Significance of the neuronal guidance molecule Semaphorin 3A for bone remodelling during orthodontic tooth movement

咬合不正和颌骨错位在学龄儿童和成人中的患病率很高。除了各种细胞因子和生长因子外，神经导向分子（NGM）因其在骨稳态中的作用以及在牙齿移动中的潜在作用而受到关注。最近，我们发现Ephrin配体和Eph受体参与牙齿移动过程中的骨重建。在最近被认为参与骨重建调节的NGM中，Semaphorin 3A（Sema 3A）是最令人感兴趣的，因为它同时诱导成骨细胞的成骨分化和干扰破骨细胞分化。为了继续我对正畸牙齿移动中神经元引导分子的分析，并阐明其潜在机制，本项目的目标是（i）研究不同的机械力是否调节牙槽骨的牙周成纤维细胞和成骨细胞中Sema 3A及其受体Neuropilin 1和PlexinA 1的表达，（ii.）研究哪些生化信号参与Sema 3A的机械调节（iii.）研究Sema 3A刺激对牙槽骨成骨细胞分化的影响，以及（iv.）阐明Sema 3A依赖性成骨细胞分化的分子机制。为了实现这一点，我将首先刺激牙周成纤维细胞和牙槽骨成骨细胞机械施加压力和张力。两种细胞类型的培养以及机械刺激在我们的实验室中得到了很好的建立。通过定量PCR和western blot分析Sema 3A及其受体的表达。其次，我将研究潜在的机械诱导途径导致Sema 3A的改变。为此，测试不同MAP激酶的磷酸化，并通过qPCR和蛋白质印迹研究推定的Sema 3A转录因子Osterix的参与。第三，重组人Sema 3A将刺激牙槽骨成骨细胞，并通过定量PCR监测不同成骨细胞分化标志物的表达来分析其对成骨分化的影响。第四，Sema 3A介导的成骨细胞分化的机制将通过研究Rac 1 GT3和β-连环蛋白转录因子的参与，分别使用下拉测定和免疫荧光染色和western blot来研究。对Ephrin/Eph家族成员的研究和Sema 3A的初步数据表明，神经元引导分子在正畸牙齿移动调节中的作用可能被低估了。因此，该项目可能会照亮额外的细胞和分子事件，这可能是有用的药理学操作的目标，旨在无副作用和加速正畸牙齿移动。

Malocclusions and jaw misalignments appear with high prevalence in school children and in adults. Besides various cytokines and growth factors neuronal guidance molecules (NGM) gained attention for their roles in bone homeostasis and thus potential roles during tooth movement. Recently we have shown that Ephrin ligands and Eph receptors are involved in bone remodeling during tooth movement. Among the NGMs which have recently been implicated in the regulation of bone remodeling Semaphorin 3A (Sema3A) is the most interesting as it concurrently induces osteogenic differentiation in osteoblasts and disturbs osteoclast differentiation. To continue my analysis of neuronal guidance molecules in orthodontic tooth movement and to elucidate the underlying mechanisms the objectives of this project are (i.) to investigate if different mechanical forces modulate the expression of Sema3A and its receptors Neuropilin1 and PlexinA1 in periodontal fibroblasts and osteoblasts of the alveolar bone, (ii.) to study which biochemical signals are involved in the mechanical modulation of Sema3A (iii.) to investigate the effects of Sema3A stimulation on alveolar bone osteoblasts differentiation and (iv.) to elucidate the molecular mechanisms underlying the Sema3A dependent osteoblast differentiation. To achieve this, I will first stimulate periodontal fibroblasts and alveolar bone osteoblasts mechanically by applying compressive and tension forces. Culture of both cell types, as well as the mechanical stimulation is well established in our laboratory. The expression of Sema3A and its receptors will be analyzed by means of quantitative PCR and western blot. Second, I will investigate the potential mechanoinductive pathway leading to Sema3A alterations. To this end, different MAPKinases are tested for phosphorylation and the involvement of the putative Sema3A transcription factor Osterix will be studied by qPCR and western blot. Third, alveolar osteoblasts will be stimulated by recombinant human Sema3A and its impact on osteogenic differentiation will be analyzed by monitoring the expression of different osteoblast differentiation markers by quantitative PCR. Fourth, the mechanism underlying the Sema3A mediated osteoblast differentiation will be investigated by studying the involvement of the Rac1 GTPase and the beta-catenin transcription factor using pull down assays and immunofluorescent staining and western blot, respectively. Own research on members of the Ephrin/Eph family and preliminary data on Sema3A suggest that the role of neuronal guidance molecules in the regulation of orthodontic tooth movement might have been underestimated. Thus, this project might illuminate additional cellular and molecular events which might be useful as targets for pharmacological manipulations aiming for a side effect free and accelerated orthodontic tooth movement.