Engineering Bone Cell Migration Via Mechanotaxis

描述（由申请人提供）：最近在文献中描述了一种新的定向细胞迁移形式，称为机械趋向性（或硬性趋向性），其中速度和方向持久性都受到基质机械特性的影响。利用机械趋向性治疗，结合趋化性和触觉趋向性，可能是设计生物材料的一种新方法，这种生物材料提供了化学和机械线索的适当组合，以促进成骨细胞（或间充质干细胞）从周围健康组织选择性迁移到小骨缺损中。在进行颅面组织工程应用之前，需要更多的基础科学研究来解决骨机械趋向性的相关性，并努力确定细胞感知其局部机械环境以启动细胞运动程序的机制。本R03申请提出了一组可行性研究，以确定成骨前细胞系（MC3T3-E1）是否会通过机械趋向性在模型基质上迁移。具体来说，本提案旨在：1.)使用光聚合工艺制备具有预定机械顺应性的聚丙烯酰胺水凝胶，并随后用细胞外基质（ECM）蛋白功能化这些水凝胶，以支持MC3T3-E1的粘附和扩散；2)。量化MC3T3-E1细胞在ecm修饰的水凝胶基质上的细胞迁移速度和定向持久性，这些基质具有均匀的刚度、尖锐的刚度界面或刚度梯度；和3)。确定Rho GTPases在感应底物顺应性和调节机械策略反应中起什么作用（如果有的话）。这些目标的成功完成不仅将为细胞迁移机制提供有用的基础数据，而且可能指导未来利用这种机械趋向性范式的骨传导策略。这些策略将在申请人未来的R21和R01提案中进行探讨。

DESCRIPTION (provided by applicant): A new form of directed cell migration, called mechanotaxis (or durotaxis), in which both speed and directional persistence are influenced by substrate mechanical properties, has been recently described in the literature. Exploiting mechanotaxis therapeutically, in conjunction with chemotaxis and haptotaxis, may represent a new approach to designing biomaterials that provide the appropriate combinations of chemical and mechanical cues to promote the selective migration of osteoblasts (or mesenchymal stem cells) from surrounding healthy tissue into small bony defects. Prior to pursuing craniofacial tissue engineering applications, additional basic science studies addressing the relevance of mechanotaxis for bone, combined with efforts to determine the mechanisms by which cells sense their local mechanical environment to initiate a program of cell motility, are required. This R03 application proposes a set of feasibility studies to determine if a pre-osteoblastic cell line (MC3T3-E1) will migrate via mechanotaxis on model substrates. Specifically, this proposal aims to: 1.) Fabricate polyacrylamide hydrogels with pre-defined mechanical compliance using a photopolymerization process, and to subsequently functionalize these hydrogels with extracellular matrix (ECM) proteins to support MC3T3-E1 adhesion and spreading; 2.) Quantify cell migration speed and directional persistence of MC3T3-E1 cells on ECM-modified hydrogel substrates possessing uniform stiffness, sharp interfaces in stiffness, or gradients in stiffness; and 3.) Determine what role, if any, is played by the Rho GTPases in sensing substrate compliance and regulating the mechanotactic response. Successful completion of these aims will not only provide useful fundamental data regarding cell migration mechanisms, but may also guide future osteoconductive strategies that exploit this mechanotaxis paradigm. Such strategies will be explored in the applicant's future R21 and R01 proposals.