Osterix在正畸牙周组织改建中的表达及调控机制研究
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摘要
背景和目的
正畸牙移动是通过在机械力作用下牙周组织发生改建来完成的。其中尤以牙槽骨的改建最为重要,它是骨吸收和骨形成的动态平衡过程。目前研究证实在牙槽骨改建的过程中,牙周膜的生物力学反应起着至关重要的介导和调控作用。牙周膜是具有成骨能力的骨/牙界面,是一种变异的骨膜,具有明显的骨吸收和骨形成能力。已有研究证实牙周膜细胞中包含具有分化潜能的成纤维细胞群体,正畸机械力能诱导其表达一些成骨细胞的表型和功能蛋白,向成骨样细胞分化。牙周膜细胞分化为成骨样细胞,参与骨吸收和骨形成,是正畸骨改建的关键。
牙周膜细胞通过特定的信号转导途径对机械力做出反应,引发一系列细胞生物学反应。这是一个复杂的过程,在牙周膜细胞内及周围发生许多层网状反应,而将应力信号转变为分子事件(信号转导),引起牙周组织改建。此过程涉及多种信号转导通路,比如:骨形成蛋白2(BMP-2)、胰岛素样生长因子Ⅰ(IGF-Ⅰ)、转化生长因子β1(TGF-β1)、P物质、血管活性肽、前列腺素E2(PGE2)、前列腺素Ⅰ2(PGⅠ2)、动力敏感钙离子通道和白介素系列等。近年少量研究发现,一些核内转录因子(c-Fos、c-Jun、核心结合因子α1和转录激活因子4等)也参与了牙周膜细胞内调控途径,将细胞外物理或机械刺激转化为协调的细胞反应。
Osterix(Osx)是新近发现的调控成骨细胞分化和骨形成的一个关键转录因子。人类Osx同源物又称为Sp7(specific protein7),属于Sp/XKLF家族,是一种含有锌指结构的转录因子。Osx调控许多重要的成骨基因的表达,如骨钙素(osteocalcin,OC)、骨粘素、骨桥素(osteopontin,OPN)、骨涎蛋白(bone sialoprotein,BSP)和a1(Ⅰ)型胶原蛋白(collagenⅠ,ColⅠ)。Osx单独作用就可有效诱导鼠胚胎干细胞、骨髓基质细胞和脂肪干细胞分化为成骨细胞系。在Osx基因剔除小鼠胚胎中,成骨细胞的分化受到阻碍,各种成骨分化标志的表达水平也严重降低或缺如。其中OC作为一种高度特异性的晚期成骨细胞标志物的表达缺失,说明成骨细胞的分化、成熟被完全阻断。研究发现,15.5d的Osx(-/-)小鼠胚胎中无矿化反应,完全丧失骨形成能力;新生的Osx(-/-)小鼠因矿化缺失,呼吸困难,出生后15min内即死去。这些研究说明Osx是成骨细胞分化和骨发育不可缺少的调节因子。2002年,Nakashima等研究发现Osx在牙胚的间叶细胞中有阳性信号显示;2006年Kumamoto H等研究也发现Osx mRNA在牙胚、造釉细胞瘤牙源性组织中均有阳性表达。这些表明Osx可能在牙齿、牙周组织的发育中具有重要的作用。Osx作为成骨细胞分化的关键因子,与骨发育和牙齿发育关系的研究正在不断开展。但Osx在正畸牙齿移动牙周组织的表达和分布,机械力作用下在体外培养的人牙周膜细胞中的表达变化情况,以及在人牙周膜细胞受力后向成骨样细胞分化的过程中所起的作用,国内外尚未有报道。
本研究通过建立大鼠正畸牙齿移动的动物模型和体外培养人牙周膜细胞,结合体内外加力实验技术和质粒转染等分子生物学技术,拟从组织、细胞水平以及分子生物学角度探讨Osx在正畸牙齿移动过程中对牙周组织改建的影响及作用机制,以期为进一步系统性研究正畸力作用下牙周组织骨改建的分子生物学机制开辟一条新途径。
方法
1.观察大鼠正畸牙周组织改建过程中Osx的表达变化
以大鼠上颌右侧第一磨牙为实验牙,采用拉簧加力法建立正畸牙齿移动的动物模型。在加力0h、1h、2h、4h、8h、12h、1d、3d、5d、7d和14d后,处死大鼠,固定,脱钙,制作大鼠第一磨牙牙周组织石蜡切片,采用HE染色观察牙周组织形态学变化,并采用免疫组化方法半定量分析Osx在牙周组织的表达变化。
2.观察机械力加载下人牙周膜细胞Osx mRNA和蛋白的表达变化
