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形式的定向细胞迁移，称为机械性（或硬旋转），其中速度和定向持久性均受基质机械性质的影响。利用mechanotaxis治疗，结合趋化性和haptotaxis，可能代表了一种新的方法来设计生物材料，提供适当的化学和机械线索的组合，以促进成骨细胞（或间充质干细胞）从周围的健康组织到小骨缺损的选择性迁移。在进行颅面组织工程应用之前，需要进行额外的基础科学研究，以解决骨机械性的相关性，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。该R 03申请提出了一组可行性研究，以确定前成骨细胞系（MC 3 T3-E1）是否会通过模型基质上的机械性迁移。具体而言，本提案旨在：1.）使用光聚合工艺制造具有预定义机械顺应性的聚丙烯酰胺水凝胶，并随后用细胞外基质（ECM）蛋白质官能化这些水凝胶以支持MC 3 T3-E1粘附和铺展; 2.）量化MC 3 T3-E1细胞在ECM改性的水凝胶基底上的细胞迁移速度和方向持久性，所述ECM改性的水凝胶基底具有均匀的刚度、刚度的尖锐界面或刚度的梯度;以及3.）确定Rho GTP酶在感知底物顺应性和调节机械定向反应中所起的作用（如果有的话）。 这些目标的成功完成将不仅提供有用的基本数据细胞迁移机制，但也可能指导未来的骨传导策略，利用这种mechanotaxis范例。 申请者将在今后的R21和R 01提案中探讨这些战略。

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.