Intracellular Curvature Sensing as a Regulator of Musculoskeletal Differentiation

描述(申请人提供)：修复骨丢失、骨折不愈合、脊柱融合、全关节置换，以及肌腱、韧带和肌肉断裂的修复程序。 每年有超过200万美国人。所有这些过程的外科修复技术都有可能通过基于生物材料纳米纤维的策略进行渐进或革命性的改进。这些肌肉骨骼组织由间充质干细胞(MSCs)发育而来，涉及到由各种细胞外基质蛋白和生长因子组成的独特的利基环境。毫不奇怪，ECM的成分是肌肉骨骼组织所独有的。细胞表达大约150种不同的蛋白质，参与近700种独特的相互作用，粘附性，它们用来感知和响应独特的ECM成分。在Addese3种蛋白中，有3种直接参与了Addese3种蛋白中36%的激酶相互作用，即FAK、Src和Fyn。这项提议试图阐明与MSC附着在呈现一定直径范围的纳米纤维上有关的导致表型改变的黏附信号的各个方面。了解底层底物的几何结构如何改变黏附相关蛋白的定位和激活将提供设计标准，使合成的MSC生态位能够向肌肉骨骼谱系定向分化。未来的生物材料基质不能忽视形状等简单方面在将祖细胞引导到靶组织上所起的作用。这笔赠款旨在融合再生医学、材料科学和细胞生物学的学科，以确定一种机制，通过这种机制，骨髓间充质干细胞对纳米纤维的弯曲做出感知和反应，从而改变表型，并最终改变肌肉骨骼谱系的世系承诺。支持这项赠款的初步证据表明，纤维直径和局部粘连大小/成熟度之间存在相关性。与最大粘连对应的纤维直径也显示出RhoA活性和细胞骨架硬度的增加。此外，纳米纤维直径与MAPK活性之间存在相关性，这表明可能与谱系承诺有关。特定目标1将生产直径范围从1.5？m到500 nm的纳米纤维基板；同时保持与纳米纤维基板的所有其他几何参数的一致性，并将检查FAK与Src或Fyn的结合。特定目标2研究了每个纤维直径上存在的独特表型，即迁移、增殖和谱系承诺。特异性目标3试图将前两个目标结合在一起，并将FAK/Src家族激酶结合和激活中依赖纳米纤维直径的变化与观察到的变化的表型相关联。该提案的成功完成将为未来的生物材料体系结构和 通过鉴定细胞内曲率感应机制来促进生物学的发展。

DESCRIPTION (provided by applicant): Procedures to repair bone loss, fracture non-unions, spinal fusions, total joint replacement, as well as, ruptures to tendon, ligament and muscle affect well over 2 million Americans annually. Surgical repair techniques for all of these procedures have the potential for either incremental or revolutionary improvement through biomaterial nanofiber based strategies. The development of these musculoskeletal tissues from mesenchymal stem cells, MSCs, involves a unique niche composed of assorted extracellular matrix proteins and growth factors. Not surprising, the ECM composition is unique to the musculoskeletal tissue. Cells express approximately 150 different proteins involved in nearly 700 unique interactions, the adhesome, which they use to sense and respond to unique ECM compositions. Of all current proteins in the adhesome, three are directly involved in 36% of all kinase interactions in the adhesome, FAK, Src and Fyn. This proposal seeks to make clear the aspects of adhesion signaling leading to altered phenotype involved with MSC attachment to nanofibers presenting a range of diameters. Understanding how the geometry of the underlying substrate alters the localization and activation of adhesion related proteins will provide design criteria enabling the generation of synthetic MSC niche's capable of directed differentiation down musculoskeletal lineages. Future biomaterial substrates cannot ignore the role that simple aspects such as shape have on directing progenitor cells to the target tissue. This grant seeks to merge the disciplines of regenerative medicine, materials science and cell biology to determine a mechanism by which MSCs sense and respond to the curvature of a nanofiber leading to altered phenotype and ultimately altered lineage commitment down musculoskeletal lineages. Preliminary evidence in support of this grant has indicated that there is a correlation between fiber diameter and focal adhesion size/maturity. The fiber diameters corresponding to the largest adhesions also demonstrated increased RhoA activity and cytoskeletal stiffness. Additionally, nanofiber diameter demonstrated a correlation over MAPK activity, indicating a possible connection to lineage commitment. Specific Aim 1 will produce nanofiber substrates that demonstrate a range of diameters from 1.5¿m to 500nm; while maintaining consistency with all other geometric parameters of a nanofiber substrate and will examine the binding of FAK to either Src or Fyn. Specific Aim 2 examines the unique phenotype present on each fiber diameter, i.e. migration, proliferation and lineage commitment. Specific Aim 3 seeks to bring together the previous two aims and correlate the nanofiber diameter dependent alterations in FAK/Src-family kinase binding and activation with the altered phenotypes observed. Successful completion of this proposal will provide design guidelines for future biomaterial architectures and advance biology through identification of an intracellular curvature sensing mechanism.