采用组织块法培养原代人牙周膜细胞,并做波形丝蛋白抗体和角蛋白抗体染色鉴定。取生长状态良好的第2-3代人牙周膜细胞,按2.5×10~5个/孔接种于6孔细胞培养板培养。待细胞融合80%,换用含2%FBS的条件培养液继续培养24h。后将六孔板置于37℃离心机加力支架中,实验组以离心机加力(631rpm,约80g相对离心力),加力时间分别为1h、2h、4h、6h、8h和12h。加力结束后,采用Real-time RT-PCR和Western blot方法分别观察Osx mRNA和蛋白表达情况;同时,采用细胞免疫荧光技术检测Osx蛋白绿色荧光在细胞内的表达定位,并统计分析Osx蛋白阳性表达的细胞数量及其核内阳性表达的细胞比例。
3.观察Osx过表达对人牙周膜细胞骨向分化的影响
取生长状态良好的第2-3代人牙周膜细胞,按2.5×10~5个/孔接种于6孔细胞培养板培养。待细胞融合80%,按阳离子脂质体Lipofeetamine ~(TM)2000说明操作,用获赠的重组质粒pcDNA3.1 flag-Osx和空载体质粒pcDNA3.1 flag转染人牙周膜细胞。采用Real-time RT-PCR和Western blot方法检测未转染、转染空质粒(pcDNA3.1 flag)、转染目的基因(pcDNA3.1 flag-Osx)三组细胞Osx mRNA和蛋白表达水平;应用试剂盒检测三组细胞碱性磷酸酶(alkaline phosphatase,ALP)活性变化;并采用RT-PCR技术观察Cbfal及相关成骨标志基因(ALP、OPN、OC、BSP和ColⅠ)的mRNA表达情况。三组细胞均加载6h离心力(631rpm)后,再次观察三组细胞的上述检测指标变化。
4.观察人重组骨形成蛋白2(rhBMP-2)作用下人牙周膜细胞Osx的表达
取生长状态良好的第3代细胞,以1×10~5个/孔的密度接种于6孔细胞培养板中。孵育24小时后,加入含rhBMP-2的培养液进行培养。实验分组1)按照不同的浓度分组:实验组用含10%FBS和浓度分别为50、100、150、200、250、300、400、600ng/ml rhBMP-2的DMEM培养液培养,诱导7d。每种浓度加6孔。2)按培养时间分组:实验组用含10%FBS和浓度为200ng/ml的rhBMP-2的培养液分别培养2d、3d、5d、7d、10d、14d和21d。每组加6孔。两对照组细胞均用含10%FBS的DMEM培养液培养。培养结束后,采用Real-time RT-PCR和Western blot方法检测各组细胞Osx mRNA和蛋白表达水平。
结果
1.大鼠正畸牙周组织改建过程中Osx的表达变化
在正常对照组,Osx在牙周膜呈弱阳性表达,分布较均匀,在靠近牙骨质侧牙周膜部分着色相对较重。正畸加力后大鼠牙周组织中Osx表达增强,表达贯穿正畸牙周组织改建的全过程。加力1d时,在压力区和张力区牙周膜Osx阳性着色均已显著增强,着色分布较均匀,且两侧着色强度无明显差异。随着加力时间的延长,Osx着色分布发生一定的改变。在张力区,靠近牙骨质和牙槽骨表面的牙周膜区域强阳性表达;在压力区,靠近牙骨质表面的牙周膜强阳性表达,而在发生明显骨吸收的牙槽骨侧无明显着色,而且张力区整体上比压力区阳性染色深,新骨形成区域阳性染色较强。
2.机械力作用下人牙周膜细胞Osx mRNA和蛋白的表达变化
Osx mRNA在正常人牙周膜细胞中的表达极其微弱。在机械力加载下,OsxmRNA的表达发生了一系列变化:细胞加力1h、2h时,Osx mRNA的表达略升高,但无统计学意义;加力4h时,表达开始明显增强(P＜0.01);随后快速增加至8h,表达显著增强(P＜0.01);后表达缓慢增加,持续到12h加力结束。
Osx蛋白在正常人牙周膜细胞中未见表达。细胞加载80g离心力1h、2h时,仍未检测到Osx蛋白表达(P＞0.05);加力4h时,呈现弱表达(P＜0.05);6h、8h时,表达逐渐增加(P＜0.01);12h时,表达显著增强,达到最高水平。变化具有时间依从性。
免疫荧光检测结果显示:正常的人牙周膜细胞内无绿色荧光显现;加力1h、2h的细胞内仍未检测到Osx阳性表达产物;加力4h后,少量细胞的细胞质内开始呈现微弱的绿色荧光(P＜0.01);加力8h时,约40%-50%的细胞发出明显的绿色荧光,细胞质、细胞核内均有阳性表达产物;加力12h时,大部分细胞(约87%)呈现强阳性表达,且主要集中在细胞核内。
3.Osx过表达对人牙周膜细胞骨向分化的影响
