Does FAK mediate implant microtopography

描述(申请人提供)：随着老龄化人口的增加，更多的人被定义为部分无牙。护理标准已经扩大到包括牙科植入物的使用。为了成功，植入物必须与周围的硬组织结合，这一过程被称为骨整合。就分子水平上的微地形对细胞的影响而言，骨整合的定义是模糊的。在骨整合过程中，这些表面如何影响成骨细胞分化的分子途径尚不清楚。可预测的种植体表面形态与临床结果的整合依赖于在细胞和分子水平上对三维生物学的明确理解。我们的假设是，牙种植体表面微形态对成骨细胞分化和运动的影响是通过粘着斑激酶(FAK)信号通路调节的。FAK是一种与整合素受体相关的细胞内酪氨酸激酶。我们的总体战略是在一系列具有良好特征的钛种植体表面微形貌(600g凹槽与50um氧化铝喷砂)上，对人腭间充质前成骨细胞进行细胞黏附和分化以及迁移研究。我们将确定微地形效应是否通过FAK进行调制。实时荧光定量聚合酶链式反应和蛋白质印迹技术将用于从野生型对照或使用siRNA-FAK、frnk显性负性结构或Y925F HA标记的FAK突变体降低FAK基因和蛋白表达水平的细胞中分离出的RNA和蛋白质。这将使我们能够确定FAK、FAK-磷酸酪氨酸或FAK-MAPK通路是否受到植入物表面微地形的调节。扫描电子显微镜将使我们能够评估静态表型变化。标记细胞的实时成像将允许实时评估动态变化。这项工作的意义在于，它将深入了解如何以受控和可预测的部位特定方式构建骨组织，从而通过改变种植表面的微地形来影响生物材料表面的传导和诱导效应。这将允许一种受控的、局部的、特定部位的组织工程方法与牙科种植治疗相结合。

DESCRIPTION (provided by applicant): As the aging population increases more individuals are being defined as partially edentulous. Standards of care have expanded to include the use of Dental implants. To be successful the implant must integrate with the surrounding hard tissues, a process known as osseointegration. The definition of osseointegration is vague with respect to cellular influences of the microtopography at the molecular level. How these surfaces influence molecular pathways of osteoblast differentiation during osseointegration is not known. Integration of predictable implant surface topography with clinical outcomes depends on a well defined understanding of the three dimensional biology at the cellular and molecular level. Our hypothesis is that the effect of Dental implant surface microtopographies on osteoblast differentiation and motility is regulated through focal adhesion kinase (FAK) signaling pathways. FAK is an intracellular tyrosine kinase that is associated with integrin receptors. Our overall strategy is to perform cell adhesion and differentiation, and migration studies with human palatal mesenchymal pre-osteoblast cells on a series of well characterized titanium implant surface microtopographies 600g grooved versus 50um Al2O3 sandblasted). We will determine if microtopography effects are modulated through FAK. Real time PCR and Western blotting strategies will be used on RNA and protein isolated from wild type controls, or cells whose level of FAK gene and protein expression has been reduced using siRNA-FAK, FRNK dominant negative constructs, or Y925F HA-tagged FAK mutants. This will allow us to determine if FAK, FAK-phosphotyrosine, or FAK-MAPK pathways are modulated by implant surface microtopographies. Scanning electron microscopy will allow us to evaluate static phenotypic changes. Live imaging of labeled cells will allow for evaluation of dynamic changes in real time. The significance of this work is that it would yield insight into how osseous tissues may be engineered in a controlled and predictable site specific manner with respect to conductive and inductive effects of the biomaterial surface by alteration of the implant surface microtopography. This would allow for a controlled, localized, site specific tissue engineering approach in association with Dental implant therapy.