A microrheometric assay of matrix mechanics

描述(申请人提供)：细胞合成、组织和重塑细胞外基质的三维网格，改变其组成、结构，并最终改变其机械性能，以满足个别组织的需要。了解(并最终操纵)细胞对细胞外基质机械特性的控制将对改进组织替代的工程至关重要，并将推动细胞外基质疾病的新治疗策略的发展。目前研究细胞介导的基质机制控制的方法受到时间和资源成本的限制。这项建议的中心目标是开发、验证和使用一种独特的生物测定方法，使快速、经济有效地测量细胞填充的细胞外基质的机械性能。所提出的方法，称为磁性扭转微观流变学，使用微米级的铁磁珠嵌入细胞基质结构(成纤维细胞种子的I型胶原凝胶)。嵌入的珠子被磁化并暴露在振荡的磁场中。珠子旋转被周围的母体抵抗，珠子牢固地连接到母体上。由此得到的扭矩和观察到的旋转之间的关系被用来表征基质的粘弹性行为。在第一个目标中，将开发和优化该分析，并根据标准技术验证其输出。在第二个目标中，将使用磁扭转微观流变学来测量在选定的基质活性介质和炎症介质单独和组合控制下细胞介导的基质粘弹性特性的调节。建议的分析在时间和大小尺度上探索复杂的、综合的细胞过程，服从于对化合物、治疗策略和分子扰动的资源高效筛选。这些研究的成功完成将为评估细胞介导的基质重塑提供一个强大的新的实验范式，为详细研究细胞建立和修改细胞外基质机制的分子和微结构机制奠定基础。这项提议的中心目标是开发一种新技术，能够快速有效地剖析控制细胞外基质机制的细胞过程。了解这些过程将改进替代组织的工程设计，并改善细胞外基质疾病的治疗。

DESCRIPTION (provided by applicant): Cells synthesize, organize, and remodel the three-dimensional latticework of the extracellular matrix, altering its composition, structure, and ultimately its mechanical properties to fulfill the needs of individual tissues. Understanding (and eventually manipulating) cellular control of extracellular matrix mechanical properties will be essential to the engineering of improved tissue replacements, and will drive the development of new treatment strategies for disorders of the extracellular matrix. Current approaches for investigating cell- mediated control of matrix mechanics are limited by time and resource costs. The central goal of this proposal is to develop, validate, and employ a unique bioassay that enables rapid, cost-effective measurement of the mechanical properties of cell-populated extracellular matrices. The proposed approach, termed magnetic twisting microrheometry, uses micron-scale ferromagnetic beads embedded within cell- matrix constructs (fibroblast-seeded collagen type I gels). The embedded beads are magnetized and exposed to an oscillatory magnetic twisting field. Bead rotations are resisted by the surrounding matrix, to which the beads are firmly coupled. The resulting relationship between applied torque and observed rotation is used to characterize the viscoelastic behavior of the matrix. In the first aim the assay will be developed and optimized, and its output validated against a standard technique. In the second aim magnetic twisting microrheometry will be employed to measure cell-mediated modulation of matrix viscoelastic properties under the control of selected matrix-active and inflammatory mediators, alone and in combination. The proposed assay probes a complex, integrated cellular process on a time and size scale amenable to resource efficient screening of compounds, treatment strategies, and molecular perturbations. Successful completion of the proposed studies could provide a powerful new experimental paradigm by which cell- mediated matrix remodeling is evaluated, setting the stage for detailed investigations into the molecular and microstructural mechanisms by which cells establish and modify extracellular matrix mechanics. The central goal of this proposal is to develop a new technology enabling rapid and efficient dissection of the cellular processes controlling extracellular matrix mechanics. Understanding these processes will improve the engineering of replacement tissues, and the treatment of extracellular matrix diseases.