转染24h后,转染目的基因pcDNA3.1 flag-Osx组Osx mRNA表达水平相对未转染细胞升高28.1倍,同时Osx蛋白亦呈现一个强烈的表达(P＜0.01)。相反,转染空质粒(pcDNA3.1 flag)组的Osx mRNA和蛋白水平相对未转染组无明显变化(P＞0.05)。加力6h后,未转染组、转染空质粒组、转染Osx组三组细胞Osx表达水平均明显升高,但转染Osx组Osx mRNA和蛋白表达增加量约为其他两组的2倍。
正常人牙周膜细胞表达ALP,转染目的基因Osx后,ALP表达活性明显升高(P＜0.05),约为正常细胞的2.5倍,但转染空质粒组ALP表达未发生明显变化。三组细胞受到80g离心力,ALP表达均升高,但转染空质粒与未转染细胞相比,受力后ALP表达无明显差异(P＞0.05),而转染Osx质粒组受力后ALP表达进一步增强(P＜0.01),增长量约为未转染组的2.3倍。
转染空质粒组与未转染组间,Cbfal及五个成骨标志基因(ALP、OPN、OC、BSP和ColⅠ)的mRNA表达变化无明显差异(P＞0.05)。与未转染组相比,细胞转染目的基因Osx后,Cbfal mRNA表达无明显变化(P＞0.05),但五个成骨标志基因的mRNA表达均明显升高(~*P＜0.05,~(**)P＜0.01)。加力6h后,三组细胞的Cbfal mRNA表达无明显变化,而ALP、OPN、OC、BSP和Col I的mRNA表达水平均升高。但转染空质粒组与未转染组相比,受力后五个成骨标志基因变化无明显差异(P＞0.05);转染Osx细胞与未转染细胞相比,受力后五个成骨标志基因mRNA表达进一步上调(~*P＜0.05,~(**)P＜0.01)。
4.rhBMP-2作用下人牙周膜细胞Osx的表达
人牙周膜细胞在不同浓度rhBMP-2诱导7d后,相对对照组,Osx mRNA和蛋白均有明显的表达,但其表达强度不同,当浓度小于200ng/ml时,随着rhBMP-2浓度的增加,Osx mRNA和蛋白的表达均逐渐显著增强,当大于200ng/ml时,随着rhBMP-2浓度的增加,Osx mRNA和蛋白的表达略增加,基本趋于平缓。
在200ng/ml rhBMP-2不同时间诱导下,Osx mRNA表达变化具有时效性,具体如下:2d时,Osx mRNA表达略增强,但仍较弱(P＞0.05);培养3d时,Osx mRNA表达开始上调(P＜0.05);后迅速增强;10d时达到最高峰(P＜0.01);后逐渐减弱,但仍显著高于正常人牙周膜细胞中的水平。200ng/ml rhBMP-2诱导人牙周膜细胞2d时,未检测到Osx蛋白的表达;培养3d、5d时,Osx蛋白开始略有表达(P＜0.05);后表达逐渐增强,持续至21d(P＜0.01),表达趋于平稳。
结论
1.大鼠正畸牙齿移动过程中,牙周组织Osx表达增强贯穿牙周组织骨改建的全过程,且变化具有一定时空规律性。从而确定Osx参与了大鼠体内牙齿移动过程中的牙周组织骨改建。
2.应力刺激诱导人牙周膜细胞骨向分化过程中,Osx基因表达增强、蛋白合成活化,从而发挥信号转导作用。从体外细胞水平证实Osx参与了正畸力诱导的人牙周膜细胞骨向分化和牙周组织骨改建过程。
3.Osx通过上调成骨表型蛋白和功能蛋白的表达,促进加载下的人牙周膜细胞向成骨样细胞分化。
4.Osx是机械刺激和人牙周膜细胞骨向分化间的一个重要分子环节,在机械力诱导人牙周膜细胞成骨分化过程中,发挥着信号级联放大和转导调控作用。
5.BMP-2信号通路可能是介导机械信号诱导Osx表达的一条重要途径。
6.本研究为进一步系统性探讨机械力作用下牙周组织骨改建的分子生物学机制开辟了一条新途径,有助于更好的理解正畸牙齿移动的机理,从而为加快牙齿移动的正畸临床研究提供理论依据。
Background and Objective
Orthodontic 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 factorⅠ(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 typeⅠ(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.
Methods
