAfter 7 days of treatment,rhBMP-2 at different concentraction all significantly upregulated the expression levels of Osx gene and protein.The Osx gene expression intensity was enhanced starting at 50ng/ml and significantly increased in a dose-dependent manner up to 200ng/ml,then showed slight increase from 200ng/ml to 600ng/ml.The expression change of protein was coincident with that of gene.At day 2 of 200ng/ml rhBMP-2 treatment,the expression levels of Osx mRNA appeared a slight increase,and then drastically increased from day 3(P＜0.05)up to day 10(P＜0.01)with a slightly decrease thereafter.At day 21,the expression was still obviously higher than control.Based on the Western blot analysis,at day 3 and 5 of rhBMP-2 treatment,a faint immunoreactive band of Osx protein began to be observed (P＜0.05).Subsequently from day 7,the expression level increased in a time-dependent manner up to day 21(P＜0.01). Conclusions1.Osx is involved in the periodontal tissue remodeling during orthodontic tooth movement in rats.2.It is proved by in vitro experiment that Osx participates in human PDL cells osteogenic differentiation and periodontal tissue remodeling induced by mechanical stress.3.Osx contributes to the mechanical stress-induced osteogenic differentiation of human PDL cells by upregulating the osteoblastic activity and the expression of osteogenic marker and functional genes.4.It is sugguested that being a molecular link,Osx probably plays an impotant signaling cascade and transmission role in the mechanical stress-induced osteogenic differentiation of human PDL cells.5.The mechanical stress upregulates the expression of Osx through BMP-2 signal transduction pathways.6.This study will contribute to a better understanding of molecule mechanism of bone remodeling during tooth movement which may set the basis for clinical work.