Unraveling the Mechanism of Mechanotransduction in Hierarchical Collagen Fiber Formation

项目摘要 胶原纤维是整个身体组织，特别是肌腱， 韧带和韧带。细胞按层次组织这些纤维，将它们从nm宽的原纤维组装成更大的 纤维和纤维束，随着机械需求的增加，尺寸和强度不断增长。受伤破坏了这一点 组织，导致功能丧失，疼痛和活动能力下降。不幸的是，胶原纤维不 受伤后再生，也不是在工程更换，造成缺乏修复选项撕裂肌腱， 韧带和韧带。我们的长期目标是了解细胞如何调节胶原纤维的形成， 设计功能性替代品，并在体内驱动全身肌肉骨骼组织的修复。作为 为了实现这一目标，本提案的目的是探索机械提示如何通过细胞传递， 收缩和拉伸激活的离子通道调节韧带成纤维细胞的分级纤维的发育。 而细胞收缩力，高度调节的整合素，粘着斑激酶（FAK），和肌动球蛋白 网络，是公认的发挥主要作用的胶原纤维排列，最近的工作表明，拉伸- 激活的离子通道TRPV 4和Piezo 1也在调节胶原蛋白中发挥独立和短暂的作用 organization.然而，这项工作在很大程度上局限于2D表面，无组织的胶原蛋白凝胶，或小鼠 这些模型都缺乏支配人类肌肉骨骼组织的较大纤维和纤维束。最近我们 开发了一种新的培养装置，引导细胞产生天然大小的分层组织的原纤维，纤维， 培养6周以上的束。这种新颖的系统提供了独特的能力，最终剖析机械 发展的线索，并研究这些力量如何引导细胞产生强大的层次纤维。我们 通过基于整合素收缩和拉伸激活离子通道的假设机械信号对于 细胞驱动的分层纤维形成，每个调节纤维成熟的不同方面， 没有动态负载。具体来说，我们假设，虽然通过FAK的细胞收缩是进行性的， TRPV 4将在原纤维水平上调节排列和重塑，而Piezo 1将 在纤维和纤维束水平通过胶原交联调节基质成熟。在目标1中，我们将评估 当细胞产生的收缩力驱动时，FAK，TRPV 4和Piezo 1在被动静态培养中的作用 纤维形成，在目标2中，我们将评估它们的贡献如何随着动态机械刺激而变化。 在这两个目标，我们将探讨如何每一个信号机制，当抑制或激活，改变胶原蛋白 在原纤维、纤维和束长度尺度上的组织化，蛋白多糖积累和胶原交联， 所有这些对整个组织功能和力学都很重要。更好地理解机械线索如何驱动细胞 生产层次纤维不仅是创造功能替代品的组成部分， 用于损伤后再生胶原纤维、减少瘢痕形成和开发最佳的治疗靶点， 再生全身肌肉骨骼组织的康复方案。

PROJECT SUMMARY Collagen fibers are the primary source of strength and function in tissues throughout the body, particular tendons, ligaments, and menisci. Cells organize these fibers hierarchically, assemble them from nm-wide fibrils, into larger fibers and fascicles, growing in size and strength with increasing mechanical demand. Injuries disrupt this organization, resulting in loss of function, pain, and decreased mobility. Unfortunately, collagen fibers do not regenerate after injury, nor in engineered replacements, creating a lack of repair options for torn tendons, ligaments, and menisci. Our long-term goal is to understand how cells regulate collagen fiber formation so to engineer functional replacements and drive repair in vivo for musculoskeletal tissues throughout the body. As a step towards this goal, the objective of this proposal is to explore how mechanical cues transmitted via cellular contraction and stretch-activated ion channels regulate ligament fibroblast’s development of hierarchical fibers. While cellular contraction forces, highly regulated by integrins, focal adhesion kinase (FAK), and the actomyosin network, are well established to play a major role in collagen fibril alignment, recent work has suggested stretch- activated ion channels, TRPV4 and Piezo1, also play independent and transient roles in regulating collagen organization. However, this work is largely confined to 2D surfaces, unorganized collagen gels, or mouse models, which all lack the larger fibers and fascicles that dominate human musculoskeletal tissues. Recently, we developed a novel culture device that guides cells to produce native-size hierarchically organized fibrils, fibers, and fascicles over 6 weeks of culture. This novel system provides the unique ability to finally dissect the mechanical cues of development and investigate how these forces guide cells to produce strong hierarchical fibers. We hypothesize mechanical signals via integrin-based contraction and stretch-activated ion channels are critical to cell-driven hierarchical fiber formation, with each regulating different aspects of fiber maturation, both with and without dynamic load. Specifically, we hypothesize that while cellular contraction via FAK is critical to progressive hierarchical development, TRPV4 will regulate alignment and remodeling at the fibril level, and Piezo1 will regulate matrix maturation via collagen crosslinking at the fiber and fascicle level. In Aim 1 we will evaluate the contribution of FAK, TRPV4, and Piezo1 in passive static culture when cell-generated contraction forces drive fiber formation and in Aim 2 we will evaluate how their contribution changes with dynamic mechanical stimulation. In both aims, we will investigate how each signaling mechanism, when inhibited or activated, alters collagen organization at the fibril, fiber, and fascicle length-scale, proteoglycan accumulation, and collagen crosslinking, all important to overall tissue function and mechanics. A better understanding of how mechanical cues drive cells to produce hierarchical fibers is integral not only to creating functional replacements, but also identifying therapeutic targets for regenerating collagen fibers after injury, reducing scar formation, and developing optimal rehabilitation protocols for regenerating musculoskeletal tissues throughout the body.