1.Observe the changes of Osx expression in periodontal tissues during orthodontic tooth movement in rats
A 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 2.5×10~5 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
differentiation
Human PDL cells between passages 2 and 3 were seeded at 2.5×10~5 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~(TM)2000,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 cells
Human PDL cells were seeded at 1.0×10~5 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 1.0×10~5 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.
Results
1.The expression of Osx in periodontal tissues after orthodontic tooth movement in rats
In 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 differentiation
At 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 cells
After 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).
Conclusions
1.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.
正畸牙移动是通过在机械力作用下牙周组织发生改建来完成的。其中尤以牙槽骨的改建最为重要,它是骨吸收和骨形成的动态平衡过程。目前研究证实在牙槽骨改建的过程中,牙周膜的生物力学反应起着至关重要的介导和调控作用。牙周膜是具有成骨能力的骨/牙界面,是一种变异的骨膜,具有明显的骨吸收和骨形成能力。已有研究证实牙周膜细胞中包含具有分化潜能的成纤维细胞群体,正畸机械力能诱导其表达一些成骨细胞的表型和功能蛋白,向成骨样细胞分化。牙周膜细胞分化为成骨样细胞,参与骨吸收和骨形成,是正畸骨改建的关键。
牙周膜细胞通过特定的信号转导途径对机械力做出反应,引发一系列细胞生物学反应。这是一个复杂的过程,在牙周膜细胞内及周围发生许多层网状反应,而将应力信号转变为分子事件(信号转导),引起牙周组织改建。此过程涉及多种信号转导通路,比如:骨形成蛋白2(BMP-2)、胰岛素样生长因子Ⅰ(IGF-Ⅰ)、转化生长因子β1(TGF-β1)、P物质、血管活性肽、前列腺素E2(PGE2)、前列腺素Ⅰ2(PGⅠ2)、动力敏感钙离子通道和白介素系列等。近年少量研究发现,一些核内转录因子(c-Fos、c-Jun、核心结合因子α1和转录激活因子4等)也参与了牙周膜细胞内调控途径,将细胞外物理或机械刺激转化为协调的细胞反应。
Osterix(Osx)是新近发现的调控成骨细胞分化和骨形成的一个关键转录因子。人类Osx同源物又称为Sp7(specific protein7),属于Sp/XKLF家族,是一种含有锌指结构的转录因子。Osx调控许多重要的成骨基因的表达,如骨钙素(osteocalcin,OC)、骨粘素、骨桥素(osteopontin,OPN)、骨涎蛋白(bone sialoprotein,BSP)和a1(Ⅰ)型胶原蛋白(collagenⅠ,ColⅠ)。Osx单独作用就可有效诱导鼠胚胎干细胞、骨髓基质细胞和脂肪干细胞分化为成骨细胞系。在Osx基因剔除小鼠胚胎中,成骨细胞的分化受到阻碍,各种成骨分化标志的表达水平也严重降低或缺如。其中OC作为一种高度特异性的晚期成骨细胞标志物的表达缺失,说明成骨细胞的分化、成熟被完全阻断。研究发现,15.5d的Osx(-/-)小鼠胚胎中无矿化反应,完全丧失骨形成能力;新生的Osx(-/-)小鼠因矿化缺失,呼吸困难,出生后15min内即死去。这些研究说明Osx是成骨细胞分化和骨发育不可缺少的调节因子。2002年,Nakashima等研究发现Osx在牙胚的间叶细胞中有阳性信号显示;2006年Kumamoto H等研究也发现Osx mRNA在牙胚、造釉细胞瘤牙源性组织中均有阳性表达。这些表明Osx可能在牙齿、牙周组织的发育中具有重要的作用。Osx作为成骨细胞分化的关键因子,与骨发育和牙齿发育关系的研究正在不断开展。但Osx在正畸牙齿移动牙周组织的表达和分布,机械力作用下在体外培养的人牙周膜细胞中的表达变化情况,以及在人牙周膜细胞受力后向成骨样细胞分化的过程中所起的作用,国内外尚未有报道。
本研究通过建立大鼠正畸牙齿移动的动物模型和体外培养人牙周膜细胞,结合体内外加力实验技术和质粒转染等分子生物学技术,拟从组织、细胞水平以及分子生物学角度探讨Osx在正畸牙齿移动过程中对牙周组织改建的影响及作用机制,以期为进一步系统性研究正畸力作用下牙周组织骨改建的分子生物学机制开辟一条新途径。
方法
1.观察大鼠正畸牙周组织改建过程中Osx的表达变化
以大鼠上颌右侧第一磨牙为实验牙,采用拉簧加力法建立正畸牙齿移动的动物模型。在加力0h、1h、2h、4h、8h、12h、1d、3d、5d、7d和14d后,处死大鼠,固定,脱钙,制作大鼠第一磨牙牙周组织石蜡切片,采用HE染色观察牙周组织形态学变化,并采用免疫组化方法半定量分析Osx在牙周组织的表达变化。
2.观察机械力加载下人牙周膜细胞Osx mRNA和蛋白的表达变化
采用组织块法培养原代人牙周膜细胞,并做波形丝蛋白抗体和角蛋白抗体染色鉴定。取生长状态良好的第2-3代人牙周膜细胞,按2.5×10~5个/孔接种于6孔细胞培养板培养。待细胞融合80%,换用含2%FBS的条件培养液继续培养24h。后将六孔板置于37℃离心机加力支架中,实验组以离心机加力(631rpm,约80g相对离心力),加力时间分别为1h、2h、4h、6h、8h和12h。加力结束后,采用Real-time RT-PCR和Western blot方法分别观察Osx mRNA和蛋白表达情况;同时,采用细胞免疫荧光技术检测Osx蛋白绿色荧光在细胞内的表达定位,并统计分析Osx蛋白阳性表达的细胞数量及其核内阳性表达的细胞比例。
3.观察Osx过表达对人牙周膜细胞骨向分化的影响
取生长状态良好的第2-3代人牙周膜细胞,按2.5×10~5个/孔接种于6孔细胞培养板培养。待细胞融合80%,按阳离子脂质体Lipofeetamine ~(TM)2000说明操作,用获赠的重组质粒pcDNA3.1 flag-Osx和空载体质粒pcDNA3.1 flag转染人牙周膜细胞。采用Real-time RT-PCR和Western blot方法检测未转染、转染空质粒(pcDNA3.1 flag)、转染目的基因(pcDNA3.1 flag-Osx)三组细胞Osx mRNA和蛋白表达水平;应用试剂盒检测三组细胞碱性磷酸酶(alkaline phosphatase,ALP)活性变化;并采用RT-PCR技术观察Cbfal及相关成骨标志基因(ALP、OPN、OC、BSP和ColⅠ)的mRNA表达情况。三组细胞均加载6h离心力(631rpm)后,再次观察三组细胞的上述检测指标变化。
4.观察人重组骨形成蛋白2(rhBMP-2)作用下人牙周膜细胞Osx的表达
取生长状态良好的第3代细胞,以1×10~5个/孔的密度接种于6孔细胞培养板中。孵育24小时后,加入含rhBMP-2的培养液进行培养。实验分组1)按照不同的浓度分组:实验组用含10%FBS和浓度分别为50、100、150、200、250、300、400、600ng/ml rhBMP-2的DMEM培养液培养,诱导7d。每种浓度加6孔。2)按培养时间分组:实验组用含10%FBS和浓度为200ng/ml的rhBMP-2的培养液分别培养2d、3d、5d、7d、10d、14d和21d。每组加6孔。两对照组细胞均用含10%FBS的DMEM培养液培养。培养结束后,采用Real-time RT-PCR和Western blot方法检测各组细胞Osx mRNA和蛋白表达水平。
结果
1.大鼠正畸牙周组织改建过程中Osx的表达变化
在正常对照组,Osx在牙周膜呈弱阳性表达,分布较均匀,在靠近牙骨质侧牙周膜部分着色相对较重。正畸加力后大鼠牙周组织中Osx表达增强,表达贯穿正畸牙周组织改建的全过程。加力1d时,在压力区和张力区牙周膜Osx阳性着色均已显著增强,着色分布较均匀,且两侧着色强度无明显差异。随着加力时间的延长,Osx着色分布发生一定的改变。在张力区,靠近牙骨质和牙槽骨表面的牙周膜区域强阳性表达;在压力区,靠近牙骨质表面的牙周膜强阳性表达,而在发生明显骨吸收的牙槽骨侧无明显着色,而且张力区整体上比压力区阳性染色深,新骨形成区域阳性染色较强。
2.机械力作用下人牙周膜细胞Osx mRNA和蛋白的表达变化
Osx mRNA在正常人牙周膜细胞中的表达极其微弱。在机械力加载下,OsxmRNA的表达发生了一系列变化:细胞加力1h、2h时,Osx mRNA的表达略升高,但无统计学意义;加力4h时,表达开始明显增强(P＜0.01);随后快速增加至8h,表达显著增强(P＜0.01);后表达缓慢增加,持续到12h加力结束。
Osx蛋白在正常人牙周膜细胞中未见表达。细胞加载80g离心力1h、2h时,仍未检测到Osx蛋白表达(P＞0.05);加力4h时,呈现弱表达(P＜0.05);6h、8h时,表达逐渐增加(P＜0.01);12h时,表达显著增强,达到最高水平。变化具有时间依从性。
免疫荧光检测结果显示:正常的人牙周膜细胞内无绿色荧光显现;加力1h、2h的细胞内仍未检测到Osx阳性表达产物;加力4h后,少量细胞的细胞质内开始呈现微弱的绿色荧光(P＜0.01);加力8h时,约40%-50%的细胞发出明显的绿色荧光,细胞质、细胞核内均有阳性表达产物;加力12h时,大部分细胞(约87%)呈现强阳性表达,且主要集中在细胞核内。
3.Osx过表达对人牙周膜细胞骨向分化的影响
转染24h后,转染目的基因pcDNA3.1 flag-Osx组Osx mRNA表达水平相对未转染细胞升高28.1倍,同时Osx蛋白亦呈现一个强烈的表达(P＜0.01)。相反,转染空质粒(pcDNA3.1 flag)组的Osx mRNA和蛋白水平相对未转染组无明显变化(P＞0.05)。加力6h后,未转染组、转染空质粒组、转染Osx组三组细胞Osx表达水平均明显升高,但转染Osx组Osx mRNA和蛋白表达增加量约为其他两组的2倍。
正常人牙周膜细胞表达ALP,转染目的基因Osx后,ALP表达活性明显升高(P＜0.05),约为正常细胞的2.5倍,但转染空质粒组ALP表达未发生明显变化。三组细胞受到80g离心力,ALP表达均升高,但转染空质粒与未转染细胞相比,受力后ALP表达无明显差异(P＞0.05),而转染Osx质粒组受力后ALP表达进一步增强(P＜0.01),增长量约为未转染组的2.3倍。
转染空质粒组与未转染组间,Cbfal及五个成骨标志基因(ALP、OPN、OC、BSP和ColⅠ)的mRNA表达变化无明显差异(P＞0.05)。与未转染组相比,细胞转染目的基因Osx后,Cbfal mRNA表达无明显变化(P＞0.05),但五个成骨标志基因的mRNA表达均明显升高(~*P＜0.05,~(**)P＜0.01)。加力6h后,三组细胞的Cbfal mRNA表达无明显变化,而ALP、OPN、OC、BSP和Col I的mRNA表达水平均升高。但转染空质粒组与未转染组相比,受力后五个成骨标志基因变化无明显差异(P＞0.05);转染Osx细胞与未转染细胞相比,受力后五个成骨标志基因mRNA表达进一步上调(~*P＜0.05,~(**)P＜0.01)。
4.rhBMP-2作用下人牙周膜细胞Osx的表达
人牙周膜细胞在不同浓度rhBMP-2诱导7d后,相对对照组,Osx mRNA和蛋白均有明显的表达,但其表达强度不同,当浓度小于200ng/ml时,随着rhBMP-2浓度的增加,Osx mRNA和蛋白的表达均逐渐显著增强,当大于200ng/ml时,随着rhBMP-2浓度的增加,Osx mRNA和蛋白的表达略增加,基本趋于平缓。
在200ng/ml rhBMP-2不同时间诱导下,Osx mRNA表达变化具有时效性,具体如下:2d时,Osx mRNA表达略增强,但仍较弱(P＞0.05);培养3d时,Osx mRNA表达开始上调(P＜0.05);后迅速增强;10d时达到最高峰(P＜0.01);后逐渐减弱,但仍显著高于正常人牙周膜细胞中的水平。200ng/ml rhBMP-2诱导人牙周膜细胞2d时,未检测到Osx蛋白的表达;培养3d、5d时,Osx蛋白开始略有表达(P＜0.05);后表达逐渐增强,持续至21d(P＜0.01),表达趋于平稳。
结论
1.大鼠正畸牙齿移动过程中,牙周组织Osx表达增强贯穿牙周组织骨改建的全过程,且变化具有一定时空规律性。从而确定Osx参与了大鼠体内牙齿移动过程中的牙周组织骨改建。
2.应力刺激诱导人牙周膜细胞骨向分化过程中,Osx基因表达增强、蛋白合成活化,从而发挥信号转导作用。从体外细胞水平证实Osx参与了正畸力诱导的人牙周膜细胞骨向分化和牙周组织骨改建过程。
3.Osx通过上调成骨表型蛋白和功能蛋白的表达,促进加载下的人牙周膜细胞向成骨样细胞分化。
4.Osx是机械刺激和人牙周膜细胞骨向分化间的一个重要分子环节,在机械力诱导人牙周膜细胞成骨分化过程中,发挥着信号级联放大和转导调控作用。
5.BMP-2信号通路可能是介导机械信号诱导Osx表达的一条重要途径。
6.本研究为进一步系统性探讨机械力作用下牙周组织骨改建的分子生物学机制开辟了一条新途径,有助于更好的理解正畸牙齿移动的机理,从而为加快牙齿移动的正畸临床研究提供理论依据。
Background and Objective
Orthodontic 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 factorⅠ(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 typeⅠ(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.
Methods
1.Observe the changes of Osx expression in periodontal tissues during orthodontic tooth movement in rats
A 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 2.5×10~5 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
differentiation
Human PDL cells between passages 2 and 3 were seeded at 2.5×10~5 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~(TM)2000,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 cells
Human PDL cells were seeded at 1.0×10~5 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 1.0×10~5 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.
Results
1.The expression of Osx in periodontal tissues after orthodontic tooth movement in rats
In 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 differentiation
At 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 cells
After 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).
Conclusions
1.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.
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